FAM222a as a therapeutic target

Abstract

The present disclosure related to methods of treating various diseases that can benefit from modulating angiogenesis and / or endothelial-to-mesenchymal transition (EndMT) pathways, such as retinopathies and cancer, by modulating family with sequence similarity 222 member A (FAM222A) expression in the endothelium (e.g., endothelial cells) of a subject. For example, FAM222A can be modulated with FAM222A inhibitors described herein, thereby reducing angiogenesis, or FAM222A activators described herein, thereby increasing angiogenesis.

Description

[0001] PATENT Attorney Docket No.: 51792-002WO2 FAM222A AS A THERAPEUTIC TARGET STATEMENT AS TO FEDERALLY FUNDED RESEARCH This invention was made with government support under Grant Nos. R01EY030140, R01EY029238, and HL125894 awarded by the National Institute of Health, the National Eye Institute, and the National Heart, Lung, and Blood Institute, respectively SEQUENCE LISITNG The instant application contains a Sequence Listing which has been submitted electronicallyin XML format and is hereby incorporated by reference in its entirety. Said XML copy, created onNovember 1, 2024, is named “51792-002WO2_Sequence_Listing)_11_1_2024.xml” and is 124,153 bytes in size. TECHNICAL FIELD The present disclosure related to methods of treating various diseases that can benefit from modulating angiogenesis and / or endothelial-to-mesenchymal transition (EndMT) pathways by modulating family with sequence similarity 222 member A (FAM222A). BACKGROUND Bromodomain and extra-terminal domain-containing epigenetic reader proteins (BETs), such as bromodomain-containing protein 4 (BRD4), orchestrate transcriptional programs induced by pathogenic stimuli, as intensively studied in cardiovascular disease and elsewhere. In endothelial cells (ECs), BRD4 directs induced proinflammatory, proatherosclerotic transcriptional responses. BET inhibitors, like JQ1, repress these effects and decrease atherosclerosis. While BET effects in pathogenic conditions have prompted therapeutic BET inhibitor development, the therapeutic potential of BET inhibition under basal conditions, including in ECs, has remained understudied. Diseases that might benefit from modulating EC function (e.g., preventing endothelial-to-mesenchymal transition (EndMT) pathways in ECs), such as cancer and various vascular diseases, may benefit from BET inhibition therapies or modulation of related pathways (e.g., EndMT pathways) because ECs play a crucial role in angiogenesis and vessel barrier stability while careful control over endothelial cell growth, proliferation, and cell cycling is also necessary. EndMT is implicated in many diseases and involves the loss of beneficial EC properties and the gain of muscle / mesenchymal properties, which may further complicate disease pathology (e.g., advance fibrosis). Thus, there remains a need in the field to identify therapeutic targets capable of modulating angiogenesis and blood vessel stability for treatment of disease. Such therapies can be successful for treating vascular-related diseases like retinopathy, peripheral artery disease (PAD), pulmonary arterial hypertension (PAH), coronary disease, cerebrovascular disease, atherosclerosis, and ischemia, or mitigate the blood supply to tumors in various cancers, such as breast cancer, colorectal cancer (CRC), esophageal cancer, gastric cancer ovarian cancer, prostate cancer, renal cell carcinoma (RCC), and non-small lung cancer (NSCLC). PATENT Attorney Docket No.: 51792-002WO2 SUMMARY OF THE INVENTION In one aspect, the disclosure provides a method of treating a retinopathy in a subject comprising administering to the subject an effective amount of a family with sequence similarity 222 member A (FAM222A) inhibitor. In some embodiments, the retinopathy is a proliferative retinopathy. In some embodiments, the retinopathy is a non-proliferative retinopathy. In some embodiments, the retinopathy is selected from the group consisting of: diabetic retinopathy, retinopathy of prematurity (ROP), hypertensive retinopathy, and central serous retinopathy. In another aspect, the disclosure provides a method of treating cancer in a subject having atumor, the method comprising administering to the subject an effective amount of a FAM222A inhibitor. In some embodiments, the tumor is an angiogenic tumor. In some embodiments, the FAM222A inhibitor reduces tumor angiogenesis. In some embodiments, the tumor is a non- angiogenic tumor. In some embodiments, the tumor is chemoresistant and / or radioresistant. In some embodiments, the tumor comprises a mesenchymal cell. In some embodiments, the mesenchymal cell is a migratory mesenchymal cell and treating the cancer reduces metastasis of the migratory mesenchymal cell. In some embodiments, the cancer is selected from the group consisting of breast cancer, colorectal cancer (CRC), esophageal cancer, gastric cancer ovarian cancer, prostate cancer, renal cell carcinoma (RCC), and non-small lung cancer (NSCLC). In some embodiments, the FAM222A inhibitor reduces angiogenesis of an endothelial cell in the subject. In some embodiments, the FAM222A inhibitor reduces endothelial cell proliferation, endothelial cell migration, and / or endothelial-to-mesenchymal transition (EndMT). In some embodiments of any of the foregoing aspects, the FAM222A inhibitor is an inhibitory nucleic acid molecule. In some embodiments, the inhibitory nucleic acid molecule is an anti-sense oligonucleotide (ASO), a small interfering RNA (siRNA), a short hairpin RNA (shRNA), a double- stranded RNA (dsRNA), or a microRNA (miRNA). In some embodiments, the inhibitory nucleic acid molecule is an ASO. In some embodiments, the ASO is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to FAM222A (e.g., any one of SEQ ID NOs: 1-9), or a portion thereof. In some embodiments, the ASO comprises 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 10-15. In some embodiments, the inhibitory nucleic acid molecule is an siRNA. In some embodiments, the siRNA is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to FAM222A (e.g., any one of SEQ ID NOs: 1-9), or a portion thereof. In some embodiments, the siRNA comprises: (a) a sense strand comprising 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 16, 18, 20, 22, 24, 26, 28, and 30; and (b) an antisense strand comprising 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, or 100% sequence PATENT Attorney Docket No.: 51792-002WO2 identity to any one of SEQ ID NOs: 17, 19, 21, 23, 25, 27, 29, and 31. In some embodiments, the siRNA comprises a sense strand comprising 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 16, and an antisense strand comprising 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 17. In some embodiments, the siRNA comprises a sense strand comprising 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 18, and an antisense strand comprising 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 19. In some embodiments, the siRNA comprises a sense strand comprising 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 20, and anantisense strand comprising 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, or 100%sequence identity to SEQ ID NO: 21. In some embodiments, the siRNA comprises a sense strand comprising 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 22, and an antisense strand comprising 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 23. In some embodiments, the siRNA comprises a sense strand comprising 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 24, and an antisense strand comprising 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 25. In some embodiments, the siRNA comprises a sense strand comprising 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 26, and an antisense strand comprising 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 27. In some embodiments, the siRNA comprises a sense strand comprising 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 28, and an antisense strand comprising 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 29. In some embodiments, the siRNA comprises a sense strand comprising 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 30, and an antisense strand comprising 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 31. In some embodiments, the inhibitory nucleic acid molecule is an shRNA. In some embodiments, the shRNA is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to FAM222A (e.g., any one of SEQ ID NOs: 1-9), or a portion thereof. In some embodiments, the inhibitory nucleic acid molecule is a dsRNA. In some embodiments, the dsRNA is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to FAM222A (e.g., any one of SEQ ID NOs: 1-9), or a portion thereof. In some embodiments, the inhibitory nucleic acid molecule is a miRNA. In some embodiments, the miRNA is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to FAM222A (e.g., any one of SEQ ID NOs: 1-9), or a portion thereof. PATENT Attorney Docket No.: 51792-002WO2 In some embodiments, the inhibitory nucleic acid molecule comprises a non-natural or modified nucleoside or nucleotide. In some embodiments, the non-natural or modified nucleoside or nucleotide comprises a 2′-O-methyl (2′-O-Me) modified nucleoside, a phosphorothioate (PS) bond between nucleosides, and a 2′-fluoro (2′-F) modified nucleoside. In some embodiments, the inhibitory nucleic acid molecule further comprises a targeting moiety. In some embodiments, the targeting moiety targets an endothelial cell (EC) in the subject. In some embodiments, the targeting moiety targets the subject’s endothelium. In some embodiments, the targeting moiety is selected from the group consisting of: platelet endothelial cell adhesion molecule (PECAM-1), intercellular adhesion molecule 1 (ICAM1), vascular cell adhesion protein 1 (VCAM1), and Von Willebrand factor (vWF). In some embodiments, the inhibitory nucleic acid molecule comprises a delivery vehicle. In some embodiments, the delivery vehicle is a lipid nanoparticle (LNP). In some embodiments, the delivery vehicle is an exosome. In some embodiments, the delivery vehicle is a micelle. In some embodiments, the delivery vehicle is a liposome. In some embodiments of any of the foregoing aspects, the FAM222A inhibitor is abromodomain-containing protein 4 (BRD4) degrader. In some embodiments, the BRD4 degrader is dBET6. In some embodiments, the BRD4 degrader is MZ1. In yet another aspect, the disclosure provides a method of treating a vascular disease in a subject comprising administering to the subject an effective amount of a FAM222A activator. In some embodiments, the vascular disease is selected from the group consisting of: peripheral artery disease (PAD), pulmonary arterial hypertension (PAH), coronary disease, cerebrovascular disease, atherosclerosis, and ischemia. In yet another aspect, the disclosure provides a method of treating PAD in a subject comprising administering to the subject an effective amount of a FAM222A activator. In yet another aspect, the disclosure provides a method of treating PAH in a subject comprising administering to the subject an effective amount of a FAM222A activator. In yet another aspect, the disclosure provides a method of treating coronary disease in a subject comprising administering to the subject an effective amount of a FAM222A activator. In yet another aspect, the disclosure provides a method of treating cerebrovascular disease in a subject comprising administering to the subject an effective amount of a FAM222A activator. In yet another aspect, the disclosure provides a method of treating atherosclerosis in a subject comprising administering to the subject an effective amount of a FAM222A activator. In yet another aspect, the disclosure provides a method of treating ischemia in a subject comprising administering to the subject an effective amount of a FAM222A activator. In some embodiments of any of the foregoing aspects, the FAM222A activator is a bromodomain and extraterminal domain-containing epigenetic reader protein (BET) inhibitor. In some embodiments, the BET inhibitor is selected from the group consisting of JQ1, iBET-151, PFI-1, bromosporine, birabresib, CPI-203, or pelabresib. In some embodiments, the BET inhibitor (e.g., BRD4 inhibitor) is JQ1. In some embodiments, the BET inhibitor (e.g., BRD4 inhibitor) is iBET-151. In some embodiments, the BET inhibitor (e.g., BRD4 inhibitor) is a combination of JQ1 and iBET-151. In PATENT Attorney Docket No.: 51792-002WO2 some embodiments, the BET inhibitor (e.g., BRD4 inhibitor) is PFI-1. In some embodiments, the BET inhibitor (e.g., BRD4 inhibitor) is bromosporine. In some embodiments, the BET inhibitor (e.g., BRD4 inhibitor) is birabresib. In some embodiments, the BET inhibitor (e.g., BRD4 inhibitor) is CPI-203. In some embodiments, the BET inhibitor (e.g., BRD4 inhibitor) is pelabresib. In some embodiments of any of the foregoing aspects, the FAM222A activator is a vector (e.g., an expression vector) that overexpresses FAM222A. In some embodiments, the vector is an adenovirus (e.g., Ad2, Ad5, Ad9, Ad15, Ad17, Ad19, Ad20, Ad22, Ad26, Ad27, Ad28, Ad30, or Ad39), a rhabdovirus (e.g., vesicular stomatitis virus), a retrovirus, a lentiviral vector, an adeno-associated vector, a poxviruse, a herpes viral vector, or a Sindbis viral vector. In some embodiments, the vectoris a lentiviral construct that overexpresses FAM222A.In some embodiments, the vector (e.g., expression vector) comprises a non-natural or modified nucleoside or nucleotide. In some embodiments, the non-natural or modified nucleoside or nucleotide comprises a 2′-O-Me modified nucleoside, a PS bond between nucleosides, and 2′-F modified nucleoside. In some embodiments, the vector (e.g., expression vector) further comprises a targeting moiety. In some embodiments, the targeting moiety targets an EC in the subject. In some embodiments, the targeting moiety targets the subject’s endothelium. In some embodiments, thetargeting moiety is selected from the group consisting of: PECAM-1,ICAM1, VCAM1,andvWF.In some embodiments, the vector (e.g., expression vector) comprises a delivery vehicle. In some embodiments, the delivery vehicle is a lipid nanoparticle (LNP). In some embodiments, the delivery vehicle is an exosome. In some embodiments, the delivery vehicle is a micelle. In some embodiments, the delivery vehicle is a liposome. In some embodiments of any of the foregoing aspects, the subject: (i) is to receive a stent or a bypass graft prior to, currently with, or subsequent to the administration of the FAM222A inhibitor or FAM222A activator; and / or (ii) is in a state of hypoxemia prior to, currently with, or subsequent to the administration of the FAM222A inhibitor or FAM222A activator. In some embodiments of any of the foregoing aspects, the method further comprises administering a second therapeutic agent to the subject.

[0002] PATENT Attorney Docket NO.: 51792-002WO2 BRIEF DESCRIPTION OF THE DRAWINGS The accompanying drawings are included to illustrate embodiments of the disclosure and further an understanding of its implementations. FIG.1A is a heatmap of endothelial cell (EC) genes under basal conditions whose mRNA was significantly repressed or induced by the pan-BET inhibitor JQ1 vs control (log2 fold change >2 or <2; FDR, <0.05). Z-score normalized expression ranges from +2.5 to −2.5. FIG.1B is a volcano plot of significantly JQ1-induced or JQ1-repressed genes vs control (log2 fold change <2 or >2; FDR, <0.05). Gene ontology (GO) pathway analysis of mRNAssignificantly modulated by JQ1, organized by adjusted log10 p-value, is shown below the volcano plot.FIG.1C is a Venn diagram of the top nine genes whose mRNA was most induced by JQ1. Family with sequence similarity 222 member A (FAM222A) was the only non-histone, JQ1-induced gene with no reported role in EC or cell cycling / proliferation. FIG.2A is a schematic of the FAM222A gene, also known as C12orf34. The schematic includes chromosome location, gene length, start site, RNA splicing variants, and coding exons. FIG.2B is a schematic of FAM222A’s genomic region and predicted transcriptional products as seen in the National Library of Medicine database. FIG.2C is a predicted FAM222A protein 3D structure in AlphaFold database. The model confidence key represents the level of evidence of the model. No specific known domains are predicted in the structure. FIG.2D is a schematic of the FAM222A protein highlighting that it has one predicted domain (34-155 aa) in the Pfam database with no identified homology with other domains and no predicted specific functional associations. FIG.2E is a schematic of FAM222A predicted subcellular localizations as seen in Compartments database. FIG.3A are bar graphs showing the fold change of FAM222A mRNA expression in human umbilical vein endothelial cells (HUVECs) treated with different concentrations of BET inhibitors (JQ1 and iBET-151) for 3 hours (n=3 per group). Data presented as mean ± SEM. Each data point represents a biological replicate. P values were performed by two-way ANOVA with Tukey post hoc test. FIG.3B is a bar graph showing relative luciferase activity in human embryonic kidney (HEK) 293T cells transfected with a human FAM222A promoter-containing (1523 bp proximal to start site) luciferase-reporter plasmid and assayed for luciferase activity without or with JQ1 treatment at the concentrations shown (3 hours, n=6 per group, *p<0.05).. Data presented as mean ± SEM. Each data point represents a biological replicate. P values were performed by two-way ANOVA with Tukey post hoc test. FIG.3C is a representative image of immunofluorescent staining for FAM222A and nuclei (DAPI) in HUVECs treated with JQ1 (3 hours, 500nM). A representative negative control staining (IgG isotype control) is shown below. Scale bar = 20μm. PATENT Attorney Docket NO.: 51792-002WO2 FIG.4 shows a representative image of immunofluorescent staining in normal human carotid artery for FAM222A, CD31, and nuclei (DAPI). Scale bar = 20μm. FAM222A mRNA expression in HUVECs, primary vascular smooth muscle cells (VSMCs, coronary artery), fibroblasts (skin), and monocyte-derived macrophages (MDMs, peripheral blood) is shown below (n=3 for all cell types, nd = non-detected) and normalized to VSMCs. FIG.5A is a representative image of immunohistochemistry (IHC) staining of healthy human carotid arteries for FAM222A and cluster of differentiation 31 (CD31). Negative control (IgG isotype control) staining of human carotid artery is shown below. Scale bar = 20μm. FIG.5B is a representative image of immunofluorescent staining of a wild-type murine aorticroot for FAM222A, CD31, and nuclei (DAPI). Negative control staining (IgG isotype control) in humanand mouse vascular tissue is shown below. FIG.5C is a bar graph of BRD2, BRD3, BRD4 mRNA expression before and after 3 hours of JQ1 treatment (500nM) (n=3 per group). Data presented as mean ± SEM. Each data point represents a biological replicate. P values were performed by two-way ANOVA with Tukey post hoc test. FIG.5D is a heatmap displaying relative FAM222A mRNA expression in HUVECs after siBRD2, siBRD3, siBRD4 or siControl (siCTL, all 50 nM) transfection, either alone or with concurrent JQ1 treatment (3 hours, 500nM, n=3 per group). FIG.5E is a bar graph of FAM222A mRNA expression before and after 3 hours of JQ1 treatment (500nM) (n=3 per group). Data presented as mean ± SEM. Each data point represents a biological replicate. P values were performed by two-way ANOVA with Tukey post hoc test. FIG.6 is a gene track for FAM222A displaying chromatin immunoprecipitation (ChiP) signals for BRD4 and RNA polymerase II (RNA Pol II) in HUVEC treated with JQ1 (500nM) (GEO dataset GSE54000). BRD4 binding sites in two distinct (a & b) cis-regulatory regions of FAM222A were evaluated by RT-qPCR (n=4 per group). Data presented as mean ± SEM. Each data point represents a biological replicate. P values were calculated by Student’s t-test. FIG.7 shows a Venn diagram of significant JQ1-induced genes (log2 fold change >1, n = 127) in ECs and number of transcription factors predicted to bind to the FAM222A promoter (+ / - 10kb from transcription starts site (TSS); n = 268; ChIP Atlas24). This overlap analysis identified early growth response 1 (EGR1) as the sole gene meeting both criteria in these datasets. The left bar graph shows EGR1 mRNA and protein expression after JQ1 treatment (3 hours, n = 3 per group). The right bar graph shows relative luciferase activity of the human FAM222A promoter (1523 bp proximal to TSS) in HEK293T cells transfected with an EGR1 overexpression vector in the presence or absence of JQ1 (3 hours, n = 4 per group). FIG.8A is a bar graph of relative EGR1 mRNA expression in HUVECs transfected with EGR1 siRNA (siEGR1) or control siRNA (siCtl) (n=3 per group). Data presented as mean ± SEM. Each data point represents a biological replicate. P values were calculated by Student’s t-test. FIG.8B is a bar graph of relative FAM222A mRNA expression in HUVECs after siEGR1 transfected alone or with siBRD4 co-transfection and in the presence or absence (DMSO) of JQ1 (3 hours, n=3 per group). PATENT Attorney Docket NO.: 51792-002WO2 FIG.9A is a bar graph of relative FAM222A mRNA expression in HUVECs stimulated by VEGF (50ng / ml) or hypoxia (O21%, 3 hours for both, n=3 per group). FIG.9B are bar graphs of FAM222A mRNA expression in HUVECs treated with TNFα (25 ng / ml), IL1b (10ng / ml), and TGFβ2 (10ng / ml) for 3 hours in the absence or presence of JQ1 (500nM) (n=3 per group). Data presented as mean ± SEM. Each data point represents a biological replicate. P values were performed by two-way ANOVA with Tukey post hoc test. FIG.10 shows a bar graph of FAM222A mRNA expression (n=5), a Western blot of FAM222A protein expression, and a representative image of immunofluorescent staining after HUVECs were transfected with FAM222A siRNA (siFAM222A) or control siRNA (siCtl). Immunofluorescent stainingwas performed two days after siRNA transfection. Scale bar = 50μmFIG.11A is a graph of the number of HUVECs 4 days after transfection with siFAM222A or siCtl (both 50nM, n=3 per group). FIG.11B is a graph quantifying apoptosis in siFAM222A-transfected or siCtl-transfected ECs (24 hours, n=5 per group) using caspase 3 / 7 activation. FIG.11C shows a representative fluorescent-activated cell sorting (FACs) analysis of early and late apoptosis in siFAM222A or siCtl transfected cells treated without or with Staurosporine (10 uM, 4 hours). Annexin V was used for staining. A quantification of apoptotic cells is shown below (n=3 per group). FIG.11D is a bar graph of cell proliferation, as measured using BrdU incorporation without or with VEGF stimulation (50ng / ml, n=6 per group). FIG.11E are bar graphs of cell growth, as assessed by cell counting. Briefly, cells were reseeded at 2 days post transfection and grown to confluence for additional 2 days with and without cell synchronization. Cell growth was measured in contact inhibited (quiescent) and serum-starved (synchronized) cells (n=3 per group). FIG.11F is a representative image and quantification of EC migration after transfection with siFAM222A or siCtl, as determined by a scratch wound assay. The assay was conducted with and without VEGF stimulation (50 ng / ml) for 18 hours. The left side of the figure shows representative images while the right side provides quantification of wound closure. Scale bar = 50 μm (n=3 per group). FIG.11G is a representative image of a scratch assay and quantification thereof. Briefly, EC migration after siFAM222A or siCtl transfection was measured by scratch wound assay in the absence or presence of Mitomycin C (MitoC) (1 ug / ml, 16 hours). Representative images (top) and quantification of wound closure (bottom) shown. Bar scale = 50 μm (n=3 per group). FIG.11H is a bar graph of cell viability in siFAM222A, siCtl, and untransfected HUVECs plated at the cell densities shown using WST-8 assays (n=4 per group). FIG.12A shows FAM222A mRNA and protein expression in HUVECs transduced with FAM222A (LentiFAM222A) or Control (LentiCtl) GFP-expressing lentivirus (n=3 per group). A representative immunofluorescent staining for FAM222A, GFP, and nuclei (DAPI) post transduction is shown. Scale bar = 20μm PATENT Attorney Docket NO.: 51792-002WO2 FIG.12B is a bar graph of the proliferation rate in HUVECs transfected with validated FAM222A overexpression (LentiFAM222A) or Control (LentiCtl) GFP-expressing lentivirus prior to performing BrdU cell proliferation assays. FAM222A-overexpressing ECs were transfected at different multiplicity of infection (MOI) before assessing cell proliferation (n= 6 per group). FIG.12C is a representative phase-contrast and fluorescent image of GFP-positive ECs in a wound closure assay, and a quantification of wound closure over time. Scale bar = 100 μm (n=3 per group, 14 hours). FIG.13A shows representative images and quantifications thereof of network tube formation in Matrigel using ECs transfected with siFAM222A or siCtl in the absence (upper) or presence (lower)of VEGF (50ng / ml). The representative images are of Calcein-labeled tubes. Scale bar = 100μm (n=6per group). FIG.13B shows representative phase-contrast images and quantifications thereof of mean spouting length in an endothelial spheroid sprouting assay, which were performed after FAM222A silencing (siFAM222A) or control (siCtl) transfection. FIG.13C shows representative fluorescent images and quantifications thereof (n = 10 per group) of mean sprout length, cumulative sprout number, and sprouts per spheroid after overexpression of FAM222A (LentiFAM222A) or control vectors (LentiCTL) in ECs following VEGF stimulation (50ng / ml). Scale bar = 100μm. FIG.13D is a representative phase-contrast image of endothelial LentiFAM222A or LentiCtl spheroids. FIG.14A is a representative image and quantification of microvessel sprouting in the ex vivo aortic ring assays of siFAM222A (vs siCtl) treated mouse aortic rings embedded in Matrigel (10 days post transfection / infection). Representative images (left) and quantification (right) of microvessel number per ring are shown. Scale bar; 100μm (n=5-7 per group). FIG.14B is a representative image and quantification of microvessel sprouting in the ex vivo aortic ring assays of lentiFAM222A (vs LentiCtl) treated mouse aortic rings embedded in Matrigel (10 days post transfection / infection). Representative images (left) and quantification (right) of microvessel number per ring are shown. Scale bar; 100μm (n=5-7 per group). FIG.14C is a representative image and quantification of ex vivo microvessel sprouting, as determined in the aortic ring assays of siFAM222A (vs siCtl) treated mouse aortic rings embedded in Collagen (8 days post transfection / infection). Representative images (left) and quantification (right) of sprouting area are shown (n=11-14 per group). FIG.14D is a representative image and quantification of ex vivo microvessel sprouting, as determined in the aortic ring assays of LentiFAM222A (vs LentiCtl) treated mouse aortic rings embedded in Collagen (8 days post transfection / infection). Representative images (left) and quantification (right) of sprouting area are shown (n=11-14 per group). FIG.14E is a representative image and quantification of in vivo Matrigel vascularization assays using plugs mixed with HUVECs treated with F. siFAM222A (vs siCtl) before injection into the flank region of wild type mice. Representative images (left) and Matrigel vascularization, using PATENT Attorney Docket NO.: 51792-002WO2 Hemoglobin (Hb) content (normalized to plug weight) 7 days post implantation (right) are shown (n= 4-5 per group). FIG.14F is a representative image and quantification of in vivo Matrigel vascularization assays using plugs mixed with HUVECs treated with LentiFAM222A (vs LentiCtl) before injection into the flank region of wild type mice. Representative images (left) and Matrigel vascularization, using Hemoglobin (Hb) content (normalized to plug weight) 7 days post implantation (right) are shown (n= 4-5 per group). FIG.15 is a bar graph of gene expression in siFAM222A or siCtl-transfected ECs that underwent VEGF-stimulation (50ng / ml, 48 hours) before measuring the mRNA levels of the VEGF-regulated genes shown on the x-axis (n=3 per group).FIG.16A is an immunoblot of total and phosphorylated Erk1 / 2, Akt, and VEGFR2 at the time points shown after 48 hours of siFAM222A- or siCtl-transfected ECs in VEGF stimulation (as performed in FIG.15). FIG.16B are bar graphs showing total and phosphorylated Erk1 / 2, Akt, and VEGFR2 over the time in siFAM222A or siCtl-transfected ECs that underwent VEGF-stimulation. Values are expressed are relative fold change (n=3 per group). FIG.16C is an immunoblot of total and phosphorylated Erk1 / 2, Akt, and VEGFR2 at the time points shown after 48 hours of ECs overexpressing FAM222A (LentiFAM222A) or control (LentiCtl). FIG.16D are bar graphs showing total and phosphorylated Erk1 / 2, Akt, and VEGFR2 over the time shown in ECs during FAM222A overexpression (LentiFAM222A) or Control (LentiCtl.) FIG.17A shows NOTCH pathway gene mRNA levels (left) and protein levels (right), as measured in siFAM222A or siCtl-transfected ECs 48 hours after VEGF-stimulation (n=3 per group). FIG.17B is a schematic diagram of the mouse neonatal oxygen-induced hyperoxia (OIR) model and representative retinal flat mount images after staining for retinal vasculature (isolectin B4) from neonatal pups injected at P17 with siCtl or siFAM222A. Briefly, At P7, pups of both sexes and their nursing mothers were exposed to hyperoxia conditions (e.g., 75% oxygen, 5 consecutive days, vessel loss phase). Upon return to room air (P12), pups underwent intravitreal injection with FAM222A or Control siRNAs (2 µg) before sacrifice at P17 (neovascularization phase). Avascular and neovascularization areas are shown in the flat mount images. FIG.17C is a heatmap of Notch target genes in HUVECs transfected with FAM222A or Control siRNAs and treated with NOTCH inhibitor DAPT (10υM) for 24 hours (n=3 per group). FIG.17D is a bar graph of mRNA expression of FAM222A in DAPT treated cells (n=3 per group). FIG.17E shows a representative image of a wound closure assay assessing cell migration in siFAM222A vs siCtl treated cells with or without DAPT treatment (10μM, 14 hours). Scale bar = 50 μm. FIG.18A is quantification of avascular and neovascularization areas as a percentage of total retina (n=11 per group). Each data point represents an individual mouse or biological replicate. In all cases, data are presented as mean ± SEM. P values by Student’s test. PATENT Attorney Docket NO.: 51792-002WO2 FIG.18B is a quantification of FAM222A mRNA, EC proliferation rate, and EC migration. Briefly, mouse brain endothelial cells (bEND.3) were transfected with FAM222A or Control siRNAs. FAM222A mRNA expression (left) was analyzed (n=3 per group) and cell proliferation (middle) and migration (right) were assessed by BrdU incorporation assay and scratch wound assay, respectively (n=6 per group). FIG.18C is a representative immunofluorescent staining of murine neonatal retina for FAM222A, the endothelial specific marker CD31, and nuclei (DAPI). Scale bar = 50μm. FIG.18D is a quantification of FAM222A, VEGF, and HES2 mRNA expression in the whole retina of P17 pups with Oxygen-induced retinopathy (OIR) that were treated with FAM222A or ControlsiRNAs (n= 3 retinas per group).FIG.19 is a graphic showing various phenotypic effects of modulating FAM222A in endothelial cells. FIG.20A is a heatmap of genes significantly altered by JQ1 (log2 fold change, >2 or <2 vs control; FDR, <0.05), with resting EC gene subsets either significantly repressed or induced by JQ1. Z-score normalized expression ranges from +2.5 to −2.5. FIG.20B is a volcano plot of significantly JQ1-induced or repressed mRNA levels (vs control, log2 fold change, < or >2; FDR, <0.05). GO pathway analysis of mRNAs significantly modulated by JQ1 is shown below. GO is organized by adjusted log10 p-value and indicates that BET regulates (JQ1-repressed) core EC properties including movement, proliferation, and adhesion while JQ1- induced genes regulate cell cycling. FIG.20C is a Venn diagram of the top nine JQ1-induced genes. FAM222A is the only non- histone, JQ1-induced gene with no known EC or cell cycling / proliferation role. FIG.20D is a quantification FAM222A mRNA expression and a representative immunofluorescent image of FAM222A staining in JQ1-treated (3 hours, 500nM) HUVECs. Notably, JQ1 induced FMA222A expression in a dose-dependent manner. **p<0.01, ***p<0.001, each vs basal expression / DMSO. Scale bar = 20μm. FIG.20E shows ChIP gene tracks for BRD4 and RNA polymerase II (RNA Pol II) within FAM222A proximal regulatory regions in JQ1-treated HUVECs (GEO dataset GSE54000). ChIP-PCR of BRD4 binding sites in two distinct (a & b) FAM222A cis-regulatory regions were evaluated by RT- qPCR (n=3 per group). FIG.20F is a heatmap displaying relative FAM222A mRNA expression in HUVEC after siBRD2, siBRD3 or siBRD4 (50 nM) treatment in the presence of siControl (siCtl) alone or concurrent JQ1 treatment (3 hours, 500nM, n=3 per group), and an FAM222A mRNA quantification in the absence or presence of siBRD4 or siCtl, with or without JQ1 treatment (RT-PCR, n=3 per group, ns = not significant). FIG.20G is a bar graph of relative FAM222A mRNA expression in HUVECs, primary VSMCs (coronary artery), fibroblasts (skin), and MDMs (peripheral blood) normalized to VSMCs. (n=3 for all cell types, nd = non-detected). PATENT Attorney Docket NO.: 51792-002WO2 FIG.20H is a representative immunofluorescent staining of a wild-type murine aortic root (upper panel) and a normal human carotid artery (lower panel) for FAM222A, CD31, and nuclei (DAPI). Scale bar = 20μm. FIG.20I is a bar graph showing FAM222A mRNA expression in HUVEC treated with different concentrations of the BET inhibitor, iBET-151 for 3 hours (n=3 per group). FIG.20J is a reporter assay showing FAM222A promoter (1523 bp proximal to start site) activity of human embryonic kidney (HEK) 293T cells treated without or with JQ1 at the concentrations shown (3 hours, n=6 per group, *p<0.05, here and throughout). FIG.20K is a quantification of BRD2, BRD3, BRD4 (left) and FAM222A (right) mRNAexpression in HUVEC transfected with BRD2, BRD3, FAM222A or Control siRNAs. In all cases, dataare presented as mean ± SEM. P values by two-way ANOVA with Tukey post hoc test; ns denotes not significant; *p<0.05, **p<0.01, ***p<0.001. FIG.21A is a quantification of cell number in HUVECs transfected with siFAM222A or siCtl (50nM) 4 days post transfection (n=3 per group). FIG.21B is a heat map of cell cycle-related gene mRNAs in ECs 2 hours after siFAM222A or siCtl transfection (n=3 per group). FIG.21C is an immunoblot for p-Rb (s807 / 811), p-Rb (s608), and Rb in ECs after siFAM222A or siCtl-transfection (24 hours) followed by serum-starvation (12 hours) and subsequent FBS stimulation (10%, 24 hours). FIG.21D are bar graphs of mRNA expression of key G1 / S-related genes in siFAM222A- or siCtl-transfected ECs synchronized at G1 phase prior to FBS stimulation (10%). Assessment was made at the indicated times (n=3 per group). FIG.21E is an immunoblot showing the protein levels of G1 / S related genes in siFAM222A- or siCtl-transfected ECs synchronized at G1 phase prior to FBS stimulation (10%). FIG.21F is a FACs analysis. Briefly, cell cycle phases of G1-synchronized ECs were assessed by propidium iodine (PI) staining to determine the percentage of siFAM222A vs siCtl-treated cells in G1, S or G2 phases before and after FBS release (right) (n=3 per group). While siFAM222A decreased the number of cells in G2 and increased the number of cells in G1 at baseline, a statistically significant decrease in cell cycle phase transition for both phases occurred only after FBS stimulation. Data presented as mean ± SEM throughout. P values by two-way ANOVA with Tukey post hoc test; ns = not significant; *p<0.05, **p<0.01, ***p<0.001. FIG.21G is a quantification of FAM222A mRNA and protein expression in HUVEC treated with FAM222A or Control siRNAs after 2 days of transfection. FIG.21H is cell cycle analysis by propidium iodide (PI) staining in HUVEC transfected with FAM222A or Control siRNAs without cell synchronization at G0 phase (n=3 per group) FIG.21I is FACS analysis showing cell cycle distribution of siFAM222A and siCtl treated cells that underwent pretreatment with nocodazole (10nM) in each of the G1, S and G2 phases (n=3 per group). FIG.21J is quantification of the cell growth following siFAM222A or siCtl treatment assessed by cell counting. Briefly, cells were reseeded at 2 days post transfection and grown to confluence for PATENT Attorney Docket NO.: 51792-002WO2 additional 2 days with and without cell synchronization. Cell growth was measured in contact inhibited (quiescent) and serum-starved (synchronized) cells (n=3 per group). Representative images of siFAM222A and siCtl treated HUVEC at day 4 post transfection. Scale bar; 50 μm. FIG.21K is quantification of FAM222A mRNA expression in HUVEC treated with FAM222A siRNA 1, FAM222A siRNA 2 or Control siRNA. The FAM222A siRNA #1 corresponds to the siRNA used in all the experiments. FAM222A mRNA expression was assessed at 2 days post transfection (n= 3 per group). FIG.22A is a bar graph of FAM222A mRNA expression, which was significantly increased in HUVECs stimulated by VEGF (50ng / ml) or hypoxia exposure (O21%), both for 3 hours. FIG.22B is a bar graph of HUVECs transfected with siFAM222A or siCtl (50nM) beforemeasuring cell proliferation using BrdU incorporation without or with VEGF stimulation (50ng / ml, n=6 per group). FIG.22C is a bar graph of apoptosis in ECs transfected with siFAM222A or siCtl (24 hours, n=6 per group) as assessed using caspase 3 / 7 activation,. FIG.22D is a representative image of a scratch wound assay and quantification thereof. Briefly, EC migration after siFAM222A or siCtl transfection was measured by scratch wound assay without or with VEGF stimulation (50ng / ml, 18 hours). Bar scale = 50 μm (n=3 per group). FIG.22E is a representative image of a scratch wound assay and quantification thereof. Briefly, ECs were treated with siFAM222A- or siCtl in the absence or presence of Mitomycin C (MitoC, 1 ug / ml, 16 hours). Scale bar = 50 μm (n=3 per group). Significance as before other than comparison of siFAM222A+MitoC vs siCtl+ MitoC, ### p<0.001. FIG.22F is a quantification of cell viability using WST-8 assays in siFAM222A, siCtl, and untransfected HUVECs plated at the cell densities shown (n=6 per group). FIG.22G is a quantification of proliferation rate in HUVECs transfected with a validated FAM222A overexpression (LentiFAM222A) GFP-expressing lentivirus. BrdU cell proliferation assays were performed as before and compared to Control (LentiCtl) overexpression constructs. FAM222A overexpressing ECs were transfected with different multiplicity of infection (MOI) of the respective vector (n= 6 per group). FIG.22H is a representative wound closure assay and quantification thereof. Briefly, EC migration was assessed by wound closure assay in the presence of FAM222A vs Ctl-overexpression. Representative phase-contrast and fluorescent pictures of GFP-positive cells (left) and quantification of wound closure over time (right) are shown. Scale bar = 100 μm (n=3 per group, 14 hours). FIG.22I is a representative image of immunofluorescence staining for FAM222A in HUVEC treated with FAM222A or Control siRNAs at day 2 after transfection. Scale bar; 20μm. FIG.22J is FACS analysis showing cell cycle distribution in HUVEC treated with FAM222A (#2) or Control siRNAs and pre-treated with nocodazole (n=3 per group). FIG.22K is a bar graph (right) and an immunoblot image showing FAM222A overexpression in HUVEC transduced with FAM222A (LentiFAM222A) vs Control (LentiCtl) GFP expressing lentivirus (n=3 per group).In all cases, data are presented as mean ± SEM. P values by Student’s test; ns denotes not significant; *p<0.05, ***p<0.001. PATENT Attorney Docket NO.: 51792-002WO2 FIG.23A is a representative network tube formation assay. Briefly, ECs transfected with siFAM222A or siCtl underwent network tube formation assay in Matrigel in the absence (top) or presence (bottom) of VEGF (50ng / ml). The representative immunofluorescent images are Calcein- labeled. Scale bar = 100μm (n=3 per group). FIG.23B is a representative spheroid sprouting assay. Briefly, endothelial sprouting of siFAM222A or siCtl-treated ECs was assessed by spheroid sprouting assay after VEGF stimulation (50ng / ml). Representative images (top) and quantification (bottom) of mean sprout length, cumulative sprout number, and sprouts per spheroid are shown. Scale bar = 100μm (n=10 per group). FIG.23C is a schematic diagram of the mouse neonatal oxygen-induced hyperoxia (OIR)model, an immunofluorescent flat mount staining of mouse retinas, and a quantification ofvascularization. Briefly, at P7, pups along with their nursing mother were exposed to hyperoxia (75% oxygen, 5 consecutive days), mimicking vessel loss phase in ROP. Upon return to room air, P12 pups were injected with FAM222A or Control siRNAs (2 µg) intravitreally before sacrifice at P17 (mimicking neovascularization phase in ROP). The representative images of retinal flat mounts are stained with isolectin B4 for retinal vasculature of neonatal pups injected at P17 with siCtl or siFAM222A. The avascular and neovascularization areas are shown. Quantification of avascular and neovascularization areas as a percentage of total retina is shown (n=3 experiments; each n represents 5-6 retinas, individually plotted). In all cases, data are presented as mean ± SEM. P values by Student’s test; *p<0.05, ***p<0.001. FIG.23D is a quantification of FAM222A mRNA expression, cell proliferation and migration assessed by RT-PCR, BrdU incorporation assay and scratch wound assay in mouse brain endothelial cells (bEND.3) transfected with FAM222A or Control siRNAs. (n=6 per group). FIG.23E is a representative image of immunofluorescent staining in murine neonatal retina for FAM222A , the endothelial specific marker CD31, and nuclei (DAPI) . Scale bar; 50μm. FIG.23F is a quantification by RT-PCR of VEGF mRNA expression in the whole retina of P17 pups with OIR treated with FAM222A or Control siRNAs (n= 3 retinas per group). FIG.24A is an immunoblot of total and phosphorylated Erk1 / 2, Akt, and VEGFR2 in siFAM222A or siCtl-transfected ECs that underwent VEGF stimulation over the time period shown. FIG.24B is an immunoblot of total and phosphorylated Erk1 / 2, Akt, and VEGFR2 in in ECs during FAM222A overexpression (LentiFAM222A) or Control (LentiCtl). ECs underwent VEGF stimulation over the time period shown. FIG.24C is a quantification of the VEGF and NOTCH target genes shown (n = 3 per group) in siFAM222A or siCtl-transfected ECs that were VEGF-stimulated. FIG.24D is a Western blot for the VEGF and NOTCH target genes shown in siFAM222A or siCtl-transfected ECs that underwent VEGF stimulation (n=3 per group). FIG.24E is a heatmap of gene expression in the siFAM222A vs siCtl transfection experiments in which ECs were also treated without (DMSO) or with the NOTCH inhibitor DAPT (10μM, 24 hours before analysis of NOTCH target gene mRNAs, n=3 per group). FIG.24F is a quantification of a wound closure assay. Briefly, cell migration of siFAM222A vs siCtl treated cells was assessed by wound closure assay with or without DAPT treatment (10μM, 14 PATENT Attorney Docket NO.: 51792-002WO2 hours). Data are presented as mean ± SEM. P values by two-way ANOVA with Tukey post hoc test; ns = not significant; *p<0.05, **p<0.01, ***p<0.001 and #p<0.05, ##p<0.01 (siFAM222A+DAPT vs siFAM2222A). FIG.24G is a bar graph representing quantification of the protein levels of total and phosphorylated Erk1 / 2, Akt and VEGFR2 as shown by fold change in HUVEC treated with FAM222A or Control siRNAs that underwent VEGF-treatment over the time period shown (n=3 per group). FIG.24H is a quantification of the total and phosphorylated protein levels of Erk1 / 2, Akt and VEGFR2 in HUVEC transduced with FAM222A or Control Lentivirus (n=3 per group). FIG.24I is a quantification of mRNA expression of VEGF and NOTCH target genes inHUVECs transfected with FAM222A or Control siRNAs that were treated with the NOTCH inhibitorDAPT (10υM) for 24 hours (n=3 per group). FIG.25A is a bar graph of FAM222A mRNA levels measured in mouse ECs isolated from non-ischemic vs ischemic limb gastrocnemius muscle at 3 and 14 days in a hind limb ischemia model (HLI, n=9 per group). FIG.25B shows a schematic diagram of an siRNA administration schedule in a murine HLI model along with a quantification of FAM222A after such administration. Briefly, after unilateral ligation and excision of the femoral artery (baseline, Day 0), FAM222A mRNA levels were determined in EC and non-EC fractions isolated after 14 days from the ischemic gastrocnemius muscle of mice injected with siFAM222A or siCtl (4 mg / kg, at Days 0 and 7, arrowheads). Hindlimb blood flow was assessed using noninvasive Doppler laser imaging at Day 0 (baseline), 3, 7, 11 and 14 post-ischemia (arrows). FIG.25C are a representative Doppler Laser ultrasound images (top) of blood flow reperfusion at Days 0 (baseline) and 7 after ischemic injury, along with a quantification (bottom) of the rate of hindlimb reperfusion (n=15 per group). FIG.25D are representative images of hematoxylin and eosin (H&E) staining of ischemic and non-ischemic gastrocnemius muscle at Day 14 post-HLI. Scale bar = 100 μm (left) and 50 μm (right). FIG.25E is a representative image of an immunofluorescent staining and quantification thereof of CD31 and CD31+ capillary density in ischemic gastrocnemius muscle tissue from siCtl and siFAM222A-treated mice. Scale bar = 100 μm (n=7-8 per group). FIG.25F is a bar graph showing the mRNA levels of angiogenesis and cell cycle-related genes in the EC fraction of the ischemic gastrocnemius muscle of siFAM222A- or siCtl-treated mice (n=6 per group). FIG.25G is a quantification of FAM222A mRNA levels in RNA-Seq studies of gastrocnemius biopsy samples from patients that were healthy (HA; n=15), had critical limb ischemia (CLI; n=16), or had chronic intermittent claudication (IC; n=20). Data are presented as mean ± SEM. P values by Student’s test; ns = not significant. FIG.25H is a bar graph showing FAM222A mRNA expression as analyzed in the EC and non-EC fraction of the murine gastrocnemius muscle (left) and in murine bone marrow derived macrophages (right) (n=9 per group). PATENT Attorney Docket NO.: 51792-002WO2 FIG.25I shows the mRNA expression analysis assessed by RT-PCR of angiogenesis and inflammation related genes in the EC fraction isolated from the gastrocnemius muscle of mice at day 3 post hind limb ischemia (n=9-10 per group). FIG.25J is a quantification of FAM222A mRNA expression in the non-EC fraction of the gastrocnemius muscle of mice at day 3 and 14 post-ischemia (n=9-10 per group). FIG.25K is representative image of immunofluorescence for aSMA, CD31, and nuclei (DAPI) tissue sections of the ischemic gastrocnemius muscle of siCtl and siFAM222A-treated mice and graphic representation of the aSMA+ arteriolar density. Arteriolar density was evaluated by counting aSMA+ cells per high-power field (HPF) Scale bar = 100 μm (n=7-8 per group). FIG.26A shows FAM222A mRNA levels (left, at the time points indicated, n=3 per group) andrepresentative immunofluorescent staining (right, before and 4 hours after FBS) in serum-starved ECs before and after FBS (10%) stimulation. Scale bar = 20μm. FIG.26B is an immunoblot from an immunoprecipitation (IP) assay in which HEK 293T cells were transfected with Flag-tagged FAM222A or empty expression vector. IP with Flag antibody was performed in the cellular nuclear fraction followed by immunoblotting for MCM3, MCM4 and MCM7. A nuclear cell lysate aliquot reserved prior to IP was separately analyzed (Input). FIG.26C is a representative scratch wound assay and quantification thereof. Briefly, HUVECs were transfected with siRNAs to FAM222A, MCM3, MCM4, MCM7, or Control prior to measuring cell migration by scratch wound assay (total of 16 hours). Representative images (left) and quantification of wound closure (right) shown. FIG.26D is a bar graph of cell proliferation in the indicated cell lines, as quantified by BrdU incorporation assay (n=6 per group). FIG.26E shows immunoblots from an IP assay. Briefly, IP was performed with an MCM3 antibody in nuclear extracts from ECs after serum-starvation overnight prior to FBS-stimulation (10%) with harvesting at 0, 4, and 8 hours. Immunoblotting for MCM3, FAM222A, MCM4, ORC1 or Lamin B1 (left) is shown. A nuclear cell lysate aliquot reserved prior to IP was also analyzed (right, Input). FIG.26F is an immunoblot for FAM222A and pre-replication complex (pre-RC) proteins in the chromatin-bound extract of ECs treated with siFAM222A or siCtl before (0) and after (8, 24 hours) FBS release. Data presented as mean ± SEM. P values by two-way ANOVA with Tukey post hoc test; **p<0.01, ***p<0.001. FIG.26G is a quantification of FAM222A, MCM3, MCM4 and MCM7 mRNA expression (left) and protein levels (right) in HUVEC treated with FAM222A, MCM3, MCM4, MCM7 or Control siRNAs (n=3 per group). FIG.26H shows the quantification of cell proliferation rate measured by BrdU incorporation assay (left, n=6 per group) by scratch wound assay for a total of 16 hours (right, n=3 per group) in HUVEC transfected with the combinations of siRNAs to FAM222A and either MCM3 or MCM4 or MCM7. FIG.26I is a quantification of cell migration measured by scratch wound assay for a total of 16 hours (right, n=3 per group) in HUVEC transfected with the combinations of siRNAs to FAM222A and either MCM3 or MCM4 or MCM7. PATENT Attorney Docket NO.: 51792-002WO2 FIG.26J is a representative image of immunofluorescent staining for FAM222A and MCM3 in HUVEC. Scale bar; 20μm. FIG.27 is our proposed model for FAM222A action in angiogenesis. EC Responses (Left): Upper Panel – Known angiogenic inputs like VEGF stimulation or hypoxia result in EC proliferation and sprouting (+), which involves tip and stalk cells, respectively. Lower Panel – With FAM222A deficiency, these angiogenic responses are significantly repressed (-). Signaling (Center Left): Upper Panel – During stimulated angiogenesis (upper panel), VEGF binds to the VEGFR2 to activate signaling pathways like ERK1 / 2 that promote tip cell migration and increase NOTCH ligand DLL4 levels. NOTCH, through its intracellular domain NICD, increases JAG1, DLL4, HES1, HES2expression while repressing VEGFR2 mRNA, resulting in increased stalk cell proliferation. NOTCHsignaling also induces lateral inhibition of the tip cell phenotype. Lower Panel - With FAM222A deficiency, VEGF responses and ERK1 / 2 activation are decreased, resulting in impaired tip cell migration. At the same time, in stalk cells, with FAM222A silencing, NOTCH signaling is hyperactivated, inducing the aberrant expression of NOTCH target genes JAG1, DLL4, HES1, HES2, thus impairing proliferation. Cell Cycle (Center Right): Upper Panel – VEGF induces normal cell cycle progression through G1, S, and G2 / M phases. Lower Panel – FAM222A deficiency causing G1 arrest and blocking EC transition to S / G2 / M phases. Transcriptional Mechanisms (Right): Upper Panel – When FAM222A levels are intact, angiogenic stimuli prompt assembly of transcriptional complexes involving MCMs, ORC1 and FAM222A, with subsequent phosphorylation of Rb by CDKs and E2F release, prompting cell cycle progression from G1 to the S phase and subsequent EC proliferation. Lower Panel -– In the absence of FAM222A, MCM complex assembly does not occur, in conjunction with decreased CDK mRNA expression and Rb phosphorylation and E2F release, resulting in G1 arrest and decreased EC proliferation. FIG.28 is a schematic displaying FAM222A predicted interactors as shown in the BioPlex Network and a table showing the Gene Ontology (GO) analysis of the predicted interacting targets ranked by significance. Fig.29A is a bar graph of FAM222A mRNA expression after HUVECs were treated with the indicated BET inhibitor for 3 hours. Fig.29B is a heat map showing the fold change expression of FAM222A mRNA after HUVECs were treated with the indicated (proteolysis-targeting chimera (PROTAC) BRD4 degrader. Fig.29C is a bar graph of FAM222A mRNA expression after HUVECs were treated with the indicated PROTAC inhibitor for 3 hours. FIG.30 are immunoblots showing direct FAM222A-MEIS2 interaction in 293T cells as assessed by immunoprecipitation assay in cells transfected with a Flag-Tagged FAM222A expression vector vs Control. FIG.31 are representative images of immunofluorescence staining for FAM222A and meis homeobox 2 (MEIS2) showing nuclear co-localization of these proteins in both HUVEC transduced with either a GFP-expressing FAM222A or Control Lentivirus. FIG.32 is immunofluorescence staining for FAM222A and MEIS2 in HUVEC treated with FAM222A or Control siRNAs. PATENT Attorney Docket NO.: 51792-002WO2 FIG.33 are representative immunoblots for FAM222A, HA, MEIS2 and Myc showing successful FAM222A and MEIS2 ectopic expression in 293T cells using Flag-HA-Tagged FAM222A or Myc-Tagged MEIS2 expression vectors. FIG.34 is an immunoblot of a Myc immunoprecipitation assay showing efficient FAM222A pull down in cells expressing Myc-tagged MEIS2 and representative images of immunofluorescence staining for HA and Myc showing nuclear co-localization. FIG.35 are immunoblots for Flag and PBX3 IP assays showing direct and reciprocal binding of FAM222A with PBX3 in cells ectopically expressing Flag-tagged FAM222A or naive cells, respectively, FIG.36 is a schematic of MEIS2 protein domains and immunoblots following myc IP assayshowing direct binding of FAM222A with MEIS2 through the MEINOX domain. Briefly, 293T cells were co-transfected with HA-tagged FAM222A and myc-tagged MEIS2 Wild type (wt) and deletion expression vectors and immunoprecipitated with myc antibody. FIG.37 is an immunoblot for a myc IP assay in cells co-expressing myc-tagged MEIS2 and HA-tagged FAM222A showing an effective pull down of MEIS2, FAM222A, PBX3 and PARP. (upper)The IP assay was repeated in cells pretreated with Ethidium Bromide and indicated an effective pull down of MEIS2, FAM222A, PBX3 independently of DNA binding. (lower) FIG.38 is a schematic showing MEIS2 / PARP1 dimer binding with PBX and subsequent gene activation and an immunoblot for a myc IP assay validating direct MEIS2 / PARP1 interaction. FIG.39 are immunoblots for Myc-Tagged MEIS2 pulldown assays and immunoprecipitation for Ha-tagged FAM222A and PBX3 shown direcet binding independent of additional stimulation (BMP9, TGFβ) FIG.40 are representative images of immunofluorescence staining for FAM222A, MEIS2 and PBX3 showing nuclear localization in 293T cells. FIG.41 is a bar graph showing FAM222A mRNA expression analysis and immunoblot for FAM222A protein expression in HUVEC transfected with single or combinations of FAM222A, meis homeobox 2 (MEIS2), meis homeobox 1 (MEIS1) or Control siRNAs and treated with TGFβ for 24 hours. FIG.42 is quantification of MEIS1 and MEIS2 mRNA expression. FIG.43 are immunoblots for MEIS1 and MEIS2. FIG.44 is a bar graph and an immunoblot showing TAGLN mRNA and protein expression in cells treated. FIG.45 is a quantification of TAGLN mRNA expression in HUVEC co-transfect with FAM222A, SMAD family member 2 (SMAD2) or SMAD family member 3 (SMAD3) siRNA in the absence or presence of TGFβ. Lower: TAGLN and TGFB2 mRNA expression analysis in siFAM222A or siCtl transfected HUVEC treated with the TGFβ receptor inhibitor SB-431542. FIG.46 are bar graphs showing FAM222A, MEIS2 and TALGN mRNA expression analysis in HUVEC transfected with single or combinations of FAM222A, MEIS2 (#2) or Control siRNAs and treated with TGFβ for 24 hours. PATENT Attorney Docket NO.: 51792-002WO2 FIG.47 is a schematic displaying the study design for a Pulmonary Hypertension model in rats injected with monocrotaline and treated with combinations of FAM222A (H3M3) or Control ASOs. Injection with FAM222A ASOs resulted in increased Right ventricle systolic pressure (RVSP) and Fulton Index compared to mice injected with Control ASOs. FIG.48 are representative immunofluorescence staining images for Von Willebrand factor (vWF) and smooth muscle action (SMA) in the lungs of FAM222A knockout (KO) and wild type (WT mice) injected with Sugen and exposed to hypoxia to induce Pulmonary Hypertension. Shown below is a quantification showing increased RVSP and Fulton Index in FAM222A KO mice compared to WT mice in the Sugen-hypoxia group. FIG.49 are representative images for Lac-Z and nuclear red immunohistochemistry staining of retinas from Lac-Z reporter FAM222A global KO mice at day 7 (P7) and day 31 (P31) postnatally indicating FAM222A expression localization before deletion. FIG.50 is immunofluorescence staining for FAM222A in the developing retina of mice at postnatal day 7 showing co-localization of FAM222A with endothelial CD31+cells. FIG.51 is a summary of FAM222A mRNA and protein expression (left) and promoter activity(right) in HUVECs under different angiogenic stimuli. Briefly, FAM222A expression is induced by VEGF (50 ng / ml.3 hours) and hypoxia (1% FO2, 3 hours), validated also by immunoblotting for FAM222A at the same conditions (right, lower). FAM222A expression is also increased by laminar flow stimulation for 24 hours in the absence or presence of JQ1 (500nM, n=3 per group). Additionally, FAM222A promoter activity is induced by angiogenic transcription factors as EGR1, E2F1 and HIF1A as shown by luciferase assay in 293T cells transfected with vectors expressing EGR1, E2F1 or HIF1A in the absence or presence of JQ1 (n=4 per group). FIG.52 is a bar graph showing FAM222A expression in the retina of normoxic mice and mice with oxygen-induced retinopathy (OIR) at day 14 and 17 postnatally, as assessed by RNA sequencing (e.g., see NCBI Accession No. GSE150703). FIG.53 is immunofluorescence staining for cytoskeleton changes after VEGF treatment, as indicated by F- actin (and vimentin in HUVEC treated with siFAM222A or siCtl siRNAs. FIG.54 is a quantification of overnight resistance in HUVEC treated with siFAM222A or siCtl siRNAs as assessed by a trans-endothelial electrical resistance (TEER) assay (n=4 per group). FIG.55 is quantification of luciferase activity in 293T cells that are ectopically expressing p31 or a control vector showing a repressive function of p53 on FAM222A promoter (n= 4 per group). FIG.56 is immunofluorescence staining for the proliferation marker ki67 (in HUVEC transfected with FAM222A or Control siRNAs after G0 synchronization (overnight starvation, -FBS) or after FBS release (10 %, 4 hours), showing decreased proliferation in siFAM222A-treated HUVECs. FIG.57 is quantification of the proliferation rate of HUVECs treated with FAM222A or ControlsiRNAs and exposed to normoxia or hypoxia for 24 hours as measure by BRDU incorporation assay (n=6 per group). FIG.58 is quantification of mRNA expression of cell-cycle related targets (E2F1, E2F2, E2F4, CCND1, CDK4, CCNE1, CDKN1A / p21, CDKN2B) in HUVEC treated with FAM222A or Control siRNAs and exposed to normoxia or hypoxia for 24 hours (n=3 per group). PATENT Attorney Docket NO.: 51792-002WO2 FIG.59 is an immunoblot for cell cycle related proteins (CDK4, CCND1, CCNE1, E2F1, MCM3) in the same conditions as in FIG.58. FIG.60 is an immunoblot for HIF1A, GAPDH and Lamin B1 in the cytoplasmic and nuclear fraction of siFAM222A or siCtl- treated HUVEC at normoxia and after 24 hours of hypoxia showing decreased HIF1A translation and activation (e.g., hydroxylation) in the absence of FAM222A. FIG.61 is a summary of FAM222A expression in human and rat pulmonary hypertension (PAH). Upper left are 2 graphs showing that FAM222A is enriched in mouse and lung ECs (Human protein atlas). Lower left are representative images of immunohistochemistry staining for FAM222A in lungs of control rats and rats with monocrotaline-induced PAH showing that FAM222A is reduced inrat PAH. Right are representative images of immunohistochemistry staining for FAM222A in lungs ofhealthy subjects and in patients with familial or idiopathic PAH indicating decreased expression in human PAH. FIG.62 is immunofluorescence staining for FAM222A and CD31 in a healthy human carotid artery and in a carotid artery with atherosclerosis. FIG.63 is a summary of single nucleotide polymorphisms (SNPs) close to FAM222A gene that are related to cardiovascular disease as displayed in the Common Metabolic Disease Knowledge Portal (CVD PK). FIG.64 is a summary of Endothelia to Mesenchymal Transition (EndMT)-related responses in HUVECs upon the absence of FAM2222A. HUVECs treated with siFAM222A siRNAs display morphologic (upper left, phase contrast imaging and immunofluorescence staining), functional (lower left, trans well migration assay) and transcriptional (middle, right) responses related to EndMT. A volcano plot showing the EndMT transcriptional signature in siFAM222A cells, a GO enrichment analysis bar plot showing upregulation of EndMT and a gene set enrichment analysis (GSEA) plot for EndMT are being generated by RNA sequencing in siFAM222A vs siCtl HUVECs at 24 and 48 hours post transfection (n=3 per group). FIG.65 is a summary of exacerbated transcriptional and functional EndMT-related responses in siFAM222A vs siCtl HUVECs treated with TGFβ2 (10ng / ml, 24 hours) or hypoxia (1% FO2, 24 hours) (n= 3 per group). FIG.66 is a quantification of resistance of HUVEC treated with siFAM222A or siCtl siRNAs that underwent EndMT stimulation (TGFβ2 (10ng / ml), IL1b (1 ng / ml)] for more than 72 hours as assessed by the TEER assay (n=4 per group) showing decreased barrier function stability in siFAM222A cells. FIG.67 is a summary of transcriptional EndMT or inflammatory-related responses in HUVECs transduced with a FAM222A (GFP-FAM) or Control (GFP) Lentivirus. FAM222A overexpression decreased the induction of mesenchymal and inflammatory genes upon TGFβ2 or TNFα stimulation (n= 3 per group). FIG.68 is a summary of targeting FAM222A using antisense-oligonucleotides (ASOs) in rat endothelial cells in vitro and in vivo. Left is mRNA and protein expression of FAM222A after treatment with a single or combination of ASOs designed to target FAM222A. H3M3 combination had the highest knockdown efficiency and used for in vivo targeting (right). FAM222A was successfully PATENT Attorney Docket NO.: 51792-002WO2 knockdown in the lung and lung endothelial cells of rats upon intravenous injections, without affecting FAM222A expression levels in other organs (heart, aorta, liver). FIG.69 is a schematic of FAM222A ASO treatment in rats (10mg / kg) for 21 days upon monocrotaline treatment, a bar graph (lower left) showing decreased mRNA expression in the lungs of FAM222A ASO treated rats and a quantification of hemodynamic measurements during right ventricle catheterization. FAM222A ASO treatment increased right ventricle systolic pressure (RVSP) and right ventricle hypertrophy (RVH) in the rat lungs (n= 5-6 per group). FIG.70 is a summary of PAH responses in the Sugen Hypoxia model using global FAM222A and endothelial specific FAM222A KO mice. Loss of FAM222A resulted in worsening PAH in mice asindicated by hemodynamic measurements in the right ventricle.DEFINTIONS Unless otherwise defined herein, scientific, and technical terms used herein have the meanings that are commonly understood by those of ordinary skill in the art. In the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The use of "or" means "and / or" unless stated otherwise. The use of the term "including," as well as other forms, such as "includes" and "included," is not limiting. As used herein, the term "about," as applied to one or more values of interest, refers to a value that falls within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of a stated reference value, unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value). As used herein, the terms “administer,” “administering,” and “administration” refer to providing or giving a subject a therapeutic agent (e.g., an inhibitor or activator of family with sequence similarity 222 member A (FAM222A)), by any effective route. Exemplary routes of administration are: intramuscularly, intravenously, intradermally, percutaneously, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, juxtascleral, intracameral, retrobulbar, intravaginally, intrarectally, topically, intratumorally, peritoneally, subcutaneously, subconjunctivally, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularly, orally, topically, locally, by inhalation, by injection, by infusion, by continuous infusion, by localized perfusion bathing target cells directly, by catheter, or by lavage. Effective ocular delivery routes are known in the art for surpassing precorneal, corneal, and blood-corneal barriers, such as topical, intravitreal, intraocular, juxtascleral, subconjunctival, intracameral, and retrobulbar. As used herein, the terms “administered in combination” and “combined administration” refer to two or more agents are administered to a subject at the same time or within an interval such that there may be an overlap of an effect of each agent on the patient. In some embodiments, they are administered within about 60, 30, 15, 10, 5, or 1 minute of one another. In some embodiments, the administrations of the agents are spaced sufficiently closely together such that a combinatorial (e.g., a synergistic) effect is achieved. PATENT Attorney Docket NO.: 51792-002WO2 As used herein, the term “delivery vehicle” refers to any substance which facilitates, at least in part, the in vivo delivery of a compound, substance, entity, moiety, cargo, or payload (e.g., a nucleic acid molecule described herein) to a targeted cell, tissue, or organ. As used herein, the term “effective amount” refers to a quantity sufficient to, when administered to a subject (e.g., human), provide beneficial or desired results, including clinical results, and, as such, an “effective amount” depends upon the context in which it is being applied. The amount of a given composition may depend of various factors, such as the given agent, the pharmaceutical formulation, the route of administration, the type of disease or disorder, the identity of the subject (e.g., age, sex, weight) being treated, and the like, but can nevertheless be routinelydetermined by one skilled in the art.As used herein, the term “inhibitory nucleic acid molecule” refers to a nucleic acid molecule that has sufficient complementarity to bind to a nucleic acid (e.g., DNA, RNA, or mRNA) encoding a target (e.g., FAM222A) and inhibit or reduce expression of the target. Exemplary inhibitory nucleic acid molecules are anti-sense oligonucleotides (ASOs), small interfering RNA (siRNAs), short hairpin RNA (shRNAs), double stranded RNAs (dsRNAs), and microRNA (miRNAs). Inhibitory nucleic acid molecules may reduce their target (e.g., FAM222A) expression by 10% or more (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or more). As used herein, the term “modified” refers to a changed state or structure of a nucleic acid molecule (e.g., ASO, siRNA, shRNA, dsRNA, or miRNA), which may be modified in any way including chemically (e.g., by the introduction of non-natural nucleosides and / or nucleotides), structurally, and functionally. As used herein, the term “pharmaceutically acceptable” refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. As used herein, the term “pharmaceutical composition” refers to a mixture containing a therapeutic agent (e.g., a FAM222A inhibitor or FAM222A activator), optionally in combination with one or more pharmaceutically acceptable excipients, diluents, and / or carriers, to be administered to a subject. “Percent (%) sequence identity” with respect to a reference polynucleotide or polypeptide sequence is defined as the percentage of nucleic acids or amino acids in a candidate sequence that are identical to the nucleic acids or amino acids in the reference polynucleotide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid or amino acid sequence identity can be achieved in various ways that are within the capabilities of one of skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For example, percent sequence identity values may be generated using the PATENT Attorney Docket NO.: 51792-002WO2 sequence comparison computer program BLAST. As an illustration, the percent sequence identity of a given nucleic acid or amino acid sequence, A, to, with, or against a given nucleic acid or amino acid sequence, B, (which can alternatively be phrased as a given nucleic acid or amino acid sequence, A that has a certain percent sequence identity to, with, or against a given nucleic acid or amino acid sequence, B) is calculated as follows: 100 multiplied by (the fraction X / Y) where X is the number of nucleotides or amino acids scored as identical matches by a sequence alignment program (e.g., BLAST) in that program’s alignment of A and B, and where Y is the total number of nucleic acids in B. It will be appreciated that where the length of nucleic acid or amino acidsequence A is not equal to the length of nucleic acid or amino acid sequence B, the percent sequenceidentity of A to B will not equal the percent sequence identity of B to A. As used herein, the term “subject” refers to any member of the animal kingdom (e.g., humans). In some embodiments, the subject is a human at any stage of development (e.g., infant, toddler, child, adolescence, or adult). Non-human subjects can include, but are not limited to, mammals, birds, reptiles, amphibians, fish, and worms. As used herein, the term “treating” or “treatment” refers to an approach for obtaining at least one of the following beneficial or desired results in a subject (e.g., a human subject): (1) slowing, inhibiting, or preventing the progression of a disease (e.g., a retinopathy or cancer) in a subject; (2) promoting the maintenance of a disease (e.g., a retinopathy or cancer) in a subject; (3) reversing the progression of a disease or condition (e.g., a retinopathy or cancer) in a subject; (4) alleviating or ameliorating one or more symptoms of a disease (e.g., a retinopathy or cancer) in a subject; or (5) reducing one or more clinical manifestations of a disease (e.g., a retinopathy or cancer) in a subject. DETAILED DESCRIPTION Described herein are methods of treating various diseases that may benefit from modulating angiogenesis and / or endothelial-to-mesenchymal transition (EndMT) pathways, including, but not limited to retinopathy, peripheral artery disease (PAD), pulmonary arterial hypertension (PAH), coronary disease, cerebrovascular disease, atherosclerosis, ischemia, and cancer. Advantageously, it is Applicant’s discovery that knocking down family with sequence similarity 222 member A (FAM222A; e.g., see National Center for Biotechnology Information (NCBI) Gene ID 84915) in the endothelium of a subject, and specific endothelial cells, can reduce various endothelial cell phenotypes associated with, inter alia, angiogenesis and endothelial cell function. Conversely, increasing expression of FAM222A in the endothelial cell of the subject can increase or enhance various endothelial cell phenotypes associated with, inter alia, angiogenesis and / or endothelial cell function. As a non-limiting example, Applicant has demonstrated that FAM222A inhibitors, such as inhibitory nucleic acid molecules, can reduce FAM222A, which consequently reduces angiogenesis, including specifically EC proliferation, growth, migration and cell cycling. Conversely, Applicant has demonstrated that FAM222A activators, such as lentiviral overexpression vectors and bromodomain- containing protein 4 (BRD4) inhibitors, can increase FAM222A, which consequently increases PATENT Attorney Docket NO.: 51792-002WO2 angiogenesis and these other EC responses. Additional details on methods of the invention, as well as exemplary FAM222A inhibitors / activators and applicable indications, are described below. Methods of Treating The disclosure provides methods of treating a disease in a subject. The method of treating includes the step of administering to the subject (e.g., a human) an inhibitor or an activator of family with sequence similarity 222 member A (FAM222A). Various FAM222A inhibitors and activators are described herein. For example, a FAM222A inhibitor may be an inhibitory nucleic acid molecule – such as ananti-sense oligonucleotide (ASO), a small interfering RNA (siRNA), a double-stranded RNA (dsRNA),a microRNA (miRNA), or a short hairpin RNA (shRNA) – that targets FAM222A (e.g., DNA, RNA, or mRNA encoding FAM222A) and reduces or inhibits FAM222A expression (e.g., FAM222A mRNA and / or protein expression). As another example, a FMA222A inhibitor may be a BRD4 degrader, such as dBET6 or MZ1. On the other hand, FAM222A activators may be, for example, a FAM222A overexpression construct (e.g., a lentiviral construct) or a bromodomain and extra-terminal domain-containing epigenetic reader protein (BET) inhibitor (e.g., a bromodomain-containing protein 4 (BRD4) inhibitor, e.g., JQ1, iBET-151, PFI-1, bromosporine, birabresib, CPI-203, or pelabresib). In some embodiments, the method of treating the disease in the subject further includes administering a second therapeutic agent to the subject. The second therapeutic agent may be a second FAM222A inhibitor, a second FAM222A activator, or any standard therapy known in the field to treat the disease of interest (e.g., a chemotherapy, in the context of treating cancer). For example, two, three, four, five, six, or more different inhibitory nucleic acid molecules (e.g., ASO, siRNA, dsRNA, miRNA, or shRNA) targeting FAM222A may be used in the methods of treatment described herein. Formulation The FAM222A inhibitor, FAM222A activator, and / or second therapeutic agent may be formulated into various compositions (e.g., a pharmaceutical composition) for administration to a subject in a biologically compatible form suitable for administration in vivo. For example, an inhibitory nucleic acid molecule may be administered in a suitable diluent, carrier, or excipient, and may further contain a preservative, e.g., to prevent the growth of microorganisms. Conventional procedures and ingredients for the selection and preparation of suitable compositions are described, for example, in Remington, J.P. The Science and Practice of Pharmacy, Easton, PA. Mack Publishers, 2012, 22nded., and in The United States Pharmacopeial Convention, The National Formulary, United States Pharmacopeial, 2015, USP 38 NF 33). Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to any other animal, e.g., to non-human animals, e.g. non-human mammals. Modification of PATENT Attorney Docket NO.: 51792-002WO2 pharmaceutical compositions suitable for administration to humans to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and / or perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions is contemplated include, but are not limited to, humans and / or other primates and mammals. Delivery Vehicle The FAM222A inhibitor, FAM222A activator, second therapeutic agent, or pharmaceutical compositions thereof, may be delivered by any suitable delivery vehicle known in the art. Forexample, a delivery vehicle for a FAM222A inhibitor (e.g., an ASO, siRNA, dsRNA, miRNA, or shRNAtargeting FAM222A) may be a vector, plasmid, or nano particle, (e.g., a micelle, a liposome, an exosome, or a lipid nano particle (LNP)). In some embodiments, a FAM222A inhibitor (e.g., an ASO, siRNA, dsRNA, miRNA, or shRNA targeting FAM222A) or FAM222A activator is formulated into and delivered by way of an LNP. A FAM222A inhibitor (e.g., an inhibitory nucleic acid molecule targeting FAM222A), a FAM222A activator (e.g., a FAM222A overexpression construct), or a pharmaceutical composition thereof may be delivered to a subject via a vector (e.g., a viral vector). Any suitable viral vector system can be used including, e.g., adenoviruses (e.g., Ad2, Ad5, Ad9, Ad15, Ad17, Ad19, Ad20, Ad22, Ad26, Ad27, Ad28, Ad30, or Ad39), rhabdoviruses (e.g., vesicular stomatitis virus), retroviruses, lentiviral vectors, adeno-associated vectors, poxviruses, herpes viral vectors, and Sindbis viral vectors. A FAM222A inhibitor (e.g., an inhibitory nucleic acid molecule targeting FAM222A), a FAM222A activator (e.g., a FAM222A overexpression construct), or a pharmaceutical composition thereof may be delivered to a subject via a liposome. Liposomes are artificially-prepared vesicles which may primarily be composed of a lipid bilayer and may be used as a delivery vehicle. Liposomes can be of different sizes such as, but not limited to, a multilamellar vesicle (MLV) which may be hundreds of nanometers in diameter and may contain a series of concentric bilayers separated by narrow aqueous compartments, a small unicellular vesicle (SUV) which may be smaller than 50 nm in diameter, and a large unilamellar vesicle (LUV) which may be between 50 and 500 nm in diameter. Liposome design may include, but is not limited to, opsonins or ligands in order to improve the attachment of liposomes to unhealthy tissue or to activate events such as, but not limited to, endocytosis. Liposomes may contain a low or a high pH in order to improve the delivery of the pharmaceutical composition. A FAM222A inhibitor (e.g., an inhibitory nucleic acid molecule targeting FAM222A), a FAM222A activator (e.g., a FAM222A overexpression construct), or a pharmaceutical composition thereof may be delivered to a subject via exosomes. Exosomes produced from cells can be collected from cell culture medium by any suitable method. Typically, exosomes can be prepared from cell culture or tissue supernatant by centrifugation, filtration or combinations of these methods. For example, using standard methods, exosomes can be prepared by differential centrifugation, that is low speed (<20000 g) centrifugation to pellet larger particles followed by high speed (>100000 g) PATENT Attorney Docket NO.: 51792-002WO2 centrifugation to pellet exosomes, size filtration with appropriate filters (for example, 0.22 micrometer filter), gradient ultracentrifugation (for example, with sucrose gradient) or a combination of these methods. A FAM222A inhibitor (e.g., an inhibitory nucleic acid molecule targeting FAM222A), a FAM222A activator (e.g., a FAM222A overexpression construct), or a pharmaceutical composition thereof may be delivered to a subject via LNPs, such as those described in International Publication No. WO2012170930, herein incorporated by reference in its entirety. As a non-limiting example, LNP formulations may contain cationic lipids, distearoylphosphatidylcholine (DSPC), cholesterol, polyethylene glycol (PEG), R-3-[(ω-methoxy poly(ethylene glycol)2000)carbamoyl)]-1,2-dimyristyloxl-propyl-3-amine (PEG-c-DOMG), distearoyl-rac-glycerol (DSG) and / or dimethylaminobutanoate(DMA). As a non-limiting example, 1-5% of the lipid molar ratio of PEG-c-DOMG as compared to the cationic lipid, DSPC and cholesterol. In another embodiment the PEG-c-DOMG may be replaced with a PEG lipid such as, but not limited to, PEG-DSG (1,2-Distearoyl-sn-glycerol, methoxypoly ethylene glycol) or PEG-DPG (1,2-Dipalmitoyl-sn-glycerol, methoxypolyethylene glycol). The cationic lipid may be selected from any lipid known in the art such as, but not limited to, (6Z,9Z,28Z,31Z)-heptatriacont- 6,9,28,31-tetraene-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA), 1,2-dilinoleyloxy-n,n- dimethyl-3-aminopropane (DLin-DMA), C 12-200, and N,N-dimethyl-2,2-di-(9Z,12Z)-9,12- octadecadien-1-yl-1,3-dioxolane-4-ethanamine (DLin-KC2-DMA). Exemplary commercial reagents useful for lipid-based delivery of nucleic acid molecules include, but are not limited to, TransIT-TKO™ (Mirus, Catalog No. MIR 2150), Transmessenger™ (Qiagen, Catalog No.301525), Oligofectamine™ and Lipofectamine™ (Invitrogen, Catalog No. MIR 12252-011 and Catalog No.13778-075), siPORT™ (Ambion, Catalog No.1631), and DharmaFECT™ (Fisher Scientific, Catalog No. T-2001-01). Dosage and Administration The actual dosage amount of a FAM222A inhibitor, FAM222A activator, or composition thereof of administered to a subject can be determined by physical and physiological factors such as body weight, severity of condition, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. Depending upon the dosage (e.g., mg / kg) and the route of administration, the number of administrations of a preferred dosage and / or an effective amount may vary according to the response of the subject. The practitioner responsible for administration can, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject. Administration may occur any suitable number of times per day, and for as long as necessary. Subjects may be adult or pediatric humans, with or without comorbid diseases. Administering to a subject (e.g., human) a FAM222A inhibitor, FAM222A activator, or composition thereof may occur through any suitable route and may depend on the type of disease being treated and / or the cell, tissue, or organ that is being targeted. Exemplary route of administration are: intramuscularly, intravenously, intradermally, percutaneously, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, PATENT Attorney Docket NO.: 51792-002WO2 intranasally, intravitreally, juxtascleral, intracameral, retrobulbar, intravaginally, intrarectally, topically, intratumorally, peritoneally, subcutaneously, subconjunctivally, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularly, orally, topically, locally, by inhalation, by injection, by infusion, by continuous infusion, by localized perfusion bathing target cells directly, by catheter, by lavage, in cremes, or in lipid compositions. In some embodiments, the FAM222A inhibitor or FAM222A activator is administered to the subject intravenously. In some embodiments, the FAM222A inhibitor or FAM222A activator is administered to the subject intraarterially. In some embodiments, the FAM222A inhibitor or FAM222A activator is administered to the subject subcutaneously. In some embodiments, the FAM222A inhibitoror FAM222A activator is administered to the subject intraocularly. In some embodiments, theFAM222A inhibitor or FAM222A activator is administered to the subject intravitreally. Effective ocular delivery routes are known in the art for surpassing precorneal, corneal, and blood-corneal barriers, such as topical, intravitreal, intraocular, juxtascleral, subconjunctival, intracameral, and retrobulbar. Any effective amount of a FAM222A inhibitor or FAM222A activator may be administered to the subject. An effective amount is one that is sufficient to achieve a treatment response such as: slowing, inhibiting, or preventing the progression of the disease; promoting the maintenance of the disease; reversing the progression of the disease; alleviating or ameliorating one or more symptoms of the disease; or reducing one or more clinical manifestations of the disease, as compared to a control (e.g. a non-treatment response). The amount of a given composition may depend on various factors, such as the type of FAM222 inhibitor or FAM222A activator being given, the pharmaceutical composition, the route of administration, the type of disease being treated, the location of the diseased cell, tissue, or organ, the identity of the subject (e.g., age, sex, weight), and the like, but can nevertheless be routinely determined by one skilled in the art (e.g., a clinician). Diseases or Conditions The methods described herein are useful for treating various diseases, particularly those that can benefit from the modulation of angiogenesis and / or endothelial cell function. For example, administering a FAM222A inhibitor can be useful for treating a retinopathy, such as diabetic retinopathy, retinopathy of prematurity (ROP), hypertensive retinopathy, and central serous retinopathy. Proliferative retinopathies, which are defined by exuberant endothelial cell growth, can be responsive to FAM222A inhibition, given their dependence on FAM222A for this growth. It is Applicant’s discovery that the inhibition of FAM222A in an endothelial cell (e.g., in the endothelium) can reduce angiogenesis by reducing the endothelial cell’s proliferation, migration, and cell cycling abilities, and can enhance the endothelial cell’s (e.g., endothelium’s) function as a barrier in a blood vessel. As a result of FAM222A inhibition, there can be reduced leakiness of a blood vessel, thereby treating the retinopathy and a significant complication of retinopathy. As another example, administering a FAM222A inhibitor can be useful for treating cancer, such as breast cancer, colorectal cancer (CRC), esophageal cancer, gastric cancer ovarian cancer, prostate cancer, renal cell carcinoma (RCC), and non-small lung cancer (NSCLC). It is Applicant’s discovery that the inhibition of FAM222A in an endothelial cell can reduce angiogenesis by reducing PATENT Attorney Docket NO.: 51792-002WO2 the endothelial cell’s proliferation, migratory abilities and cell cycling, leading to a reduction in the sprouting and network formation of tumor’s vasculature. Reducing the vasculature in a tumor can deprive the tumor of its blood supply, causing the tumor to shrink and, therefore, providing treatment. While this inhibition of blood supply is important in the growth of many cancers, other specific cancers and tumors involve endothelial cells and endothelial cell growth. Moreover, inhibiting FAM222A can reduce an endothelial cell’s endothelial-to-mesenchymal transition (EndMT), a phenotypic conversion of endothelial cells that is associated with metastasis and therapy resistant tumors (e.g., chemoresistant and radioresistant tumors). Taken together, administering a FAM222A inhibitor can treat cancer in a subject by reducing tumor angiogenesis, reducing metastasis (e.g., of an endothelialcell or mesenchymal cell), and overcoming chemoresistance and radioresistance.On the other hand, administering a FAM222A activator can be useful for treating various vascular diseases or conditions that can benefit from increased angiogenesis, such as peripheral artery disease (PAD), pulmonary arterial hypertension (PAH), coronary disease, cerebrovascular disease, atherosclerosis, and ischemia. In these diseases, limiting, reversing and protecting from EndMT is beneficial. It is Applicant’s discovery that activating FAM222A in an endothelial cell can increase angiogenesis, preserve / restore endothelial function, and protect against EndMT, thereby increasing blood supply to a subject’s tissues and reducing complications of the vascular disease, such as arterial fibrosis. In some embodiments, the FAM222A inhibitor or FAM222A activator is administered to a subject (e.g., a patient) who is receiving or about to receive a stent or bypass graft as a means of medical treatment for a disease (e.g., vascular disease) or complication. The FAM222A inhibitor or FAM222A activator may be administered to the subject (e.g., patient) before (e.g., 1 hour, 6 hours, 24 hours, 2 days 3, days, 4 days, 5 days, 6 days 1 week, 2 weeks, 3 weeks, 1 month, or more), during (e.g., during an operation), or soon after (e.g., within 24 hours) implementation of the stent or bypass graft. This is commonly performed with other agents and would be readily apparent by a clinician in view of the present invention. Overexpression of FAM222A on a stent can accelerate growth of endothelium to cover the stent, which is an important protective step. Treating a graft with a FAM222A inhibitor can protect grafts from the in-growth and occlusion by smooth muscle cells, as happens in bypass grafts and the specific kind of atherosclerosis heart transplant patients get, which is defined by smooth muscle cell in growth. In some embodiments, the FAM222A activator is administered to a subject (e.g., a patient) who is in a state of hypoxemia. Given that increases in FAM222A levels is a compensatory responses to clinical situations in which levels of oxygen are low, in these situations, a FAM222A activator can have an even greater effect given both endogenous as well as exogenous FAM222A increases. FAM222A Activators A FAM222A activator may be a small molecule that increases FAM222A expression. For example, the FAM222A activator may be a bromodomain and extraterminal domain-containing epigenetic reader protein (BET) inhibitor. Exemplary BET inhibitors are JQ1, iBET-151, PFI-1, bromosporine, birabresib, CPI-203, or pelabresib. In some embodiments, the FAM222A activator is PATENT Attorney Docket NO.: 51792-002WO2 JQ1. In some embodiments, the FAM222A activator is iBET-151. In some embodiments, the FAM222A activator is a combination of JQ1 and iBET-151. In some embodiments, the FAM222A activator is PFI-1. In some embodiments, the FAM222A activator is bromosporine. In some embodiments, the FAM222A activator is birabresib. In some embodiments, the FAM222A activator is CPI-203. In some embodiments, the FAM222A activator is pelabresib. In some embodiments, the FAM222A activator is a BRD4 inhibitor (e.g., JQ1 and / or iBET-151). A FAM222A activator may be a nucleic acid molecule (e.g., a vector) that expresses FAM222A and / or another protein known to increase FAM222A expression in a cell. Exemplary proteins that can increase FAM222A expression are early growth response 1 (EGR1) and bonemorphogenic protein 9 (BMP9). In some embodiments, the nucleic acid molecule (e.g., vector)expresses FAM222A. In some embodiments, the nucleic acid molecule (e.g., vector) expresses EGR1. In some embodiments, the nucleic acid molecule (e.g., vector) expresses BMP9. In some embodiments, the nucleic acid molecule (e.g., vector) is one that increases BMP9 expression. In some embodiments, the nucleic acid molecule (e.g., vector) expresses FAM222A and EGR1. In some embodiments, the nucleic acid molecule (e.g., vector) expresses FAM222A and BMP9. In some embodiments, the nucleic acid molecule (e.g., vector) expresses EGR1 and BMP9. In some embodiments, the nucleic acid molecule (e.g., vector) expresses FAM222A, EGR1, and BMP9. Any combination of FAM222A and other proteins known to increase FAM222A expression in envisioned. In some embodiments, the nucleic acid molecule is a vector (e.g., an expression vector). In a preferred embodiment, the vector contains at least one (e.g., one, two, or three) strong promoters that drive expression of FAM222A and / or the protein(s) known to increase FAM222A expression (e.g., EGR1 and / or BMP9). Any suitable vector (e.g., viral vector) system can be used including, e.g., adenoviruses (e.g., Ad2, Ad5, Ad9, Ad15, Ad17, Ad19, Ad20, Ad22, Ad26, Ad27, Ad28, Ad30, or Ad39), rhabdoviruses (e.g., vesicular stomatitis virus), retroviruses, lentiviral vectors, adeno- associated vectors, poxviruses, herpes viral vectors, and Sindbis viral vectors. In some embodiments, the FAM222A activator is a lentiviral construct that overexpresses FAM222A. In some embodiments, the FAM222A activator is a lentiviral construct that overexpresses EGR1. In some embodiments, the FAM222A activator is a lentiviral construct that overexpresses BMP9. In some embodiments, the FAM222A activator is a lentiviral construct that overexpresses FAM222A and EGR1. In some embodiments, the FAM222A activator is a lentiviral construct that overexpresses FAM222A and BMP9. In some embodiments, the FAM222A activator is a lentiviral construct that overexpresses EGR1 and BMP9. In some embodiments, the FAM222A activator is a lentiviral construct that overexpresses FAM222A, EGR1, and BMP9. Any combination of FAM222A and other proteins known to increase FAM222A expression in envisioned to be expressed in the vector. A FAM222A activator may be a soluble protein that activates FAM222A (e.g., EGR1 or BMP9), or soluble FAM222A itself. Any combination of FAM222A and other proteins known to increase FAM222A expression (e.g., EGR1 or BMP9) can be administered to a subject. PATENT Attorney Docket NO.: 51792-002WO2 FAM222A Inhibitors In some embodiments, the FAM222A inhibitor may be a BRD4 degrader, such as dBET6 (Selleckchem Catalog No. S8762) or MZ1 (Selleckchem Catalog No. S8889). dBET6 is a highly cell- permeable degrader of BET bromodomains. These degraders are potent in most cancer cell lines with cellular potency in the sub-nanomolar range. For example, treatment with 100 nM of dBET6 leads todegradation of BRD4 after 1 hour. MZ1 has been shown to induce apoptosis of ovarian cancer cellsand inhibits periostin-mediated ovarian cancer cell migration and invasion. In some embodiments, the FAM222A inhibitor may be an inhibitory nucleic acid molecule that targets FAM222A (e.g., targets DNA, RNA, or mRNA encoding FAM222A) and reduces or inhibitsFAM222A expression (e.g., FAM222A mRNA and / or protein expression). Exemplary inhibitory nucleicacid molecules are ASOs, siRNAs, dsRNAs, miRNAs, and shRNAs; however, any nucleic acid molecule capable of reducing FAM222A mRNA and / or protein expression is envisioned for use of the methods described herein. In some instances, the inhibitory nucleic acid molecule may be referred as an RNA inhibitory (RNAi) molecule. For any of the inhibitory nucleic acid molecules described herein (e.g., ASO, siRNA, dsRNA, miRNA, shRNA, or other inhibitory nucleic acid molecule is capable of reducing expression of a target gene(e.g., FAM222A). In some embodiments, the inhibitory nucleic acid molecule contains at least some sequence complementarity to FAM222A. In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence complementary to at least 15 contiguous nucleotides set forth within any one of SEQ ID NOs: 1-9. In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence complementary to at least 16 contiguous nucleotides set forth within any one of SEQ ID NOs: 1-9. In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence complementary to at least 17 contiguous nucleotides set forth within any one of SEQ ID NOs: 1-9. In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence complementary to at least 18 contiguous nucleotides set forth within any one of SEQ ID NOs: 1-9. In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence complementary to at least 19 contiguous nucleotides set forth within any one of SEQ ID NOs: 1-9. In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence complementary to at least 20 contiguous nucleotides set forth within any one of SEQ ID NOs: 1-9. In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence complementary to at least 21 contiguous nucleotides set forth within any one of SEQ ID NOs: 1-9. In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence complementary to at least 22 contiguous nucleotides set forth within any one of SEQ ID NOs: 1-9. In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence complementary to at least 23 contiguous nucleotides set forth within any one of SEQ ID NOs: 1-9. In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence complementary to at least 24 contiguous nucleotides set forth within any one of SEQ ID NOs: 1-9. In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence complementary to at least 25 contiguous nucleotides set forth within SEQ ID NOs: 1-9. In some embodiments, the inhibitory nucleic acid molecule PATENT Attorney Docket NO.: 51792-002WO2 comprises or consists of a sequence complementary to at least 26 contiguous nucleotides set forth within any one of SEQ ID NOs: 1-9. In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence complementary to at least 27 contiguous nucleotides set forth within any one of SEQ ID NOs: 1-9. In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence complementary to at least 28 contiguous nucleotides set forth within any one of SEQ ID NOs: 1-9. In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence complementary to at least 29 contiguous nucleotides set forth within any one of SEQ ID NOs: 1-9. In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence complementary to at least 30 contiguous nucleotides set forthwithin any one of SEQ ID NOs: 1-9.SEQ ID NOs: 1-9 refer to NCBI Accession numbers, XM_054373596.1, XM_054373595.1, XM_054373594.1, XM_054373593.1, XM_024449229.2, XM_017020055.2, XM_047429749.1, XM_047429748.1, and NM_032829.3, respectively. In some embodiments, the inhibitory nucleic acid is an ASO targeting FAM222A. In some embodiments, the inhibitory nucleic acid is an siRNA targeting FAM222A. In some embodiments, the inhibitory nucleic acid is an dsRNA targeting FAM222A. In some embodiments, the inhibitory nucleic acid is an miRNA targeting FAM222A. In some embodiments, the inhibitory nucleic acid is an shRNA targeting FAM222A. Each of these modalities is described further below. FAM222A sequences are readily available on public databases, e.g., see NCBI Gene ID 84915; HUGO Gene Nomenclature Committee (HGCN) ID 25915; or Ensembl ID: ENSG00000139438. Anti-Sense Oligonucleotide (ASO) ASOs are single (ss) nucleic acid molecules made of DNA, RNA, or both DNA and RNA (e.g., a chimeric) that are complementary to a target gene of interest and prevent translation of the target’s mRNA into a protein. Upon hybridization to a target mRNA, RNase H will degrade the mRNA by hydrolyzation, resulting in reduced mRNA and protein levels of the target. In some embodiments, the ASO includes a nucleotide sequence of 12 to 50 nucleotides in length (e.g., 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or 51 nucleotides in length). It is within the scope of the disclosure that any length, known and previously unknown in the art, may be employed. The nucleotide sequence of the ASO may contain sufficient complementary to a portion of a target gene of interest (e.g., FAM222A) such that the ASO can hybridize with the target gene of interest. In some embodiments, the ASO is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to the target gene of interest (e.g., FAM222A), or a portion thereof. In some embodiments, the ASO is complementary to the target gene of interest (e.g., FAM222A), or a portion thereof. In some embodiments, the ASO comprises a sequence that is complementary to at least 13 contiguous nucleotides set forth within any one of SEQ ID NOS: 1-9. In some embodiments, the ASO comprises a sequence that is complementary to at least 14 contiguous nucleotides set forth within any PATENT Attorney Docket NO.: 51792-002WO2 one of SEQ ID NOS: 1-9. In some embodiments, the ASO comprises a sequence that is complementary to at least 15 contiguous nucleotides set forth within any one of SEQ ID NOS: 1-9. In some embodiments, the ASO comprises a sequence that is complementary to at least 16 contiguous nucleotides set forth within any one of SEQ ID NOS: 1-9. In some embodiments, the ASO comprises a sequence that is complementary to at least 17 contiguous nucleotides set forth within any one of SEQ ID NOS: 1-9. In some embodiments, the ASO comprises a sequence that is complementary to at least 18 contiguous nucleotides set forth within any one of SEQ ID NOS: 1-9. In some embodiments, the ASO comprises a sequence that is complementary to at least 19 contiguous nucleotides set forth within any one of SEQ ID NOS: 1-9. In some embodiments, the ASO comprises a sequence that iscomplementary to at least 20 contiguous nucleotides set forth within any one of SEQ ID NOS: 1-9.In some embodiments, the nucleotide sequence of the ASO may contain sufficient complementary to an exon sequence of a target gene of interest (e.g., an exon of FAM222A). In some embodiments, the nucleotide sequence of the ASO may contain sufficient complementary to an intron sequence of a target gene of interest (e.g., an intron of FAM222A). In some embodiments, the ASO of the disclosure may contain sufficient complementarity to a pre-mRNA transcript or an mRNA transcript encoding FAM222A. The target gene of interest may be FAM222A (e.g., see NCBI Gene ID 84915; HGCN ID 25915; or Ensembl ID: ENSG00000139438; or any of NCBI Accession numbers XM_054373596.1, XM_054373595.1, XM_054373594.1, XM_054373593.1, XM_024449229.2, XM_017020055.2, XM_047429749.1, XM_047429748.1, and NM_032829.3). Different ASOs can be combined for decreasing the protein expression of a target gene of interest (e.g., FAM222A). A combination of two ASOs may be used in a method of the invention, such as two different ASOs, three different ASOs, four different ASOs, or five different ASOs targeting the same gene of interest (e.g., FAM222A, or variants thereof). TABLE 1. EXEMPLARY ASO SEQUENCES A = adenine; C = cytosine; G = guanine; T = thymine In some embodiments, the ASO contains at least 13 contiguous nucleotides set forth within any one of SEQ ID NOs: 10-15 (e.g., see Table 1). In some embodiments, the ASO contains at least 14 contiguous nucleotides set forth within any one of SEQ ID NOs: 10-15 (e.g., see Table 1). In some embodiments, the ASO contains at least 15 contiguous nucleotides set forth within any one of SEQ ID PATENT Attorney Docket NO.: 51792-002WO2 NOs: 10-15 (e.g., see Table 1). In some embodiments, the ASO contains the sequence of any one of SEQ ID NOs: 10-15 (e.g., see Table 1). In some embodiments, the ASO sequence may contain at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to any one or more of SEQ ID NOs: 10-15 (e.g., see Table 1), or a complementary sequence thereof. In some embodiments, the ASO sequence may contain the sequence of any one or more of SEQ ID NOs: 10- 15 (e.g., see Table 1), or a complementary sequence thereof. small interfering RNA (siRNA) AsiRNA is a single-stranded (ss) or double-stranded (ds) nucleic acid molecule made ofDNA, RNA, or both DNA and RNA (e.g., a chimeric) that is complementary to a target gene of interest and prevents translation of the target’s mRNA into a protein. Once an siRNA molecule enters a cell, it is incorporated into an RNA-induced silencing complex (RISC). Upon siRNA hybridization to a target mRNA, the RISC complex will cleave the target mRNA, thereby inactivating the target mRNA, resulting in reduced mRNA and protein levels of the target. In some embodiments, the siRNA includes a nucleotide sequence of about 10 to about 30 nucleotides in length (e.g., 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, or 31 nucleotides in length). It is within the scope of the disclosure that any length, known and previously unknown in the art, may be employed. In some embodiments, the siRNA contains an antisense strand. In some embodiments, length for an antisense strand of the siRNA is between 10 and 30 nucleotides (e.g., 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, or 30 nucleotides), 15 and 25 nucleotides (e.g., 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, or 25 nucleotides), or 18 and 23 nucleotides (e.g., 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, or 23 nucleotides). In some embodiments, the antisense strand is 17 nucleotides. In some embodiments, the antisense strand is 18 nucleotides. In some embodiments, the antisense strand is 19 nucleotides. In some embodiments, the antisense strand is 20 nucleotides. In some embodiments, the antisense strand is 21 nucleotides. In some embodiments, the antisense strand is 22 nucleotides. In some embodiments, the antisense strand is 23 nucleotides. In some embodiments, the antisense strand is 24 nucleotides. In some embodiments, the antisense strand is 25 nucleotides. In some embodiments, the antisense strand is 26 nucleotides. In some embodiments, the antisense strand is 27 nucleotides. In some embodiments, the antisense strand is 28 nucleotides. In some embodiments, the antisense strand is 29 nucleotides. In some embodiments, the antisense strand is 30 nucleotides. In some embodiments, the siRNA contains a sense strand. In some embodiments, the sense strand of the siRNA is between 10 and 30 nucleotides (e.g., 10 nucleotides, 11 nucleotides, 12 PATENT Attorney Docket NO.: 51792-002WO2 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, or 30 nucleotides), or 14 and 23 nucleotides (e.g., 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, or 23 nucleotides). In some embodiments, the sense strand is 15 nucleotides. In some embodiments, the sense strand is 16 nucleotides. In some embodiments, the sense strand is 17 nucleotides. In some embodiments, the sense strand is 18 nucleotides. In some embodiments, the sense strand is 19 nucleotides. In some embodiments, the sense strand is 20 nucleotides. In some embodiments, thesense strand is 21 nucleotides. In some embodiments, the sense strand is 22 nucleotides. In someembodiments, the sense strand is 23 nucleotides. In some embodiments, the sense strand is 24 nucleotides. In some embodiments, the sense strand is 25 nucleotides. In some embodiments, the sense strand is 26 nucleotides. In some embodiments, the sense strand is 27 nucleotides. In some embodiments, the sense strand is 28 nucleotides. In some embodiments, the sense strand is 29 nucleotides. In some embodiments, the sense strand is 30 nucleotides. In some embodiments, the sense and antisense strands of an siRNA molecule are completely complementary. In some embodiments, the sense and antisense strands of an siRNA molecule are completely complementary to the extent that their lengths overlap with one another. Depending on the sequence of the first and second strand, complementarity need not be complete or perfect, which means that the first and second strand are not 100% base-paired due to mismatches. One or more mismatches may be present within the ds siRNA without impacting the siRNA’s ability to reduced expression of a target gene of interest. The nucleotide sequence of an siRNA may contain sufficient complementary to a portion of a target gene of interest (e.g., FAM222A mRNA) such that the siRNA can hybridize with the target gene of interest. In some embodiments, the siRNA is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to the target gene of interest (e.g., FAM222A mRNA), or a portion thereof. In some embodiments, the siRNA is complementary to the target gene of interest (e.g., FAM222A mRNA), or a portion thereof. In some embodiments, the nucleotide sequence of the siRNA may contain sufficient complementary to an exon sequence of a target gene of interest (e.g., an exon of FAM222A). In some embodiments, the nucleotide sequence of the siRNA may contain sufficient complementary to an intron sequence of a target gene of interest (e.g., an intron of FAM222A). In some embodiments, the siRNA of the disclosure may contain sufficient complementarity to a pre-mRNA transcript or an mRNA transcript encoding FAM222A. The FAM222A mRNA molecule that is targeted by the siRNA may be any one of SEQ ID NOs: 1-9. In some embodiments, the siRNA molecule includes 0-7 nucleotide 3’ overhangs or 0-4 nucleotide 5’ overhangs. In some embodiments, the siRNA molecule has a single uracil (e.g., U) overhang at each 3’ end of the siRNA. In some embodiments, the siRNA molecule has a double uracil (e.g., UU) overhang at each 3’ end of the siRNA. In some embodiments, the siRNA molecule has a single thymine (e.g., T) overhang at each 3’ end of the siRNA. In some embodiments, the siRNA PATENT Attorney Docket NO.: 51792-002WO2 molecule has a double thymine (e.g., TT) overhang at each 3’ end of the siRNA. In some embodiments, the siRNA molecule has a cytosine and thymine (e.g., CT) overhang at each 3’ end of the siRNA. Different siRNAs can be combined for decreasing the expression of a target gene of interest (e.g., FAM222A). A combination of two siRNAs may be used in a method of the invention, such as two different siRNAs, three different siRNAs, four different siRNAs, or five different siRNAs targeting the same gene of interest (e.g., FAM222A, or variants thereof). In some embodiments, the siRNA sequence may contain at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to any one or more ofSEQ ID NOs: 16-31 (e.g., see Table 2), or a complementary sequence thereof. In someembodiments, the siRNA sequence may contain the sequence of any one or more of SEQ ID NOs: 16-31 (e.g., see Table 2), or a complementary sequence thereof. In some embodiments, the siRNA contains at least 15 contiguous nucleotides set forth within any one of SEQ ID NOs: 16-31 (e.g., see Table 2). In some embodiments, the siRNA contains at least 16 contiguous nucleotides set forth within any one of SEQ ID NOs: 16-31 (e.g., see Table 2). In some embodiments, the siRNA contains at least 17 contiguous nucleotides set forth within any one of SEQ ID NOs: 16-31 (e.g., see Table 2). In some embodiments, the siRNA contains at least 18 contiguous nucleotides set forth within any one of SEQ ID NOs: 16-31 (e.g., see Table 2). In some embodiments, the siRNA contains at least 19 contiguous nucleotides set forth within any one of SEQ ID NOs: 16-31 (e.g., see Table 2). In some embodiments, the siRNA contains at least 20 contiguous nucleotides set forth within any one of SEQ ID NOs: 16-31 (e.g., see Table 2). In some embodiments, the siRNA contains 21 contiguous nucleotides set forth within any one of SEQ ID NOs: 16-31 (e.g., see Table 2). TABLE 2. EXEMPLARY siRNA SEQUENCES PATENT Attorney Docket NO.: 51792-002WO2 A = adenine; C = cytosine; G = guanine; U = uracil. In some embodiments, the siRNA comprises a sequence that is complementary to at least 15 contiguous nucleotides set forth within any one of SEQ ID NOS: 1-9. In some embodiments, the siRNA comprises a sequence that is complementary to at least 16 contiguous nucleotides set forth within any one of SEQ ID NOS: 1-9. In some embodiments, the siRNA comprises a sequence that is complementary to at least 17 contiguous nucleotides set forth within any one of SEQ ID NOS: 1-9. In some embodiments, the siRNA comprises a sequence that is complementary to at least 18 contiguous nucleotides set forth within any one of SEQ ID NOS: 1-9. In some embodiments, the siRNA comprises a sequence that is complementary to at least 19 contiguous nucleotides set forth within any one of SEQ ID NOS: 1-9. In some embodiments, the siRNA comprises a sequence that is complementary to at least 20 contiguous nucleotides set forth within any one of SEQ ID NOS: 1-9. In some embodiments, the siRNA comprises a sequence that is complementary to at least 21 contiguous nucleotides set forth within any one of SEQ ID NOS: 1-9. In some embodiments, the siRNA comprises a sequence that is complementary to at least 22 contiguous nucleotides set forth within any one of SEQ ID NOS: 1-9. In some embodiments, the siRNA comprises a sequence that is complementary to at least 23 contiguous nucleotides set forth within any one of SEQ ID NOS: 1-9. In some embodiments, the siRNA comprises a sequence that is complementary to at least 24 contiguous nucleotides set forth within any one of SEQ ID NOS: 1-9. In some embodiments, the siRNA comprises a sequence that is complementary to at least 25 contiguous nucleotides set forth within any one of SEQ ID NOS: 1-9. In some embodiments, the siRNA comprises a sequence that is complementary to at least 26 contiguous nucleotides set forth within any one of SEQ ID NOS: 1-9. In some embodiments, the siRNA comprises a sequence that is complementary to at least 27 contiguous nucleotides set forth within any one of SEQ ID NOS: 1-9. In some embodiments, the siRNA comprises a sequence that is complementary to at least 28 contiguous nucleotides set forth within any one of SEQ ID NOS: 1-9. In some embodiments, the siRNA comprises a sequence that is complementary to at least 29 contiguous nucleotides set forth within any one of SEQ ID NOS: 1-9. In some embodiments, the siRNA comprises a sequence that is complementary to at least 30 contiguous nucleotides set forth within any one of SEQ ID NOS: 1-9. Double-stranded RNA (dsRNA) A dsRNA of the disclosure is a ds nucleic acid molecule made of DNA, RNA, or both DNA and RNA (e.g., a chimeric) that is complementary to a target gene of interest and prevents translation of the target’s mRNA into a protein. Typically, a dsRNA is longer than an siRNA and is processed within a cell to form an siRNA molecule. The siRNA is then incorporated into RISC. Upon siRNA hybridization to a target mRNA, the RISC complex will cleave the target mRNA, thereby inactivating the target mRNA, resulting in reduced mRNA and protein levels of the target. PATENT Attorney Docket NO.: 51792-002WO2 In some embodiments, the dsRNA includes a sense strand and an antisense strand, each containing a nucleotide sequence of about 25 to about 5000 nucleotides in length, or longer (e.g., 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, about 150, about 155, about 160, about 165, about 170, about 175, about 180, about 185, about 190, about 195, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, about 300, about 310, about 320, about 330, about 340, about 350, about 360, about 370, about 380, about 380, about 400, about 425, about 450, about 475, about 500, about 525, about 550, about 575, about 600, about625, about 650, about 675, about 700, about 725, about 750, about 775, about 800, about 825, about850, about 875, about 900, about 925, about 950, about 975, about 1000, about 1100, about 1200, about 1300, about 1400, about 1500, about 1600, about 1700, about 1800, about 1900, about 2000, about 2200, about 2400, about 2600, about 2800, about 3000, about 3250, about 3500, about 3750, or about 4000 nucleotides in length). In some embodiments, the dsRNA includes a sense strand and an antisense strand, each containing a nucleotide sequence of 25 to 4000 nucleotides in length, or longer (e.g., 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 380, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2200, 2400, 2600, 2800, 3000, 3250, 3500, 3750, or 4000 nucleotides in length). It is within the scope of the disclosure that any length, known and previously unknown in the art, may be employed for the current invention. In some embodiments, the sense and antisense strands of a dsRNA molecule are completely complementary. In some embodiments, the sense and antisense strands of a dsRNA molecule are completely complementary to the extent that their lengths overlap with one another. Depending on the sequence of the first and second strand, complementarity need not be complete or perfect, which means that the first and second strand are not 100% base-paired due to mismatches. One or more mismatches may be present within the ds dsRNA without impacting the dsRNA’s ability to reduced expression of a target gene of interest. The nucleotide sequence of a dsRNA may contain sufficient complementary to a portion of a target gene of interest (e.g., FAM222A mRNA) such that the dsRNA can hybridize with the target gene of interest. In some embodiments, the dsRNA is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to the target gene of interest (e.g., FAM222A mRNA), or a portion thereof. In some embodiments, the dsRNA is complementary to the target gene of interest (e.g., FAM222A mRNA), or a portion thereof. In some embodiments, the nucleotide sequence of the dsRNA may contain sufficient complementary to an exon sequence of a target gene of interest (e.g., an exon of FAM222A). In some embodiments, the nucleotide sequence of the dsRNA may contain sufficient complementary to an intron sequence of a target gene of interest (e.g., an intron of FAM222A). In some embodiments, the PATENT Attorney Docket NO.: 51792-002WO2 dsRNA contains sufficient complementarity to a pre-mRNA transcript or an mRNA transcript encoding FAM222A. The target gene of interest (e.g., FAM222A) may be any one of SEQ ID NOs: 1-9. The target gene of interest may be FAM222A (e.g., see NCBI Gene ID 84915; HGCN ID 25915; or Ensembl ID: ENSG00000139438). Different dsRNAs can be combined for decreasing the protein expression of a target gene of interest (e.g., FAM222A). A combination of two dsRNAs may be used in a method of the invention, such as two different dsRNAs, three different dsRNAs, four different dsRNAs, or five different dsRNAs targeting the same gene of interest (e.g., FAM222A, or variants thereof). micro RNA (miRNA)A miRNA is a single stranded (ss) nucleic acid molecule made of DNA, RNA, or both DNA and RNA (e.g., a chimeric) that is complementary to a target gene of interest and prevents translation of the target’s mRNA into a protein. Once a miRNA molecule enters a cell, it is incorporated into RISC. Upon miRNA hybridization to a target mRNA, the RISC complex will cleave the target mRNA, thereby inactivating the target mRNA, resulting in reduced mRNA and protein levels of the target. In some embodiments, the miRNA includes a nucleotide sequence of 6 to 30 nucleotides in length (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 nucleotides in length). It is within the scope of the disclosure that any length, known and previously unknown in the art, may be employed. The nucleotide sequence of the miRNA may contain sufficient complementary to a portion of a target gene of interest (e.g., FAM222A mRNA) such that the miRNA can hybridize with the target gene of interest. In some embodiments, the miRNA is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to the target gene of interest (e.g., FAM222A mRNA), or a portion thereof. In some embodiments, the miRNA is complementary to the target gene of interest (e.g., FAM222A mRNA), or a portion thereof. In some embodiments, the nucleotide sequence of the miRNA may contain sufficient complementary to an exon sequence of a target gene of interest (e.g., an exon of FAM222A). In some embodiments, the nucleotide sequence of the miRNA may contain sufficient complementary to an intron sequence of a target gene of interest (e.g., an intron of FAM222A). In some embodiments, the miRNA of the disclosure may contain sufficient complementarity to a pre-mRNA transcript or an mRNA transcript encoding FAM222A. The target gene of interest (e.g., FAM222A) may be any one of SEQ ID NOs: 1-9. The target gene of interest may be FAM222A (e.g., see NCBI Gene ID 84915; HGCN ID 25915; or Ensembl ID: ENSG00000139438). Different miRNAs can be combined for decreasing the protein expression of a target gene of interest (e.g., FAM222A). A combination of two or more miRNAs may be used in a method of the invention, such as two different miRNAs, three different miRNAs, four different miRNAs, or five different miRNAs targeting the same gene of interest (e.g., FAM222A, or variants thereof) PATENT Attorney Docket NO.: 51792-002WO2 short hairpin RNA (shRNA) A shRNA is a ss or ds nucleic acid molecules made of DNA, RNA, or both DNA and RNA (e.g., a chimeric) that is complementary to a target gene of interest and prevents translation of the target’s mRNA into a protein. Once an shRNA molecule enters a cell, it is incorporated into RISC. Upon shRNA hybridization to a target mRNA, the RISC complex will cleave the target mRNA, thereby inactivating the target mRNA, resulting in reduced mRNA and protein levels of the target. In some embodiments, the shRNA includes a nucleotide sequence of 60 to 100 nucleotides in length (e.g., 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides in length). AshRNA includes a variable hairpin loop structure and a stem sequence. In someembodiments the stem sequence may be 10 to 50 nucleotides in length (e.g., 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length). In some embodiments, the hairpin size is between 4 to 50 nucleotides in length, although the loop size may be larger without significantly affecting silencing activity. The shRNA molecule may contain mismatches, for example G-U mismatches between two strands of the shRNA stem without decreasing potency. In some embodiments, the shRNA is designed to include one or several G-U pairings in the hairpin stem to stabilize hairpins during propagation in bacteria, for example. The nucleotide sequence of the shRNA may contain sufficient complementary to a portion of a target gene of interest (e.g., FAM222A mRNA) such that the shRNA can hybridize with the target gene of interest. In some embodiments, the shRNA is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to the target gene of interest (e.g., FAM222A mRNA), or a portion thereof. In some embodiments, the shRNA is complementary to the target gene of interest (e.g., FAM222A mRNA), or a portion thereof. In some embodiments, the nucleotide sequence of the shRNA may contain sufficient complementary to an exon sequence of a target gene of interest (e.g., an exon of FAM222A). In some embodiments, the nucleotide sequence of the shRNA may contain sufficient complementary to an intron sequence of a target gene of interest (e.g., an intron of FAM222A). In some embodiments, the shRNA of the disclosure may contain sufficient complementarity to a pre-mRNA transcript or an mRNA transcript encoding FAM222A. The target gene of interest (e.g., FAM222A) may be any one of SEQ ID NOs: 1-9. The target gene of interest may be FAM222A (e.g., see NCBI Gene ID 84915; HGCN ID 25915; or Ensembl ID: ENSG00000139438). Different shRNAs can be combined for decreasing the protein expression of a target gene of interest (e.g., FAM222A). A combination of two or more shRNAs may be used in a method of the invention, such as two different shRNAs, three different shRNAs, four different shRNAs, or five different shRNAs targeting the same gene of interest (e.g., FAM222A, or variants thereof). Modifications to Inhibitory Nucleic Acid Molecules It is contemplated that any of the nucleic acid molecules disclosed herein (e.g., inhibitory nucleic acid molecules or expression constructs, e.g., vectors) may be used in the methods disclosed PATENT Attorney Docket NO.: 51792-002WO2 herein in an unmodified or in a modified form. Unmodified nucleic acid molecules contain nucleobases that include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleic acid molecules are described in more detail below. Modifications may be achieved by systematically adding or removing linked nucleosides to generate longer or shorter sequences. Modifications may be achieved by incorporating, for example, one or more alternative nucleosides, alternative 2’ sugar moieties, and / or alternative internucleoside linkages, which are described further below. Typically, these types of modifications are introduced to optimize the molecule’s efficacy or biophysical properties (e.g., increasing serum stability or circulating half-life,increasing thermal stability, enhancing transmembrane delivery, reduce immunogenicity, and / ortargeting to a particular location or cell type). Modification may further be achieved by covalently or non-covalently conjugating a moiety (e.g., a targeting moiety, a hydrophobic moiety, a cell penetrating peptide, or a polymer) to the 5’ end and / or 3’ end of the nucleic acid molecule, as described in more detail below. Nucleoside Modifications Modification of a nucleic acid molecule described herein (e.g., an inhibitory nucleic acid molecule or expression construct, e.g., vector) may include one or more of the following nucleoside modifications: 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2- aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (-C=C-CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8- thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5- bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7- methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and / or 3-deazaguanine and 3-deazaadenine. The nucleic acid molecule may also include nucleobases in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, and / or 2-pyridone. Further modification of the nucleic acid molecule may include nucleobases disclosed in US 3,687,808; Kroschwitz, J.I., ed. The Concise Encyclopedia of Polymer Science and Engineering, New York, John Wiley & Sons, 1990, pp.858-859; Englisch et al., Angewandte Chemie, International Edition 30:613, 1991; and Sanghvi, Y.S., Chapter 16, Antisense Research and Applications, CRC Press, Gait, M.J. ed., 1993, pp.289-302. Sugar Modifications Modifications of a nucleic acid molecule described herein (e.g., an inhibitory nucleic acid molecule or expression construct, e.g., vector) may also include one or more of the following 2’ sugar modifications: 2’-O-methyl (2’-O-Me), 2′-methoxyethoxy (2′-O-CH2CH2OCH3, also known as 2′-O-(2- methoxyethyl) or 2′-MOE), 2′-dimethylaminooxyethoxy, i.e., a O(CH2)2ON(CH3)2 group, also known PATENT Attorney Docket NO.: 51792-002WO2 as 2′-DMAOE, and / or 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethylamino- ethoxy-ethyl or 2′-DMAEOE), i.e., 2′-O-CH2OCH2N(CH3)2. Other possible 2′-modifications that can modify the inhibitory nucleic acid molecules described herein include all possible orientations of OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl. Other potential sugar substituent groups include, e.g., aminopropoxy (-OCH2CH2CH2NH2), allyl (-CH2-CH=CH2), -O-allyl (-O-CH2-CH=CH2) and fluoro (F).2′-sugar substituent groups may be in the arabino (up) position or ribo (down) position. In some embodiments, the 2′-arabino modification is 2′-F. Similar modifications may also be made at other positions on the nucleic acid molecule,particularly the 3′ position of the sugar on the 3′ terminal nucleoside or in 2′-5′ linked oligonucleotidesand the 5′ position of 5′ terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Internucleoside Linkage Modifications Modification of a nucleic acid molecule described herein (e.g., an inhibitory nucleic acid molecule or expression construct, e.g., vector) may include one or more of the following internucleoside modifications: phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates, 5'-alkylene phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3' to 3', 5' to 5' or 2' to 2' linkage. Conjugates It is contemplated that any of the nucleic acid molecules disclosed herein (e.g., inhibitory nucleic acid molecules or expression constructs, e.g., vectors) may be modified via the addition of an auxiliary moiety, e.g., a cell penetrating peptide (CPP), a polymer, a hydrophobic moiety, or a targeting moiety. The auxiliary moiety may be present as a 5’ terminal modification (e.g., covalently bonded to a 5’-terminal nucleoside), a 3’ terminal modification (e.g., covalently bonded to a 3’-terminal nucleoside), or an internucleoside linkage (e.g., covalently bonded to phosphate or phosphorothioate in an internucleoside linkage). CPPs are known in the art (e.g., TAT or Arg8) (Snyder and Dowdy, 2005, Expert Opin. Drug Deliv.2, 43-51). Specific examples of CPPs are provided in WO2011157713, which is incorporated herein by reference in its entirety. A nucleic acid molecule may include covalently attached neutral polymer-based auxiliary moieties. Neutral polymers include poly(C1-6 alkylene oxide), e.g., poly(ethylene glycol) and poly(propylene glycol) and copolymers thereof, e.g., di- and triblock copolymers. A nucleic acid molecule containing a hydrophobic moiety may exhibit superior cellular uptake, as compared to a nucleic acid molecule lacking the hydrophobic moiety. A hydrophobic moiety is a PATENT Attorney Docket NO.: 51792-002WO2 monovalent group (e.g., a bile acid (e.g., cholic acid, taurocholic acid, deoxycholic acid, oleyl lithocholic acid, or oleoyl cholenic acid), glycolipid, phospholipid, sphingolipid, isoprenoid, vitamin, saturated fatty acid, unsaturated fatty acid, fatty acid ester, triglyceride, pyrene, porphyrine, texaphyrine, adamantine, acridine, biotin, coumarin, fluorescein, rhodamine, Texas-Red, digoxygenin, dimethoxytrityl, t-butydimethylsilyl, t-butyldiphenylsilyl, cyanine dye (e.g., Cy3 or Cy5), Hoechst 33258 dye, psoralen, or ibuprofen) covalently linked to the nucleic acid backbone (e.g., 5’-terminus) of the inhibitory nucleic acid molecule. A targeting moiety is selected based on its ability to target a desired or selected cell population, tissue, or organ that expresses the corresponding binding partner (e.g., either thecorresponding receptor or ligand) for the selected targeting moiety. Exemplary targeting moieties fordelivery include platelet endothelial cell adhesion molecule (PECAM-1), intercellular adhesion molecule 1 (ICAM1), vascular cell adhesion protein 1 (VCAM1), and Von Willebrand factor (vWF). Synthesis of Nucleic Acid Molecules Anucleic acid molecule described herein (e.g., an inhibitory nucleic acid molecule orexpression construct, e.g., vector) may be synthesized using any technique or method known in the art. For example, a nucleic acid molecule may be prepared using a phosphoramidite-based synthesis cycle. This synthesis cycle includes the steps of (1) de-blocking a 5’-protected nucleotide to produce a 5’-deblocked nucleotide, (2) coupling the 5’-deblocked nucleotide with a 5’-protected nucleoside phosphoramidite to produce nucleosides linked through a phosphite, (3) repeating steps (1) and (2) one or more times as needed, (4) capping the 5’-terminus, and (5) oxidation or sulfurization of internucleoside phosphites. The reagents and reaction conditions useful for the oligonucleotide synthesis are known in the art. A nucleic acid molecule described herein (e.g., an inhibitory nucleic acid molecule or expression construct, e.g., vector) may be linked to solid support as a result of solid-phase synthesis. Cleavable solid supports that may be used are known in the art. Non-limiting examples of the solid support include, e.g., controlled pore glass or macroporous polystyrene bonded to a strand through a cleavable linker (e.g., succinate-based linker) known in the art (e.g., UnyLinkerTM). A nucleic acid linked to solid support may be removed from the solid support by cleaving the linker connecting a nucleic acid and solid support. EXAMPLES Example 1. FAM222A, part of the BET-regulated basal endothelial transcriptome, is a novel determinant of endothelial biology and angiogenesis Abstract Bromodomain and extra-terminal domain-containing epigenetic reader proteins (BETs), including BRD4, orchestrate transcriptional programs induced by pathogenic stimuli, as intensively studied in cardiovascular disease and elsewhere. In endothelial cells (ECs), BRD4 directs induced pro-inflammatory, pro-atherosclerotic transcriptional responses; BET inhibitors, like JQ1, repress PATENT Attorney Docket NO.: 51792-002WO2 these effects and decrease atherosclerosis. While BET effects in pathogenic conditions has prompted therapeutic BET inhibitor development, BET action under basal conditions, including ECs, has remained understudied. To understand BET action in basal endothelial transcriptional programs, we first analyzed EC RNA-Seq data in the absence versus presence of JQ1 before using BET regulation to identify novel determinants of EC biology and function. RNA-Seq datasets of human umbilical vein ECs without and with JQ1 treatment were analyzed. After identifying C12orf34, also known as Family with Sequence Similarity 222 Member A (FAM222A), as a previously unreported, basally expressed, potently JQ1-induced EC gene, FAM222A was studied in endothelial and angiogenic responses in vitro using siRNA silencing andlentiviral overexpression, in vitro, ex vivo and in vivo, including aortic sprouting, Matrigel plug assaysand murine neonatal oxygen- induced retinopathy (OIR). Resting EC RNA-Seq data indicates BETs direct transcriptional programs underlying core endothelial properties including migration, proliferation and angiogenesis. BET inhibition in resting ECs also significantly induced a subset of mRNAs, including FAM222A - a unique BRD4-regulated gene with no reported EC role. Silencing endothelial FAM222A significantly decreased cellular proliferation, migration, network formation, aorta sprouting and Matrigel plug vascularization through coordinated modulation of VEGF and NOTCH mediator expression in vitro, ex vivo, or in vivo; lentiviral FAM222A overexpression had opposite effects. In vivo, siFAM222A significantly repressed retinal revascularization in neonatal murine oxygen-induced retinopathy through similar angiogenic signaling modulation. In conclusion, BET control over the basal endothelial transcriptome includes FAM222A, a novel, BRD4-regulated, key determinant of endothelial biology and angiogenesis. Introduction The bromodomain and extra-terminal domain-containing protein family (BETs), including BRD2, BRD3 and BRD4, are epigenetic reader proteins that orchestrate specific transcriptional programs involved in differentiation, identity, and induced cell state transitions, as implicated in cancer, atherosclerosis, myocardial hypertrophy and pulmonary hypertension1. BETs control gene expression by binding to specific acetylated lysine residues on chromatin histone tails, thus enabling assembly of transcriptional machinery assembly, including RNA Polymerase II (Pol II)2,3. BET control over pathogenic transcription has prompted pursuit of BET inhibitors as therapeutic agents, including in cardiovascular disease1. In the endothelium, cytokine stimulation induces BRD4- regulated inflammatory and pro-atherosclerotic gene expression; BET inhibitors repress these same responses in vitro and in vivo, as reported by us and others2,4,5. Despite a focus on BETs in pathologic transcriptional programs, BETs also modulate basal gene expression, as seen with TNFα stimulation of endothelial cells (ECs) causing loss of BRD4-dependent transcription of specific genes expressed under basal conditions1,2. Although largely unstudied, BET action in basal transcription is an important component of BET biology in physiologic versus pathologic conditions as well as the potential therapeutic BET inhibitor effects6. PATENT Attorney Docket NO.: 51792-002WO2 Initially, we sought to define the basal BET-regulated endothelial transcriptome, analyzing RNA-Seq data in human umbilical vein ECs before and after exposure to the well-validated, highly specific pan-BET inhibitor JQ1. In addition to using JQ1 as a tool to probe BET action in ECs under basal conditions, we also reasoned since BET regulate induced, canonical pro-inflammatory and pro- atherosclerotic endothelial genes, BETs might similarly control important genes under basal conditions, providing an orthogonal approach to identifying novel determinants of EC function. Interrogation and gene ontogeny (GO) analysis of basal HUVEC RNA-Seq data when BET activity was intact (no JQ1) vs inhibited (with JQ1) established BETs govern key endothelial transcriptional programs, including angiogenesis. This data also underscored another understudiedaspect of BET transcriptional action, namely genes whose expression was induced, not repressed,after BET inhibition. Further consideration of basally expressed endothelial mRNAs most induced after BET inhibition and with unknown function pointed to Chromosome 12 Open Reading Frame 34 (C12orf34), also known as Family with Sequence Similarity 222 Member A (FAM222A). Subsequent data presented here establish FAM222A is abundantly expressed in ECs and essential for EC proliferation, migration, and angiogenesis by modulating VEGF and NOTCH mediator expression. In vivo, FAM222A silencing repressed retinal neovascularization in the neonatal murine oxygen-induced retinopathy (OIR), a well-established angiogenesis model of retinopathy of prematurity (ROP)7,8. Taken together, this data expands insight into BET control over endothelial transcription and identifies FAM222A as a key novel determinant of endothelial biology and angiogenesis. Methods Ethical Approval: Institutional approval was obtained for all mouse studies, as performed in C57Bl / 6J mice (Jackson Laboratories), maintained in a pathogen-free facility with standard light / dark cycling and ad libitum food containing 20% protein, 4.5% fat, PicoLab Rodent Diet20 , #5053, DietLabs and water access (Animal protocol #2016N000115, Harvard Medical School Institutional Animal Care and Use Committee). Data Sharing: Our published basal HUVEC RNA-seq and ChIP-seq datasets with or without JQ1 (500nM, 3 hours) are available (accession numbers GSE53999 and GSE539998)2. Statistical analysis: Data is shown as mean ± standard error of the mean (SEM) unless otherwise noted. Unpaired Student’s t test was used for single comparisons. One-way ANOVA followed by Tukey post-hoc test was used to determine the significance of one independent variable between more than two groups. Comparisons between two groups and two nominal variables were performed using Two- way ANOVA followed by Tukey post-hoc test. Differences were considered statistically significant at p<0.05. Statistical analysis was performed using GraphPad Prism. Cell Transfection: HUVECs were transfected with small-interference RNAs (siRNAs, Sigma; sequences in Table 3) for FAM222A, BRD2, BRD3, BRD4, EGR1 (early growth response 1), or scrambled control overnight using RNAi Max (13778075, Invitrogen, MA) as described in the manufacturer’s protocol. The FAM222A siRNA no.2 was a SMARTpool siRNA (L-015010 to 02-0005, Dharmacon). bEND.3 cells were transfected with siRNAs for FAM222A or control overnight using PATENT Attorney Docket NO.: 51792-002WO2 Lipofectamine 2000 (11668019, Invitrogen, MA) as above. All siRNAs were used for transfection at 50 nmol / L. TABLE 3. siRNA SEQUENCES USED Cell culture and reagents: Human umbilical vein endothelial cells (HUVEC; Lonza) were cultured in endothelial cell growth medium EGM-2 (Lonza, CC-3162) on 0.1% gelatin-coated dishes. Mouse brain endothelial cells (bEND.3, ATCC, CRL-2299) were cultured in Dulbecco’s Modified Eagle Medium / F12 (1:1) (DMEM; Gibco, 11320-033) supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin / streptomycin (P / S). Cells that were utilized for experiments were passaged no more than six times. HEK293T cells (ATCC, CRL-3216) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS, 1% L-glutamine (2mM final) and 1% P / S. HUVEC were stimulated with VEGF 165 (Sigma, V5765) at a concentration of 50ng / ml following overnight serum starvation (0% FBS). Similarly, TNFα (Peprotech, 300-01A), IL1b (Peprotech, 100-35B) and TGFβ2 (R&D, 302-B2) were used at a final concentration of 25 ng / ml and 10ng / ml following overnight serum starvation. To inhibit the NOTCH signaling pathway, the γ-secretase specific inhibitor N-[N-(3,5- Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT, Sigma, D5942) in DMSO was added in a final concentration of 10 uM. The pan-BET inhibitors JQ1 or iBET-151 in DMSO were added at different concentrations. Mitomycin C (Sigma, M4287) was used at 1ug / ml for all proliferation blocking conditions. Staurosporine (Abcam, ab120056) was added in final concentration of 10uM for 4 hours to induce apoptosis. Equal volume of vehicle was added in control conditions accordingly. Luciferase Reporter Assay: The 1523 bp promoter region (1186 bp proximal to start site) of the human FAM222A gene was synthesized by Genewiz (MA, USA). The synthesized nucleotides were subcloned into pGL4 basic vector (Promega, WI, USA) and confirmed by DNA sequencing. The human EGR1 (NM_001964, RC209956) expression vector was synthesized by Origene (MD, USA). HEK293T cells were seeded onto 6-well plates at 500,000 cells per well and 24 hours after seeding the cells were transfected with the FAM222A reporter plasmid, an internal control reporter plasmid and the EGR1 expression vector using Lipofectamine 3000 (L3000, Invitrogen, MA, USA) for 24 hours. JQ1 (500 nM) was added for 3 hours. Cells were lysed and luciferase activities were PATENT Attorney Docket NO.: 51792-002WO2 determined with a standard 96-well plate reads using the Dual-Luciferase Reporter Assay System (E1910, Promega). RT-qPCR: Extracted total RNA (Trizol) underwent spectrophotometric purity and concentration measurement (Nanodrop) before reverse transcription (High-Capacity cDNA Reverse Transcription, Applied Biosystem, USA). SYBR-green master mix was used for RT-qPCRs reactions and normalized to housekeeping gene 36B4 or HPRT1; relative transcript expression was determined using 2-ΔΔCT methodology. Primer sequences are in Table 4. TABLE 4. PRIMER SEQUENCES USED FOR RT-qPCR PATENT Attorney Docket NO.: 51792-002WO2 PATENT Attorney Docket NO.: 51792-002WO2 Western Blot Analysis: Whole cell lysates were extracted with RIPA buffer (Boston Bioproducts) containing Complete Protease Inhibitor Cocktail Tablets (Roche) and the phosphatase inhibitor vanadate (Biolabs). The extracted protein lysates underwent concentration measurement using the micro BCA kit (Thermofisher). After heating (Laemmli buffer, 10’, 100°C), total protein (10- 20 μg) underwent gradient gel electrophoresis (4-20%) before transfer to activated PVDF membranes (Bio-Rad) blocking with BSA (5%, 1 hour) and overnight incubation with the indicated antibodies as listed in the Table 5. Protein signal detection was done via chemiluminescence using appropriate horseradish peroxidase-conjugated secondary antibodies. TABLE 5. PRIMARY ANTIBODIES USED FOR WESTERN BLOT ANALYSIS PATENT Attorney Docket NO.: 51792-002WO2 Published human RNA-seq datasets and pathway enrichment analysis: We reanalyzed our public RNA-seq dataset from resting HUVEC treated with JQ1(500nM) for 3 hours (GSE53999)1. Normalization and differential expression analysis were performed by using Limma-Voom. Genes with adjusted p-value (FDR) < 0.05 and log2 fold change (>0.1) were called as differentially expressed genes (DEGs) for each comparison. The DEGs were visualized using hierarchical clustering plot. Top differentially expressed genes (log2 fold change, >2 or <2, FDR<0.05) were shown in volcano plot (ggplot2 package) and heatmap (d3heatmap package). Differentially expressed genes (DEGs) were identified as being at least 1.5-fold change 3 and adjusted p-value < 0.05 (false discovery rate). DEGs were subjected to gene set enrichment analyses by using Metacore and DAVID functional annotation tool. The significant values for the canonical pathways were calculated by Fisher exact test and the top 10 pathways were visualized in box plots. Chromatin Immunoprecipitation (ChIP)-qPCR: Resting HUVEC were with JQ1(500nM) for 3 hours before performing Chromatin Immunoprecipitation (ChIP) using standard approaches with modifications. Briefly, cells were crosslinked with 1% formaldehyde for 15 min at room temperature and then, the reaction was stopped by incubating with glycine (0.125 M) for 5 min. The cells were harvested in PBS before centrifugation, then resuspended in hypotonic lysis buffer. After centrifugation to isolate nuclei, resuspension in SDS lysis buffer was followed by Mnase enzyme treatment (20 min, 37°C) and sonification (Diagenode). Samples, diluted in ChIP buffer, were incubated with anti-BRD4 (Cell Signaling,13440) or anti-IgG antibodies (overnight, 4°C). Before antibody addition, input samples were collected. Immunocomplexes were isolated (DynabeadsTM Protein A, ThermoFisher), washed and eluted. Crosslinking was reversed by incubating the samples PATENT Attorney Docket NO.: 51792-002WO2 overnight at 65°C before performing RNAase A (20mg / mL) and Proteinase K (20mg / mL, both ThermoFisher) treatments. DNA was isolated by phenol / chloroform extraction (ThermoFisher). BRD4 enrichment in cis-regulatory regions of FAM222A was evaluated by RT-qPCR using specific primers sequences (Table 6). TABLE 6. PRIMER SEQUENCES USED FOR ChIP-qPCR Immunofluorescence and Immunohistochemistry staining: For immunofluorescence staining, HUVEC were seeded on vitronectin-coated cell culture chamber slides, fixed in 4% paraformaldehyde (PFA, Cat#: BM-155, Boston Bio Products) and permeabilized using 0.3% triton-X100. The cells were blocked with 5% normal bovine serum (Cat#:9048-46-8, American Bio) for 1 hour and immunostained with primary antibodies against FAM222A (1:200, Cat#: PA5-59093, Thermo Fisher) overnight at 4 oC and appropriate Alexa Fluor conjugated secondary antibodies (1:300, Invitrogen) for 30 min at room temperature. Coverslips were mounted using ProLong Gold Antifade Mountant after DAPI counterstaining (Cat#: 4083, Cell Signaling). Human healthy carotid arteries and murine aortas were fixed with 4% PFA and then embedded in OCT. The cryostat sections of tissue (8 μm) were permeabilized using 0.3% triton- X100, blocked with 5% normal bovine serum and stained with antibodies against FAM222A (1:200, Cat#: PA5-59093, Thermo Fisher) and CD31 (1:30, Cat#: 550274, BD Pharmingen) overnight followed by appropriate Alexa Fluor conjugated secondary antibody (1:300) immunostaining. All nuclei were counterstained with DAPI. Imaging was performed by BIDMC confocal imaging and IHC core facility. Images were acquired on a Carl Zeiss LSM 880 confocal microscope using Zen black software version 2.3 SP1 (BIDMC confocal imaging and IHC core facility). Objective lenses 10x 0.45 NA and 20x 0.8 NA were used for image acquisition. Immunohistochemistry staining of frozen human carotid artery cross sections arteries was performed using a rabbit polyclonal anti-FAM222A (Thermo Fisher) or a mouse monoclonal anti- CD31 (DAKO). Immunostaining was amplified using peroxidase-conjugated streptavidin complexes (Vector Laboratories) and peroxidase was detected using AEC or DAB (Vector Laboratories) substrate. Sections were lightly counterstained with hematoxylin and examined with a bright field microscope (Nikon Optiphot-2 equipped with a Nikon digital camera DXM 1200F). Results BETs regulate the EC transcriptome under basal conditions, including FAM222A, an essential determinant of EC proliferation and migration To investigate BET endothelial action under basal conditions, we analyzed existing RNA-Seq datasets (GSE53999)2,9in human umbilical vein ECs (ECs, here and throughout) under standard PATENT Attorney Docket NO.: 51792-002WO2 maintenance conditions and without or with BET inhibitor JQ1 treatment (500 nM2), identifying basally expressed genes (RPKM>1) whose mRNA levels were significantly altered by BET inhibition. BETs significantly regulated mRNA expression of EC genes under basal conditions, with JQ1 repressing a significant cassette of EC genes while also significantly increasing mRNA levels of another distinct subset of basally expressed genes (FIG.1A). To better understand these BET-dependent, divergent EC transcriptional programs, we analyzed those mRNAs repressed or induced significantly (≥ log 2- fold mRNA change) by JQ1 after 3 hours treatment (false discovery rate, FDR, <0.01; FIG.1B, Upper Panel). In GO pathway analyses (FIG.1B, Lower Panel), basally expressed EC gene transcripts significantly repressed by JQ1 were predominantly involved in functional EC properties of cell adhesion, cell cycling and apoptosis (FIG.1B). A smaller but significant subgroup of basally expressed EC genes manifest significant mRNA induction after JQ1 treatment, with GO analysis indicating predominate regulation of angiogenesis, including major angiogenic signaling pathways like NOTCH (FIG.1B). BET inhibition increasing gene expression, a form of trans induction, has remained largely understudied and poorly understood, despite its relevance to BET action and potential responses to therapeutic BET inhibitors1,4,10. As such, we further investigated basally expressed EC genes significantly induced by JQ1. Given the prior GO data (FIG.1C), this basal EC RNA-Seq dataset of JQ1-induced genes was analyzed for non-structural proteins without known involvement in cellular proliferation, migration or EC biology. Among the nine most JQ1-induced EC genes, GADD45B and TENT5C have reported roles in cellular proliferation and proliferation11,12, H2BC5 is a histone protein13, while five other genes – HEXIM1, ID2, FOS, VIP, and C9 – have reported EC effects, including vascular morphogenesis (FIG.1C)14-18. Of note, most of these JQ1-induced genes, specifically GADD45B, TENT5C, HEXIM1, ID2 and FOS, are reported as involved in NOTCH signaling, in keeping with the GO analysis11,19-23. The open reading frame protein C12orf34, also known as FAM222A, emerged as the sole gene significantly expressed in ECs under basal conditions, potently induced by JQ1, and lacking a known role in migration, proliferation or EC biology, prompting our further study of this novel gene consisting of a 1,359 base pair coding region, 3 exons, 452 amino acids, and no obvious predicted domains or protein family association (FIG.2). In line with RNA-Seq data, FAM222A mRNA expression increased in a dose-dependent manner in ECs after BET inhibition with JQ1 or the structurally distinct BET inhibitor I-BET (FIG.3A). JQ1 also increased activity of a human FAM222A promoter luciferase reporter in a dose-dependent manner after EC transfection (FIG.3B) and FAM222A protein levels, as evident in immunofluorescent EC staining, which was primarily nuclear (FIG.3C). Before further investigating FAM222A, we sought evidence for FAM222A’s potential relevance in human tissue. Immunohistochemistry staining demonstrated FAM222A protein in the endothelial layer of human carotid artery specimens (FIG.4, Upper Panel; FIG.5A), as also seen in mouse aorta (FIG.5B). FAM222A expression was restricted predominantly to ECs as compared to other vascular and inflammatory cell types, including primary human coronary artery smooth muscle cells (SMCs), fibroblasts, and monocyte-derived macrophages (FIG.4, Lower Panel). Given this data, we moved on to investigate BET regulation of FAM222A, testing if BRD2, BRD3 and / or BRD4 explained the increased FAM222A mRNA expression by the pan- PATENT Attorney Docket NO.: 51792-002WO2 BET inhibitor JQ1, using specific, validated small interference RNA (siRNAs) to each BET isoform (FIG.5C). Only siBRD4, and not siBRD2 or siBRD3, significantly decreased FAM222A mRNA levels (FIG.5D), as seen under both basal conditions and after JQ1 exposure (FIG.5E). In line with BRD4 acting through chromatin binding, analysis of our existing Chromatin Immunoprecipitation (ChIP) sequencing datasets in ECs indicated BRD4 binds to the FAM222A promoter regulatory regions at two distinct sites in either the absence of presence of JQ1 while JQ1 treatment increased RNA Pol II recruitment to these same FAM222A promoter locations (FIG.6). Given BRD4 binding on the FAM222A promoter either with or without JQ1, we hypothesized additional transcriptional regulators act in cooperation with BRD4 to direct FAM222A expression. Byoverlapping data of EC genes significantly induced by JQ1 treatment (log2fc>1) with transcriptionmediators predicted to bind to the FAM222A promoter (ChIP Atlas, + / - 10kb from TSS24), early growth response (EGR)-1 was identified as the sole gene with both JQ1 induction and predicted FAM222A promoter binding (FIG.7, Venn diagram). Subsequent in silico analysis demonstrated two distinct EGR1 binding sites on the FAM222A promoter region25,26. Indeed, JQ1 treatment increased significantly both mRNA and protein levels of EGR1, an established pro-angiogenic mediator (FIG.7, lower left)27. Moreover, overexpression of EGR1 increased FAM222A promoter activity in transfected ECs under basal conditions and after JQ1 treatment (FIG.7, lower right) while repressing EGR1 expression with a validated siRNA (FIG.8A) significantly decreased JQ1’s effects on FAM222A expression, which declined further after siBRD4 transfection (FIG.8B). Having identified FAM222A as an EGR1-regulated gene basally expressed in ECs and further induced after BET inhibition as well as GO predicted action, we next investigated FAM222A in key functional EC responses involving growth, proliferation, and migration, with and without pro- angiogenic stimuli of VEGF and hypoxia. Both VEGF stimulation (50 ng / ml, 3 hours) and hypoxia (1% O2, 3 hours) significantly induced FAM222A mRNA expression in ECs (FIG.9A) while the cytokines TNFα, TGFλ32, and IL1λ3 had no effect on basal FAM222A expression (FIG.9B). These VEGF and hypoxia effects on FAM222A mRNA did not further increase JQ1-stimulated induction of FAM222A mRNA (data not shown), as also the case with concomitant JQ1 and TNFα, TGFλ32 or IL1λ3 (FIG. 9B). We next assessed EC growth in the presence or absence of specific, validated FAM222A siRNA (90% decrease in FAM222A mRNA and protein, FIG.10). siFAM222A significantly decreased EC growth vs siControl-transfected cells (siCtl, FIG.11A), without increasing apoptosis, as seen in caspase 3 / 7 (FIG.11B) and Annexin V / staurosporine treatment assays (FIG.11C). Silencing FAM222A decreased EC proliferation significantly in BrdU incorporation assays under both basal and VEGF-stimulated conditions (vs siCtl, FIG.11D). siFAM222A also decreased VEGF-induced proliferation in synchronized ECs, which was independent of contact-inhibition (FIG.11E). In scratch wound assays, siFAM222A decreased EC migration and wound closure significantly under both basal (-38%) and VEGF-stimulated conditions (-48%, both vs siCtl, FIG.11F, middle and right, respectively). Next, to test if siFAM222A decreased wound closure by impairing EC proliferation or migration, scratch assays were repeated in mitotically-inactive, mitomycin C-treated ECs, which did not alter siFAM222A-mediated decreased wound closure (-46% vs siCtl, Suppl. FIG.11G). PATENT Attorney Docket NO.: 51792-002WO2 siFAM222A-treated ECs did manifest a modest but significant decrease in cell viability (WST-8 assays, FIG.11H), in keeping with the significant decrease in proliferative and migratory capacity seen after FAM222A silencing. To further test FAM222A involvement in EC proliferation and migration, we undertook gain-of- function experiments using Green fluorescent protein (GFP)-Lentiviral FAM222A overexpression, which increased FAM222A mRNA (380-fold) and protein (6-fold) significantly (FIG.12A). FAM222A- overexpressing ECs had significantly increased proliferation in a multiplicity of infection (MOI)- dependent manner (FIG.12B) and wound closure rates (FIG.12C). Thus, in both loss- and gain-of function studies, FAM222A was found to significantly affect EC proliferation and migration under basaland VEGF stimulated conditions.FAM222A regulates angiogenic responses in vitro, ex vivo and in vivo through coordinated modulation of VEGF and NOTCH pathways Given FAM222A involvement in VEGF-stimulated EC responses, we further explored FAM222A in network formation and vascular sprouting as key angiogenic responses28,29. Silencing FAM222A decreased EC capillary-like tube formation (Matrigel assays) under both basal (47%) and VEGF-stimulated (52%) conditions (FIG.13A). siFAM222A also decreased VEGF-induced vessel sprout length (-82%) and sprout number (-80%) in EC spheroids (FIG.13B). FAM222A overexpression had the opposite effects on EC sprouting, with LentiFAM222A-infected ECs forming more (+ 117% sprout number) and longer (+ 54% sprout length) vascular sprouts (FIG.13C and FIG. 13D), with distinct changes on “tip-like” EC appearance vs non-infected ECs. Although Matrigel as a medium for investigating angiogenesis in vitro has some potential limitations28,30, we used this approach to connect with our prior data and experience in two other models of angiogenesis, namely an ex vivo aortic ring31-33sprouting angiogenesis model and in in vivo Matrigel plugs34. We first compared the effect of FAM222A silencing vs overexpression on sprouting of murine Matrigel-embedded aortic rings. In keeping with effects seen in vitro, siFAM222A treatment decreased aortic ring microvessel formation (-76%, FIG.14A) significantly while FAM222A overexpression had the opposite effects (+191%, FIG.14B). Similar results were seen after repeating FAM222A silencing or overexpression in sprout assays performed in collagen (FIG.14C and FIG. 14D). We next tested if ECs with FAM222A silencing (siFAM222A) or overexpression (LentiFAM222A) or respective controls, when mixed with Matrigel prior to flank injection into wild-type mice, altered vessel plug in-growth (Day 7)34. siFAM222A-treated EC / Matrigel demonstrated significantly decreased vessel growth into Matrigel plugs, as measured using hemoglobin (Hb) content (-55%, FIG.14E, vs siControls). In contrast, FAM222A overexpressing EC / Matrigel plugs had increased vascularization (vs LentiCtl ECs, +29, FIG.14F). These findings for FAM222A in EC proliferation, migration and vascularization prompted the question of FAM222A involvement in angiogenic signaling pathways. VEGF promotes angiogenesis via carefully coordinated signaling pathways involving multiple proteins, including activating phosphorylation of the VEGF receptor 2 (VEGFR2) and key distal mediators of mitogen-activated protein kinase / extracellular-signal-regulated kinase-1 / 2 (Erk1 / 2) and phosphatidylinositol 3-kinase PATENT Attorney Docket NO.: 51792-002WO2 (PI3-K) / Akt35. Given FAM222A’s nuclear localization, we investigated if FAM222A controlled expression of VEGF-induced mediators involved in EC proliferation, sprouting, and survival. Indeed, FAM222A silencing decreased mRNA levels of VEGF, the VEGF co-receptor NRP1, the pro- angiogenic growth factor ANG1, its agonist TIE2, and the angiogenic transcriptional factors EGR1 and TAL1 (FIG.15), all of which are implicated in VEGF- directed EC responses36,37. Concurrently, siFAM222A also increased expression of ANG2 (FIG.15), a known anti-angiogenic mediator that opposes TIE2-ANG1 effects38. Moreover, the distal VEGF-induced Erk1,2 signaling proteins, including MAPK3 (Erk1), MAPK8 and MAPK14 were also decreased in siFAM222A-treated ECs (FIG.15). Interestingly, siFAM222A increased VEGFR2 mRNA levels (FIG.15) while expression of PI3K / Aktpathway genes were unchanged (data not shown). These changes suggested FAM222A controlledexpression of these distal VEGF mediators would alter VEGF signaling, which we studied next. After siFAM222A transfection in ECs (48 hours), the expected VEGF-induced phosphorylation of VEGFR2, Erk1 / 2 and Akt were all significantly suppressed (-86%, - 56%, -25%, respectively, vs siCtl; FIG.16A and FIG.16B). While siFAM222A did not alter Erk1 / 2 and Akt protein levels, basal Erk1 / 2 and Akt phosphorylation were marginally decreased by siFAM222A (Time 0, FIG 16A). Consistent with the mRNA findings, basal VEGFR2 protein levels were also increased by siFAM222A (FIG.16A), suggesting a potential compensatory response to impaired VEGF signaling after loss of FAM222A. To further test FAM222A involvement VEGF signaling, we next investigated VEGF- induced angiogenic responses after FAM222A overexpression in ECs, using our previously validated lentiviral constructs (FIG.12A). VEGF stimulation of FAM222A overexpressing ECs resulted in angiogenic responses that were the opposite of those seen after FAM222A silencing, with increased activating phosphorylation of VEGFR2, Akt, and Erk1 / 2 (FIG.16C), without altering basal expression of these same proteins (FIG.16D). VEGF stimulation also activates NOTCH signaling, which offsets angiogenic responses39,40. Given FAM222A’s coordinated modulation of multiple angiogenic mediators, we next considered if this extended to the NOTCH pathway. Silencing FAM222A in ECs promoted NOTCH-mediated signals that limit angiogenesis, increasing mRNA levels of NOTCH receptors NOTCH2 and NOTCH4 and the NOTCH ligands Jagged 1 (JAG1) and Delta- like 4 (DLL4) (FIG.17A, left). NOTCH receptors NOTCH1, NOTCH2 and NOTCH4 were all found to be activated, as indicated by increased levels of their intracellular domains (ICD), which depends on NOTCH cleavage by ψ-secretase,26 and decreased levels of their transmembrane subunits (NTM; FIG.17A, left). NOTCH-ICD also increases the anti- angiogenic mediators HES1 and HES2,41,42 whose mRNA and protein levels were also increased by siFAM222A (FIG.17A). In keeping with siFAM222A increasing NOTCH signaling, repeating these experiments in the presence of the ψ-secretase–specific NOTCH inhibitor DAPT [N- [N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butylester] reversed the siFAM222A effects on all NOTCH signaling mediator mRNAs other than JAG1 (FIG.17C), doing so without altering FAM222A expression (FIG.17D). Functionally, DAPT treatment significantly rescued decreases in EC migration and wound closure after siFAM222A treatment (FIG.17E). Together these findings identify FAM222A as determining endothelial proliferation, migration and angiogenic responses in vitro through its coordinated modulation of angiogenic mediators involved in VEGF and NOTCH pathways. PATENT Attorney Docket NO.: 51792-002WO2 Given that no validated FAM222A-deficient mouse currently exists, we tested the effects of FAM222A deficiency on angiogenesis in vivo using siFAM222A in the neonatal oxygen-induced retinopathy (OIR) model, given its extensive validation, representation of both physiologic and pathologic angiogenesis, and accepted replication of retinopathy of prematurity (ROP) in human newborns7. In OIR, postnatal day 7 (P7) mouse pups, and their nursing mothers, undergo hyperoxia (75% O2) exposure for 5 days (P7-P12), which causes retinal capillary depletion, before being returned to room air (P12-P17), which induces pathologic neovascularization (FIG.17B and FIG. 18A), as used by us and others28,43-45. After first confirming siFAM222A decreased FAM222A expression in mouse brain ECs (bEND.3), murine EC proliferation and migration (FIG.18B), andFAM222A protein levels in mouse retinal ECs (FIG. 18C), intra-vitreal siFAM222A or siCtl injections inOIR were performed (P12) before initiating the vascular proliferation phase (P12-P17), followed by retinal harvesting (P17). Retinas from siFAM222A-treated pups exhibited significantly reduced vaso- obliteration and neovascularization vs siCtl-treated mice (P17, FIG.18A). In keeping with in vitro findings, siFAM222A intravitreal injection also significantly decreased retinal VEGF mRNA levels while increasing gene expression of the angiogenic repressor HES2 (FIG.18D). Taken together, the data presented here demonstrate BETs regulate the basal endothelial transcriptome, which includes BRD4 control over FAM222A, a previously unreported protein essential for endothelial biology and function that exerts coordinated effects on proliferation, migration, and angiogenesis, as evident in vitro, ex vivo and in vivo (FIG.19). Discussion The endothelium’s key role in both homeostasis and pathogenesis involves dynamic modulation of endothelial transcriptional programs. BET epigenetic reader proteins help direct these coordinated changes in gene expression by facilitating transcriptional complex assembly, as reported in pathogenic, stimulated EC responses in multiple cardio-pulmonary conditions2,5,46. Here, we used RNA-Seq datasets in the absence or presence of BET inhibition to investigate the largely unexplored issue of BET-dependent transcription in ECs under basal conditions. This transcriptome data revealed BET transcriptional regulation of genes involved in core aspects of basal EC function, including angiogenesis as well as distinct basally expressed endothelial genes induced after BET inhibition. Further analysis of these basally expressed, JQ1-induced EC mRNAs led to identification of FAM222A as a unique, novel, BRD4-regulated determinant of EC biology and angiogenesis. FAM222A knockdown suppressed VEGF-stimulated EC migration, proliferation and sprouting while FAM222A overexpression had the opposite effects. In vivo, silencing FAM222A disrupted retinal revascularization in the OIR model by modulating expression of these same proteins involved in controlling angiogenesis. In addition to providing new insight into dynamic BET action in the endothelium, these studies establish FAM222A as a novel, essential determinant of EC biology and angiogenesis. BETs have received considerable attention although primarily for their role directing induced, pathologic transcriptional programs1,2, like in myocardial pressure overload47and atherosclerosis, including TNFα-induced endothelial NFκB activation2. Importantly, when these stimuli induce BRD4- PATENT Attorney Docket NO.: 51792-002WO2 mediated transcription, it comes with concomitant loss of BRD4 activity directing gene expression under basal, pre-stimulus conditions48. Although largely overlooked, this BRD4-regulated, basal transcriptional program is integral to understanding BET biology, including potential clinical effects of BET inhibitors under study in atherosclerosis, pulmonary hypertension, cancer, inflammatory conditions, and other diseases5,49-53. Prior studies of BET involvement in angiogenesis reflects the complexity of these issues, including differences between stimulated and quiescent endothelium as well as different BET inhibitors. While some reports suggest BET inhibitors decrease angiogenesis, as studied primarily in cancer54, others find BET inhibition restores angiogenic responses, as seen with apabetalone55, with different distal mediators invoked in the responses seen, mostly under stimulatednot resting conditions.Here, studying EC RNA-Seq data under basal conditions with and without the BET inhibitor JQ1, BETs are shown to control important functional endothelial properties of cell adhesion, migration, and angiogenesis, as seen in GO analyses of basally expressed genes repressed by JQ1 exposure (FIG.1A and FIG.1B). This data also highlighted mRNAs significantly induced by BET inhibition, as seen here with BET inhibitors and siBRD4, responses predicted to also occur through poorly understood endogenous mechanisms that regulate BRD4 activity. Given BET control over canonical, pro-inflammatory, pro- atherosclerotic mediators induced by specific stimuli, we hypothesized similar BET regulation of basal endothelial biology also occurs, which would also provide an orthogonal approach to identifying novel determinants of EC function. This proved to be the case; analysis and annotation of basally expressed, non-structural, JQ1-induced genes with no reported roles in ECs, migration or proliferation pointed to FAM222A, also known as C12orf34 (FIG.1C). Subsequent studies presented here establish FAM222A as basally and predominately expressed in ECs, with mRNA and protein levels potently increased by BET inhibition involving BRD4 as well as EGR1 (FIG 4, FIG.5D, FIG.7, and FIG.8B). Cell proliferation and migration are integral to angiogenesis56,57. Pro-angiogenic stimuli, like VEGF and hypoxia, increased FAM222A mRNA and protein levels while silencing FAM222A decreased VEGF-induced EC migration, proliferation, vascular spouting in vitro, ex vivo and in Matrigel plug revascularization in vivo (FIG.9A, FIG.11, FIG.12C, FIG.13, and FIG, 14); FAM222A overexpression had opposite effects. FAM222A silencing did not alter activation of p53 or p53- mediated p21 (data not shown) nor induce apoptosis, excluding these as explanations for decreased cell proliferation and migration after FAM222A silencing. FAM222A deficiency (48 hours) causes transcriptional modulation of players in VEGF and NOTCH pathways underlying angiogenesis. While VEGF drives EC spatial guidance, sprout initiation, and stalk-cell proliferation, NOTCH counters these responses, decreasing EC activity and inducing repressive stalk cell features, including increases in NOTCH-dependent angiogenic repressors HES1, HES2, and HEY239,58,59. siFAM222A modulates expression of all these mediators – decreasing pro-angiogenic targets and growth factors like VEGF, ANG1 and TIE2 while increasing angiogenic repressors. Such coordinated control implicates FAM222A as a key proximal determinant of angiogenic transcriptional programs. Transcriptional responses to FAM222A silencing also disrupts VEGF-induced phosphorylation, decreasing pro- mitogenic MAPK signaling while increasing anti-angiogenic NOTCH effects42. The pharmacologic PATENT Attorney Docket NO.: 51792-002WO2 NOTCH cleavage inhibitor DAPT blocked siFAM222A effects on NOTCH target gene expression and partially reversed siFAM222A-mediated decreased EC migration, suggesting that aberrant NOTCH hyperactivation, a reported cause of decreased EC proliferation57,60, may also contribute to FAM222A’s effects on angiogenesis. The only siFAM222A-induced alteration not aligned with decreased angiogenesis was an increase in VEGFR2 levels, which might represent a compensatory response, assuming these changes align with VEGR2 activity, as requires further study. In vivo, silencing FAM222A decreased angiogenesis in the neonatal OIR angiogenesis model, with changes that replicate in vitro responses. OIR replicates retinopathy of prematurity (ROP) in newborns, with initial vaso-obliteration followed by ischemia / hypoxia-induced neovascularization7,43.Silencing FAM222A during OIR decreased retinal revascularization and VEGF expression whileincreasing angiogenic repressor HES2 mRNA levels, thus altering EC proliferation and migration during OIR’s second, neovascularization phase. Given in vitro evidence that FAM222A overexpression promotes angiogenesis, increased FAM222A levels may promote disorders characterized by excess angiogenesis, like ROP, other proliferative retinopathies, and vascular tumors, as warrants further consideration. FAM222A involvement in other angiogenesis models, like hind limb ischemia, and conditions like peripheral arterial disease (PAD)61, as also warrants investigation, as prompted by these findings. In support of our using BET regulation / BRD4 localization in ECs as a strategy for identifying novel determinants of EC biology, only one prior FAM222A-focused report exists, which suggested FAM222A expression is restricted to neurons and might promote Alzheimer’s Disease (AD)62. The data provided here clearly extends FAM222A action beyond the central nervous system (CNS) and establishes its involvement in non- pathologic processes. At the same time, our data also raises questions if FAM222A in brain ECs might be involved in these reported CNS effects. Of note, in support of FAM222A involvement outside the CNS, its induction after BET inhibition, and its cardiovascular relevance, FAM222A is among the mRNAs most induced in the myocardium of BET inhibitor-treated mice undergoing transaortic constriction47. Whether this FAM222A signal derives from myocardial ECs or cardiomyocytes, which contain less FAM222A than ECs (data not shown), is unclear but also requires investigation. Taken together, we establish here BET control over dynamic endothelial gene expression involves basal transcriptional programs. Using basal BET regulation and BRD4 action for endothelial gene discovery enabled identification of FAM222A, which is demonstrated to be a novel, key determinant of endothelial cell migration, proliferation, and angiogenesis in vitro, ex vivo and in vivo. Highlights • Despite most reports focusing on BET epigenetic reader proteins BRD2, BRD3, BRD4 in controlling pathogenic gene expression, as supported by BET inhibitors decreasing endothelial inflammation, atherosclerosis, and pulmonary hypertension, BETs also direct the basal endothelial cell (EC) transcriptome, as defined here in RNA-Seq data from ECs under basal conditions in the absence versus presence of the specific pan-BET inhibitor JQ1. PATENT Attorney Docket NO.: 51792-002WO2 • The BET-regulated endothelial transcriptional program under basal conditions includes genes involved in core aspects of endothelial biology as well as a subset of mRNAs whose basal expression is increased significantly after BET inhibition (BET inhibitors, siBRD4). • Analysis of BET-regulated mRNAs in ECs under basal conditions identifies Chromosome 12 Open Reading Frame 34 (C12orf34), also known as Family with Sequence Similarity 222Member A (FAM222A), as a unique, basally-expressed, BET-regulated gene in ECs with no previously identified role in endothelial biology; data presented here demonstrates FAM222A is essential for EC migration, proliferation, vascular sprouting and angiogenic responses in vitro and ex vivo by modulating expression of key VEGF and NOTCH pathway mediators. 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BET and HDAC inhibitors induce similar genes and biological effects and synergize to kill in Myc-induced murine lymphoma. Proc Natl Acad Sci U S A.2014;111(26):E2721-30. doi:10.1073 / pnas.1406722111 49. Latif AL, Newcombe A, Li S, et al. BRD4-mediated repression of p53 is a target for combination therapy in AML. Nat Commun.2021;12(1):241. doi:10.1038 / s41467-020- 20378-8 50. Ding N, Hah N, Yu RT, et al. BRD4 is a novel therapeutic target for liver fibrosis. Proc Natl Acad Sci U S A.2015;112(51):15713-15718. doi:10.1073 / pnas.1522163112 PATENT Attorney Docket NO.: 51792-002WO2 51. Tian B, Zhao Y, Sun H, Zhang Y, Yang J, Brasier AR. BRD4 mediates NF-kappaB-dependent epithelial-mesenchymal transition and pulmonary fibrosis via transcriptional elongation. Am J Physiol Lung Cell Mol Physiol.2016;311(6):L1183-L1201. doi:10.1152 / ajplung.00224.2016 52. Li Z, Guo J, Wu Y, Zhou Q. The BET bromodomain inhibitor JQ1 activates HIV latency through antagonizing Brd4 inhibition of Tat-transactivation. Nucleic Acids Res. 2013;41(1):277-287. doi:10.1093 / nar / gks976 53. Song S, Liu L, Yu Y, et al. Inhibition of BRD4 attenuates transverse aortic constriction- and TGF-beta-induced endothelial-mesenchymal transition and cardiac fibrosis. J Mol Cell Cardiol. 2019;127:83-96. doi:10.1016 / j.yjmcc.2018.12.002 54. Bid HK, Kerk S. BET bromodomain inhibitor (JQ1) and tumor angiogenesis. Oncoscience.2016;3(11-12):316-317. 55. Mohammed SA, Albiero M, Ambrosini Samuele and Gorica E, et al. The BET protein inhibitor apabetalone rescues diabetes-induced impairment of angiogenic response by epigenetic regulation of thrombospondin-1. Antioxid Redox Signal.2022;36(10-12):667- 684. 56. Chavkin NW, Genet G, Poulet M, et al. Endothelial cell cycle state determines propensity for arterial-venous fate. Nat Commun.2022;13(1):5891. doi:10.1038 / s41467-022-33324- 7 57. Noseda M, Niessen K, McLean G, Chang L, Karsan A. Notch-dependent cell cycle arrest is associated with downregulation of minichromosome maintenance proteins. Circ Res. 2005;97(2):102-104. doi:10.1161 / 01.RES.0000174380.06673.81 58. Pitulescu ME, Schmidt I, Giaimo BD, et al. Dll4 and Notch signalling couples sprouting angiogenesis and artery formation. Nat Cell Biol.2017;19(8):915-927. doi:10.1038 / ncb3555 59. Lamalice L, le Boeuf F, Huot J. Endothelial cell migration during angiogenesis. Circ Res. 2007;100(6):782-794. doi:10.1161 / 01.RES.0000259593.07661.1e 60. Wei Y, Gong J, Thimmulappa RK, Kosmider B, Biswal S, Duh EJ. Nrf2 acts cell- autonomously in endothelium to regulate tip cell formation and vascular branching. Proc Natl Acad Sci U S A. 2013;110(41):E3910-8. doi:10.1073 / pnas.1309276110 61. Ryan TE, Yamaguchi DJ, Schmidt CA, et al. Extensive skeletal muscle cell mitochondriopathy distinguishes critical limb ischemia patients from claudicants. JCI Insight. 2018;3(21). doi:10.1172 / jci.insight.123235 62. Yan T, Liang J, Gao J, et al. FAM222A encodes a protein which accumulates in plaques in Alzheimer’s disease. Nat Commun.2020;11(1):411. doi:10.1038 / s41467-019-13962-0 Example 2. FAM222A is a novel determinant of cell cycling and angiogenesis Abstract The bromodomain and extra-terminal domain-containing epigenetic reader proteins (BETs) orchestrate pathogenic transcriptional programs, including BRD4-mediated pro-atherosclerotic PATENT Attorney Docket NO.: 51792-002WO2 responses. Although understudied, BETs also regulate basal, resting transcription, of potential relevance to BET-inhibitor therapies. We leveraged BET-dependent activity in resting human endothelial cell (ECs) as an orthogonal strategy for understanding BET action and identifying novel players in cell biology. In resting cells, RNA-Seq analysis establishes BETs direct core EC properties, including cell cycling while the BET inhibitor JQ1 also potently induces specific mRNAs. Family with Sequence Similarity 222 Member A (FAM222A) emerged as a unique, basally expressed, previously unidentified JQ1-induced gene. FAM222A is essential for cell cycling, G1 transition and angiogenesis in vitro; in vivo, silencing FAM222A represses revascularization in oxygen-induced retinopathy (OIR) and hind limb ischemia (HLI). Mechanistically, FAM222A controls G1 initiation / transition by binding tospecific mini-chromosome maintenance (MCM) proteins. These findings establish FAM222A as anovel determinant of cell cycling and angiogenesis. Introduction The bromodomain and extra-terminal domain-containing protein family (BETs), which includes BRD2, BRD3 and BRD4, are epigenetic reader proteins that coordinate transcriptional programs by binding to specific acetylated lysine residues on histone tails, thus enabling assembly of transcriptional machinery, including RNA Polymerase II (Pol II) and Positive transcriptional elongation factor-b (PTEF-b)1,2. BETs, which control transcriptional programs in differentiation, cell identity and multiple cell state transitions, have been implicated in multiple diseases, including cancer and atherosclerosis3. In the endothelium, BRD4 coordinate stimulus-induced gene expression, like in response to cytokine stimulation with BET inhibitors repressing atherosclerosis and inflammation in vitro and in vivo, as shown by us and others1,4,5. While attention has focused on BETs in governing stimulated transcription in pathologic settings, BETs also modulate gene expression under basal conditions1; BET action in resting cells, prior to their stimulus-induced redeployment to super enhancer regions and transcriptional program execution, has remained largely unstudied, despite its relevance to BET biology, transcriptional mechanisms, and BET inhibition as a therapeutic strategy. BET-directed basal versus stimulated transcription may be especially pertinent in the endothelium, given rapid, dynamic endothelial cell (EC) responses involved in systemic homeostasis and multiple diseases6-8. As such, we sought to define BET action across the endothelial transcriptome in resting human umbilical vein ECs before and after exposure to the well-validated BET inhibitor JQ1. Moreover, given BET regulation of canonical pro-atherosclerotic, pro-inflammatory induced endothelial genes, we also hypothesized identifying BET-regulated novel EC genes would provide an orthogonal approach to finding previously unreported determinants of EC biology and function. Interrogation of HUVECs RNA-Seq data in the absence versus presence of JQ1 demonstrated BET action in govern dynamic transcriptional programs in resting ECs. This analysis also highlighted a subset of genes significantly induced, not repressed, by BET inhibition, a largely overlooked response offering insight into transcriptional mechanisms and BET inhibitor effects. As such, we further annotated and considered those genes expressed in resting ECs whose mRNA was most induced by BET inhibition, that were largely unknown or without a known endothelial role. This PATENT Attorney Docket NO.: 51792-002WO2 process pointed to Chromosome 12 Open Reading Frame 34 (C12orf34), also known as Family with Sequence Similarity 222 Member A (FAM222A). As presented here, subsequent studies establish FAM222A as abundantly expressed in ECs, necessary for cell cycling, and essential for EC proliferation, migration, and angiogenesis, with coordinated transcriptional regulation of VEGF and NOTCH pathways. In vivo, silencing FAM222A repressed retinal neovascularization in murine angiogenesis models of oxygen-induced retinopathy (OIR) in neonates and decreased blood flow recovery in the hind limb ischemia (HLI) in adults. Mechanistically, FAM222A controls early cell cycle G1 progression by interacting directly with DNA replication factors. Taken together, this data expands insight into BET modulation of transcription and establishes FAM222A as a novel player in endothelialbiology, cell cycling, and angiogenesis.Methods Institutional approval was obtained for all mouse studies, as performed in C57Bl / 6J mice (Jackson Laboratories), which were maintained in a pathogen-free facility with standard light / dark cycling and ad libitum food and water access, under animal protocol approval (#2016N000115) granted by the Harvard Medical School Institutional Animal Care and Use Committee (IACUC). Data Sharing: Our published resting HUVEC RNA-seq dataset with or without JQ1 (500nM, 3 hours) is available (e.g., see NCBI accession number GSE53999)1. ChIP-seq and Chem-seq data (including microarray, aligned and raw data) are also available (e.g., see NCBI accession numbers GSE53998 and GSE54000) as is the RNA-seq dataset from human gastrocnemius biopsy samples (e.g., see NCBI accession number GSE120642)9. Statistical analysis: Data are shown as mean ± SEM unless otherwise noted. Unpaired Student’s t test was used for single comparisons. One-way ANOVA followed by Tukey post-hoc test was used to determine the significance of one independent variable between more than two groups. Comparisons between two groups and two nominal variables were performed using Two- way ANOVA followed by Tukey post-hoc test. Differences were considered statistically significant at p<0.05. Statistical analysis was performed using GraphPad Prism. Additional detailed experimental methods, including statistical approaches to dataset analyses are provided in the Supplemental Materials and Methods section. Results BETs Modulate the Resting HUVEC Transcriptome To investigate BET action in unstimulated HUVECs, we analyzed existing RNA-Seq datasets (GSE53999)1,10in resting ECs without or with BET inhibitor JQ1 stimulation, identifying basally expressed genes (RPKM>1) whose transcription was significantly BET-dependent. BET epigenetic reader proteins regulated mRNA expression of HUVEC genes under basal conditions, with JQ1 (500 nM) repressing a cassette of basally expressed EC genes (FIG.20A). In contrast, BET inhibition also significantly increased mRNA levels of a specific basally expressed EC gene subset (FIG.20A). To better understand these BET-dependent, divergent EC transcriptional programs, we analyzed those mRNAs repressed or induced significantly (≥log 2-fold mRNA change) by JQ1 with 3 hours of PATENT Attorney Docket NO.: 51792-002WO2 treatment (false discovery rate, FDR, <0.01; FIG.20B, Upper Panel) before performing gene ontology (GO) pathway analyses (FIG.20B, Lower Panel). Basally expressed EC gene transcripts significantly repressed by JQ1 were associated predominantly with basal functional EC properties of migration, proliferation and adhesion (FIG.20B). A smaller subgroup of basally expressed EC genes manifest significant mRNA induction by JQ1, with GO analysis suggesting their primary regulation of cell cycling (FIG.20B). BET inhibition increasing transcription has been understudied and the mechanisms involved poorly understood, especially in the vasculature, despite potential implications, including BET inhibition for therapeutic benefit. As such, we further considered basally expressed EC genessignificantly induced by JQ1. GO analysis indicated cell cycling as the pathway most regulated amongJQ1-induced EC genes (FIG.20C). We also used this dataset to identify novel non-structural proteins without known roles in EC biology, cell cycling or cellular proliferation. Indeed, among EC-expressed genes most induced by JQ1, two – GADD45B and TENT5C – have known involvement in cell cycling and cellular proliferation; five genes have reported endothelial roles while H2BC5 is a histone protein (FIG.20C). FAM222A, also known as C12orf34, was the sole gene significantly expressed in resting ECs, further induced by JQ1 and with no known role in ECs or in cell cycling, prompting further investigation into this mostly unknown protein. In line with RNA-Seq data, FAM222A mRNA expression increased in a concentration- dependent manner in ECs after BET inhibition with JQ1 (FIG.20D, left) or the structurally distinct BET inhibitor I-BET (FIG.20I). JQ1 also increased activity of a human FAM222A promoter luciferase reporter construct in a concentration-dependent manner (FIG.20J) and FAM222A immunofluorescent EC staining (FIG.20D, right). Analysis of existing endothelial chromatin immunoprecipitation- sequencing (ChIP-Seq) datasets treated without and with JQ1 reveals BRD4 association with two distinct FAM222A promoter regulatory regions as also seen on ChIP-PCR for FAM222A on immunoprecipitated BRD4 also demonstrating FAM222A mRNA association, which interestingly is not altered by JQ1 (FIG.20E). Given JQ1 is a pan-BET inhibitor, we next investigated which BET isoforms direct FAM222A mRNA expression using specific, validated small interference RNA (siRNA) to BRD2, BRD3 or BRD4 (FIG.20K) transfected into ECs before JQ1 stimulation, as before. Only siBRD4, and not siBRD2 or siBRD3, decreased JQ1 induction of FAM222A mRNA (FIG.20F, left), significantly repressing both FAM222A basal expression and JQ1-induced FAM222A mRNA levels (FIG.20F, right). FAM222A expression was predominantly restricted to ECs as compared to vascular cell types such as primary human coronary artery smooth muscle cells (SMCs), fibroblasts, and monocyte-derived macrophages (FIG.20G). Immunohistochemistry staining demonstrated FAM222A protein in mouse aorta and human carotid artery endothelium (FIG.20H). Having identified FAM222A as a novel, basally expressed EC gene induced by BET inhibition, we next investigated FAM222A’s functional role in ECs, starting with cell cycling given the prior GO analysis. Silencing FAM222A decreases cell cycle progression To study FAM222A in endothelial cell cycling, we first assessed EC growth in the presence or absence of specific, validated siRNA to FAM222A (90% mRNA, protein decrease; FIG.21G). In the PATENT Attorney Docket NO.: 51792-002WO2 presence of siFAM222A, EC growth significantly decreased vs siControl-transfected cells (siCtl, FIG. 21A). We then analyzed expression of key cell cycle-related genes in siFAM222A versus siCtl transfected ECs, under basal and serum-starved (G0 phase) conditions. In resting ECs, siFAM222A significantly repressed cyclin-dependent kinases (CDKs, CDK4, 2, 1) and cyclins (CCNE1, CCND1, CND2, CCNA2, CCNB1) mRNA while inducing expression of CDK inhibitors (CDKN2A, CDKN2B) (vs siCtl, FIG.21B). siFAM222A also repressed E2 factor (E2F) family of transcription factors mRNAs (FIG.21B) - critical regulators of cell cycle progression and distal targets of retinoblastoma (Rb) protein activity10. While siFAM222A did not alter Rb mRNA or protein levels (FIG.21C), activating Rb phosphorylation (s807 / 811, s608) was decreased, under both serum-starved, and FBS-stimulatedconditions (FIG. 21C). Rb s807 / 811 and s608 phosphorylation releases sequestered E2F proteins,enabling G1 / S cell cycle phase progression. In keeping with siFAM222A interrupting G1 / S progression, siFAM222A-treated ECs demonstrated significantly reduced expression of targets that promote G1 / S progression, namely CDK4, CCND1, CDK2, CCNE1, as evident under serum-starved (Time 0) and FBS-stimulated conditions (FIG.21D). While siFAM222A-mediated decreases in protein levels of G1 / S targets were restricted to CDK4 and Cyclin D1 during serum-starvation (Time 0), all these G1 / S regulators were decreased after FBS-stimulation of siFAM222A-treated ECs (24 hours, FIG.21E). To further characterize FAM222A action during cell cycling, we analyzed the cell cycle phase distribution in siFAM222A vs siCtl transfected, serum-starved ECs before and after FBS stimulation. Although siFAM222A had no significant basal effect, after serum stimulation (24 hours), FAM222A silencing significantly increased ECs in G1 (+33%) with while decreasing transition to G2 (-67.6%), all vs similarly synchronized siCtl-treated cells (FIG.21F). Interestingly, siFAM222A had similar but less potent cell cycle distribution effects in unsynchronized, unstimulated ECs (FIG.21H), which persisted after nocodazole-induced G2 arrest (24 hours post-stimulation, FIG.21I) and reproduced with another FAM222A siRNA (FIG.21J and FIG.21K). Given this evidence establishing FAM222A in cell cycle control, including G1 / S transition, we next considered FAM222A in key functional EC responses involving cell cycling, including EC migration and proliferation, as induced by pro-angiogenic stimuli of VEGF and hypoxia. FAM222A, induced by VEGF and hypoxia, regulates angiogenic responses including EC proliferation and migration Both VEGF stimulation (50 ng / ml, 3 hours) and hypoxia (1% O2, 3 hours) significantly induced FAM222A mRNA expression in ECs (FIG.22A). Given EC proliferation and migration during angiogenesis, we next studied these responses in the absence or presence of FAM222A siRNA with and without VEGF stimulation. Silencing FAM222A decreased EC proliferation significantly in BrdU incorporation assays under basal and VEGF-stimulated conditions (vs siCtl, FIG.22B), without increasing apoptosis (Fig.3C). siFAM222A also decreased VEGF-induced proliferation in synchronized ECs (FIG.22B) and independent of contact-inhibition (FIG.21J). In scratch wound assays, siFAM222A also decreased EC migration and wound closure significantly under both basal (-38%) and VEGF-stimulated conditions (-48%, vs siCtl, FIG.22D). PATENT Attorney Docket NO.: 51792-002WO2 Next, we tested if decreased wound closure after siFAM222A was due to impaired EC proliferation or migration. In repeat scratch assays in ECs after mitotic inactivation by mitomycin C treatment, siFAM222A-mediated decreased wound closure remained unchanged (-46% vs siCtl, FIG.22E). As expected, given the significantly decreased proliferative and migratory capacity of siFAM222A-treated ECs, a modest but significant decrease in cell viability also occurred (WST-8 assays, FIG.22F). To further examine FAM222A in EC proliferation and migration, we overexpressed FAM222A using a Lentiviral construct, which increased FAM222A mRNA (380-fold) and protein (6-fold) levels (FIG.22K). FAM222A-overexpressing ECs demonstrated significantly increased proliferation in a multiplicity of infection (MOI)-dependent manner (FIG.22G) and wound closure rates (FIG.22H).Thus, both loss- and gain-of function studies reveal FAM222A helps maintain EC proliferative andmigratory capacity at rest and after VEGF stimulation. FAM222A regulates EC network formation, sprouting, and in vivo angiogenesis Given FAM222A involvement in VEGF-stimulated EC responses, we explored further FAM222A in key angiogenic responses of network formation and vascular sprouting11. Silencing FAM222A decreased EC capillary-like tube formation under both basal (47%) and VEGF-stimulated (52%) conditions in Matrigel-based tube formation assays (FIG.23A). siFAM222A also decreased vessel sprout length (-52%) and sprout number (-76%) in EC spheroids (FIG.23B). To test FAM222A effects on angiogenesis in vivo, we first employed the neonatal oxygen- induced retinopathy (OIR) model, which mimics retinopathy of prematurity12, in the absence or presence of siFAM222A. In OIR, postnatal day 7 (P7) mouse pups, and their nursing mothers, are exposed to hyperoxia (75% O2) for 5 days (P7-P12), causing retinal capillary depletion, before returning to room air (P12-P17), which induces pathologic neovascularization (schematic, FIG.23C), as used by us and others11,13-15. After first confirming siFAM222A decreased FAM222A expression in mouse brain ECs (bEND.3), murine EC proliferation and migration (FIG.23D), and FAM222A protein levels in mouse retinal ECs (FIG.23E), intravitreal siFAM222A or siCtl injections were performed at P12 before initiating the vascular proliferation phase (P12-P17), with retinal harvesting at P17. Retinas from siFAM222A-treated pups exhibited significantly reduced vaso-obliteration and neovascularization vs siCtl-treated mice (P17, FIG.23C). siFAM222A intravitreal injection also significantly decreased retinal VEGF mRNA levels (FIG.23F). This data establishes FAM222A helps determine angiogenic responses in vivo during neonatal OIR, raising questions about how FAM222A modulates angiogenic signaling. Silencing FAM222A coordinately modulates angiogenic signaling, suppressing VEGF while increasing NOTCH responses VEGF promotes angiogenic responses via carefully orchestrated signaling pathways involving multiple proteins, including activating phosphorylation of the VEGF receptor 2 (VEGFR2) and key distal mediators’ mitogen-activated protein kinase / extracellular-signal-regulated kinase-1 / 2 (Erk1 / 2) and phosphatidylinositol 3-kinase (PI3-K) / Akt16. We interrogated EC proliferation, sprouting, and survival responses after FAM222A silencing or overexpression and subsequent induced PATENT Attorney Docket NO.: 51792-002WO2 transcriptional changes (4 days post-FAM222A manipulation). siFAM222A transfection in ECs significantly suppressed VEGF-induced phosphorylation of VEGFR2 (-86%), Erk1 / 2 (-56%), and Akt (25%), (FIG.24A and FIG.24G). While siFAM222A did not alter Erk1 / 2 and Akt protein levels, baseline VEGFR2 levels were increased (FIG.24A). Conversely, after lentiviral FAM222A overexpression in ECs, VEGF stimulation increased activating phosphorylation (FIG.24B), but not basal expression, FIG.24H) of VEGFR2, Akt, and Erk1 / 2. Thus, FAM222A regulated targets help promote VEGF-induced signaling in ECs. VEGF stimulation also activates counterbalancing NOTCH signaling, which offsets angiogenesis17,18. Silencing FAM222A in ECs increased NOTCH-mediated anti-angiogenicresponses, with increased mRNA (FIG. 24C) and protein (FIG. 24D) levels of NOTCH receptorsNOTCH2 and NOTCH4, the NOTCH ligands Delta-like 4 (DLL4) and Jagged 1 (Jag 1), the NOTCH1 intracellular domain (ICD) while decreasing NOTCH1 trans membrane subunit (NTM) levels, as mediated by increased ψ-secretase NOTCH cleavage (FIG.24D)19. Consistent with this, NOTCH-ICD regulated anti-angiogenic mediators HES1 and HES2 mRNA (FIG.24C) and protein (FIG.24D) also occurred after siFAM222A treatment. Evidence for siFAM222A increasing NOTCH signaling was also supported by the ψ-secretase-specific inhibitor N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S- phenylglycine t-butyl ester (DAPT) reversing these siFAM222A effects on all NOTCH signaling mediators (other than JAG1) mRNAs (FIG.24E), without altering FAM222A expression (FIG.24G). Functionally, DAPT treatment significantly rescued siFAM222A-induced decreases in EC migration (FIG.24F) and wound closure (FIG.24H). Of note, DAPT blocked decreased EC migration after FAM222A silencing occurred without changing cell proliferation (FIG.24I). Together, these findings identify FAM222A as a key modulator of endothelial migration through coordinated effects on VEGF and NOTCH pathways. FAM222A promotes angiogenesis and restoration of blood flow in a hind limb ischemia model Angiogenesis is implicated in both physiologic and pathologic settings, as evident in in vivo models. While in OIR, hyperoxia causes EC VEGF leakage and distinct phases culminating in structurally abnormal vessels in neonatal mice, in hind limb ischemia (HLI), femoral artery ligation (FAL) and subsequent release in adult mice induces adaptative remodeling, defined temporal transcriptional patterns, and a structurally normal microvasculature11,20,21. To investigate FAM222A in angiogenesis beyond OIR, we first characterized then studied FAM222A in HLI. As expected, under basal conditions, FAM222A is more highly expressed in ECs isolated from gastrocnemius muscle versus non-ECs or bone marrow derived macrophages (BMDM) from these same mice (FIG.25H). During the initial FAL response (Day 3), when inflammation predominates20, as also evident here (FIG.25I), FAM222A mRNA decreased significantly in ECs isolated from ischemic gastrocnemius muscle (FIG.25A). However, at Day 14 post-FAL, when angiogenesis occurs, FAM222A mRNA was increased 4.3-fold in ECs (FIG.25A). This reversed expression pattern, evident in isolated ECs but not other cell types (FIG.25J), suggested endothelial FAM222A may promote the pro-angiogenic phase of HLI. PATENT Attorney Docket NO.: 51792-002WO2 To investigate FAM222A manipulation in HLI, we performed siFAM222A intramuscular injections at Day 0 and 7 post-FAL (schematic, FIG.25B). FAM222A mRNA levels at Day 14 (but not Day 0) were significantly reduced in EC (75% reduction) but not non-EC fractions (FIG.25B), which have lower FAM222A levels at baseline (FIG.25H). In siFAM222A-treated mice, blood flow restoration was significantly decreased at Day 14 (FIG.25C, top) and throughout the study (FIG.25C, bottom), all vs siCtl-injected mice. In ischemic muscle fibers, decreased endothelial FAM222A was associated with centralized nuclei, suggestive of regenerative tissue21(FIG.25D), decreased capillary density on CD31 staining, indicating delayed perfusion recovery and angiogenesis (FIG.25E), and decreased arterial density on αSMA (FIG.25K), indicating impaired arteriogenesis in siFAM222A-treated ischemic hindlimbs. To consider FAM222A regulation of angiogenic pathways during HLI,expression of key angiogenic mediators was assessed in gastrocnemius-isolated ECs from siFAM222A vs siCtl-treated mice. Silencing FAM222A during HLI (Day 14) significantly modulated expression of multiple important angiogenic mediators in VEGF and NOTCH signaling pathways (FIG. 25F). In addition to significantly decreased VEGF mRNA, as also seen in neonatal OIR (FIG.23C), angiogenic repressors NOTCH2, HES2 and HEY2 mRNAs were significantly increased (FIG.25F). ECs isolated from ischemic gastrocnemius of siFAM222A-treated mice also exhibited significantly decreased expression of key cell cycle regulators (FIG.25F), all consistent with prior in vitro EC findings (FIG.21B). Thus, in a second distinct, adult in vivo mouse model of angiogenesis, FAM222A’s role in angiogenesis, with VEGF, NOTCH and cell cycling pathways effects, is further established. While studies of FAM222A in humans will be necessary, it is noteworthy that analysis of published RNA-Seq datasets from gastrocnemius muscle biopsies, which include ECs, as demonstrated by endothelial markers, from affected limbs in PAD patients with critical limb ischemia (CLI) or intermittent claudication (IC) reveals increased FAM222A expression as compared to control biopsy samples from healthy volunteers (FIG.25G)9,22. FAM222A facilitates G1 phase initiation by interacting with DNA replication factors Given FAM222A regulation of cell cycling genes is seen in vitro (FIG.21) and during HLI in vivo (FIG.25F), we further investigated mechanisms for FAM222A action in cell cycling. We first determined FAM222A expression patterns over time in serum-stimulated G0-synchronized ECs. With serum stimulation, both FAM222A mRNA and protein levels increased in synchronized ECs within 4 hours, remaining elevated for 12 hours before decreasing significantly by 24 hours (FIG.26A). This data, combined with the evidence that siFAM222A disrupts expression of key cell cycle mediators (FIG.21B), suggests FAM222A involvement in endothelial G1 initiation and subsequent G1 / S transition. As such, we investigated FAM222A regulation by or control over early cell cycling proteins acting proximal to Rb and Cyclins / CDKs, as reported for pre-replicative complexes (pre-RCs)22-25, including mini-chromosome maintenance3,7,4(MCM3, MCM7, MCM4) proteins. MCMs are known cell proliferation regulators although largely unstudied in ECs26and have no reported role in angiogenesis. Using a validated Flag-tagged FAM222A construct (FIG.26B) transfected into human embryonic kidney 293T cells, FAM222A pull down also co-immunoprecipitated endogenous MCM3, PATENT Attorney Docket NO.: 51792-002WO2 MCM7, and MCM4 (FIG.26B). Functionally, silencing MCM3, MCM4, or MCM7 with validated siRNAs (FIG.26G) significantly decreased EC wound closure, replicating siFAM222A effects (FIG.26C). EC proliferation was most decreased by siMCM3; siMCM7 had significant but lesser effects while siMCM4 did not alter proliferation despite decreasing EC migration (FIG.26D). Combining siFAM222A transfection with any of these individual siMCMs had no additional effect on proliferation (FIG.26H) or migration (FIG.26I) beyond siFAM222A alone. To further elucidate FAM222A-MCM protein interaction during the G1 phase, MCM3 immunoprecipitation was performed before and after serum stimulation (0, 4, 8 hours). As reported24,27, MCM3 immunoprecipitation also pulled down MCM4 and origin replication 1 (ORC1) both before and after FBS stimulation. However, MCM3 pulldown also co-immunoprecipitated FAM222A in the nuclear fraction, peaking at 4 hours post-serumstimulation, aligning with FAM222A involvement in early G1. FAM222A also co-localized with MCM3 in EC nuclei (FIG.26J). As such, we tested if FAM222A silencing alters MCM complex assembly on chromatin during the G1 phase. In siFAM222A-treated ECs, MCM3, MCM4 and MCM7 chromatin association was significantly reduced during the early G1 phase (vs siCtl) while ORC1, a critical regulator of DNA replication whose chromatin binding precedes RC formation and MCM loading, remained unchanged (FIG.26F)27,28. These results demonstrate FAM222A is essential for MCM loading on chromatin in early G1 cell cycling, thus facilitating G1 / S phase transition and subsequent Rb and CDK recruitment and activation (FIG.27). Independent of MCM loading in mediating FAM222A’s angiogenic effects, extensive in vitro and in vivo evidence provided here establishes FAM222A, identified through its BRD4 regulation, as an important, novel protein essential for endothelial biology and function in vitro and in vivo, through its effects on cell cycling, proliferation, migration, and angiogenesis, with relevance for physiologic, pathologic, and BET inhibitor-induced responses (FIG.27). Discussion The endothelium’s central role in physiology and multiple disease states involves regulation of endothelial transcriptional programs. Epigenetic mechanisms including BET epigenetic reader proteins, facilitate transcriptional complex assembly, as reported in pathogenic, stimulated EC responses1,5,29. Here, we began by investigating the largely unexplored issue of BET action in resting ECs, using BET inhibition to uncover BET-dependent gene expression. While this analysis provided insight into BET action in the endothelium, we hypothesized that BET regulation could be an orthogonal approach to identify novel determinants of endothelial biology. While RNA-Seq data in resting ECs exposed to the BET inhibitor JQ1 establishes BETs control specific genes involved in basal EC function, it also revealed BET inhibition induced expression of a subset of EC genes, another largely unstudied response. Further analysis of JQ1-induced EC genes pointed to FAM222A as a unique, previously unidentified determinant of EC biology, as supported by subsequent in vitro and in vivo data. FAM222A controls cell cycling in ECs, with silencing FAM222A regulating expression of multiple key cell cycle mediators and inducing G1 arrest. VEGF-stimulated EC migration, proliferation and sprouting are suppressed by FAM222A knockdown and promoted by FAM222A overexpression, coordinately modulating both VEGF and NOTCH signaling by altering expression of PATENT Attorney Docket NO.: 51792-002WO2 mediators in these pathways (FIG.24). In vivo, silencing FAM222A disrupted retinal revascularization in OIR and post-ischemic blood flow restoration in HLI. Together these studies provide new information regarding endothelial BET action and identify FAM222A as a novel, essential determinant of EC biology and angiogenesis. Although still incompletely understood, BET action has been primarily studied in directing induced, pathologic responses, prompting pursuit of BET inhibitors as therapeutic agents. In addition to its established role in cancer, BETs orchestrate gene expression in cardiovascular settings, like cardiomyocytes during pressure overload30and ECs executing TNFα-induced NFκB transcription1. Indeed, BET inhibitors decrease myocardial hypertrophy, atherosclerosis, and pulmonary arterialhypertension (PAH) in preclinical models, even reaching a clinical cardiovascular outcome trial thatfailed to meet its primary cardiovascular endpoint31. In ECs, TNFα stimulation prompts BRD4 binding to super enhancer regions to direct expression of the NF-kB inflammatory program. Importantly, BRD4 action in response to stimuli involves its relocation from its activity orchestrating basal gene expression, understudied effects relevant to better understanding both BET mechanisms and clinical responses to BET inhibitors being pursued for cardiovascular disease, cancer, and other disorders. As delineated here, gene ontology (GO) RNA-Seq pathway analysis of JQ1-treated resting ECs establishes BET control of endothelial genes involved in proliferation, migration, and other functional properties, with transcriptional resting EC heat maps showing striking shifts before and after JQ1 exposure (FIG.20A). This data also reveals EC genes whose mRNA is induced significantly after BET inhibition, as seen with pharmacologic inhibitors and BRD4 silencing as well as expected through endogenous BET regulation. GO analyses implicate JQ1-induced EC targets in cell cycle regulation. Given BET control over canonical pro-inflammatory, pro-atherosclerotic mediators, we leveraged BET regulation as a distinct approach to identifying novel regulators of EC biology in resting cells. Annotation of basally expressed EC genes most induced by JQ1 (> 2-fold), excluding genes for structural proteins, those with known EC effects and / or roles in cell cycling, pointed to FAM222A, also known as C12orf34. Subsequent studies established FAM222A as basally and predominately expressed in ECs (human and mouse) and further induced by BET inhibition, and specifically BRD4. Despite the focus on inhibiting BETs to disrupt pathologic transcriptional responses32-35, limited other examples have been reported of genes like FAM222A with induced expression after BET inhibition. BET inhibitors increase HIV gene transcription, suggesting a potential approach to HIV latency36,37. In cancer cells, BET inhibitor-induced genes overlapped targets induced by histone deacetylase inhibitors (HDACi) and included HEXIM1, which represses PTEF-b activity38. In adipocytes, JQ1 increases SOCS3 mRNA, perhaps releasing PTEF-b from HEXIM1 inhibition39. Other studies suggest BET inhibitors increase primarily expression of histones and non- polyadenylated genes40,41. None of these mechanisms explain BET inhibition increasing levels of FAM222A, a non-histone, polyadenylation-signal containing protein not regulated by HEXIM1 or HDACi (data not shown). While specific mechanisms for BRD4 and FAM222A interaction warrant further study, we focused here on elucidating FAM222A function. PATENT Attorney Docket NO.: 51792-002WO2 In line with GO analysis of JQ1-induced EC genes, FAM222A is essential for cell cycle control, a critical process governed by three key protein families: cyclins, CDKs, and CDK inhibitors10. After endothelial FAM222A silencing, expression of cyclins and CDKs decrease while CDK inhibitors increase, disrupting Rb signaling and causing G1 arrest. siFAM222A did not alter p53 or p53- mediated p21 activation (data not shown) nor induce apoptosis, excluding these as explaining siFAM222A-mediated cell cycle arrest. Cell cycling is integral to EC migration, proliferation, and angiogenesis, as relevant in health and multiple diseases26,42, prompting our studying FAM222A in these responses. Silencing FAM222A decreased VEGF-induced EC migration and proliferation while pro-angiogenic stimuli, like VEGF andhypoxia, further increased FAM222A mRNA and protein levels. Similar FAM222A effects occurred invivo in neonatal OIR and HLI angiogenesis models. OIR replicates retinopathy of prematurity (ROP), with initial vaso-obliteration followed by ischemia / hypoxia-induced neovascularization, with variable and structurally abnormal blood vessel formation12,13. Silencing FAM222A during OIR decreased retinal revascularization and VEGF expression, altering EC proliferation and migration during OIR’s second, hypoxic phase. In HLI, initial inflammation is followed by qualitatively normal micro-vessel formation11,21,43. During HLI, endothelial FAM222A expression was initially decreased in the inflammatory phase before increasing in the angiogenic phase20, suggesting dynamic FAM222A changes during angiogenesis. Moreover, FAM222A deficiency during HLI delayed post-ischemic blood flow restoration and reduced angiogenic properties of ischemic gastrocnemius muscle. Interestingly, FAM222A displayed increased expression in transcriptional profiling data of tissue from patients with critical limb ischemia9, which suggests FAM222A as a compensatory pro-angiogenic response. FAM222A deficiency (48 hours) causes transcriptional changes that alter dynamic VEGF and NOTCH pathways that control angiogenesis. While VEGF drives EC spatial guidance, sprout initiation, and stalk-cell proliferation, NOTCH counters these responses, decreasing EC activity and inducing repressive stalk cell features, including increases in NOTCH-dependent angiogenic repressors HES1, HES2, and HEY217,19,44. siFAM222A modulates expression of these mediators, decreasing pro-angiogenic targets while inducing angiogenic repressors, supporting FAM222A as a key determinant of angiogenic transcriptional programs. Transcriptional shifts through FAM222A silencing also disrupts VEGF-induced phosphorylation, decreasing pro-mitogenic MAPK signaling while increasing anti-angiogenic NOTCH responses45. Although the pharmacologic NOTCH inhibitor DAPT blocked siFAM222A effects on NOTCH target gene expression, DAPT only partially reversed siFAM222A-mediated decreased EC migration, suggesting that aberrant NOTCH hyperactivation, reported as causing cell cycle arrest and decreased EC proliferation26,46, is unlikely to explain FAM222A’s effects on these responses. Silencing FAM222A might also induce compensatory responses that limit NOTCH inhibitors overcoming siFAM222A effects. Although these additional potential mechanisms warrant further study, the similar pattern of modulated VEGF and NOTCH target gene expression seen in vitro and ex vivo in ECs from the ischemic hindlimbs of siFAM222A- treated mice supports FAM222A’s involvement in angiogenesis. PATENT Attorney Docket NO.: 51792-002WO2 Given FAM222A’s role in EC biology, cell cycling and angiogenic responses in vitro and in vivo, we pursued intracellular mechanisms for FAM222A action. siFAM222A altered cell cycling, cell cycle-dependent transcription, and cell cycle progression, suggesting its interaction with cell cycle mediators that precede G1 phase initiation. MCM proteins, increasingly recognized as cell cycle determinants, form a pre-replication complex (pre-RC) along with other DNA replication factors, like ORC1, in early G1 replication licensing27,47. During G1-S transition, CDKs and other mediators are recruited to the pre-RC, enabling initiation of DNA replication through subsequent CDK phosphorylation25. MCM inactivation decreases cell proliferation and cell cycle progression, as reported in cancer, neuroinflammation and DNA damage48,49. Although decreased MCM2 and MCM6have been associated with NOTCH-mediated cell cycle arrest2 6, MCMs have no reported role inangiogenesis and their involvement vascular biology remains mostly unexplored. Here, we show FAM222A interacts with specific MCMs to exert distinct EC responses. MCM3, MCM4, and MCM7 facilitate EC migration while MCM7 and especially MCM3 help control EC proliferation. We show FAM222A binds directly to MCM3, MCM4 and MCM7, primarily during early G1 (4 hours), in keeping with FAM222A promoting EC proliferation through MCM interaction while disrupting MCM-FAM222A association expected to block replication licensing and initiation of DNA replication (FIG.27). FAM222A as a protein is largely unknown, suggesting the approach employed here, using BET regulation to identify novel mediators, may offer unique, broadly applicable opportunities for discovery. The evidence presented for FAM222A expression and action in EC biology, cell cycling, and angiogenesis extends the only other prior FAM222A-focused report, which suggested FAM222A expression was restricted to neurons and might promote Alzheimer’s Disease (AD)50. In that study, FAM222A was reported to localize primarily to amyloid plaque centers while hippocampal FAM222A deficiency did not cause neuronal death50. While the data provided here extends FAM222A action outside of the central nervous system (CNS), it also raises the question if FAM222A in brain ECs or the functional effects we show might contribute to its CNS role and / or underlie any FAM222A effects in AD. Of note, in support of FAM222A’s broader, non-CNS roles and BET inhibitor induction for FAM222A demonstrated here, FAM222A is found among the genes most induced in myocardial samples from BET inhibitor-treated mice undergoing transaortic constriction30. Whether this FAM222A signal derives from myocardial ECs or cardiomyocytes, which contain less FAM222A than ECs (data not shown), is unclear but warrant investigation. Taken together, we establish here BET control of dynamic endothelial gene expression includes transcriptional programs in resting ECs as well as genes whose mRNAs are induced by BET inhibition or BRD4 silencing. Building off BET control over induced pathogenic EC targets, an orthogonal approach using BET regulation and BRD4 chromatin localization to find novel genes enabled identification of FAM222A, demonstrated here to be a previously undescribed, key determinant of endothelial cell cycling, migration, proliferation, and angiogenesis in vitro and in vivo, with relevance to physiologic and pathologic settings involving cell cycling, endothelial function and angiogenesis. PATENT Attorney Docket NO.: 51792-002WO2 References 1. Brown, J. D. et al. NF-kappaB directs dynamic super enhancer formation in inflammation and atherogenesis. Mol Cell 56, 219-231 (2014). 2. Wu, T., Kamikawa, Y. F. & Donohoe, M. E. Brd4’s Bromodomains Mediate Histone H3 Acetylation and Chromatin Remodeling in Pluripotent Cells through P300 and Brg1. Cell Rep 25, 1756-1771 (2018). 3. Borck, P. C., Guo, L. W. & Plutzky, J. BET Epigenetic Reader Proteins in Cardiovascular Transcriptional Programs. Circ Res 126, 1190-1208 (2020). 4. Tsujikawa, L. M. et al. Apabetalone (RVX-208) reduces vascular inflammation invitro and in CVD patients by a BET-dependent epigenetic mechanism. Clin Epigenetics 11, 102 (2019).5. Zhang, M. et al. The BD2 domain of BRD4 is a determinant in EndoMT and vein graft neointima formation. Cell Signal 61, 20-29 (2019). 6. Dejana, E., Hirschi, K. K. & Simons, M. The molecular basis of endothelial cell plasticity. Nat Commun 8, 14361 (2017). 7. Jeong, H. W. et al. Transcriptional regulation of endothelial cell behavior during sprouting angiogenesis. Nat Commun 8, 726 (2017). 8. Gao, Y. & Galis, Z. S. Exploring the Role of Endothelial Cell Resilience in Cardiovascular Health and Disease. Arterioscler Thromb Vasc Biol 41, 179-185 (2021). 9. Ryan, T. E. et al. Extensive skeletal muscle cell mitochondriopathy distinguishes critical limb ischemia patients from claudicants. JCI Insight 3, (2018). 10. Giacinti, C. & Giordano, A. RB and cell cycle progression. Oncogene 25, 5220-843 (2006). 11. Nowak-Sliwinska, P. et al. Consensus guidelines for the use and interpretation of angiogenesis assays. Angiogenesis 21, 425-532 (2018). 12. Stahl, A. et al. The mouse retina as an angiogenesis model. Invest Ophthalmol Vis Sci 51, 2813-2826 (2010). 13. Liu, H. et al. Epac1 inhibition ameliorates pathological angiogenesis through coordinated activation of Notch and suppression of VEGF signaling. Sci Adv 6, eaay3566 (2020). 14. Sun, Y. et al. Nuclear receptor RORα regulates pathologic retinal angiogenesis by modulating SOCS3-dependent inflammation. Proc Natl Acad Sci U S A 112, 10401-10406 (2015). 15. Sun, Y. et al. SOCS3 in retinal neurons and glial cells suppresses VEGF signaling to prevent pathological neovascular growth. Sci Signal 8, ra94 (2015). 16. Hoeben, A. et al. Vascular endothelial growth factor and angiogenesis. Pharmacol Rev 56, 549- 580 (2004). 17. Jakobsson, L. et al. Endothelial cells dynamically compete for the tip cell position during angiogenic sprouting. Nat Cell Biol 12, 943-953 (2010). PATENT Attorney Docket NO.: 51792-002WO2 18. Phng, L. K. & Gerhardt, H. Angiogenesis: a team effort coordinated by notch. Dev Cell 16, 196- 208 (2009). 19. Pitulescu, M. E. et al. Dll4 and Notch signalling couples sprouting angiogenesis and artery formation. Nat Cell Biol 19, 915-927 (2017). 20. Lee, C. W. et al. Temporal patterns of gene expression after acute hindlimb ischemia in mice: insights into the genomic program for collateral vessel development. J Am Coll Cardiol 43, 474- 482 (2004). 21. Paoni, N. F. et al. Time course of skeletal muscle repair and gene expression following acute hind limb ischemia in mice. Physiol Genomics 11, 263-272 (2002).22. Forsburg, S. L. Eukaryotic MCM proteins: beyond replication initiation. Microbiol Mol Biol Rev68, 109-131 (2004). 23. Ghosh, S., Vassilev, A. P., Zhang, J., Zhao, Y. & DePamphilis, M. L. Assembly of the human origin recognition complex occurs through independent nuclear localization of its components. J Biol Chem 286, 23831-23841 (2011). 24. Kuipers, M. A. et al. Highly stable loading of Mcm proteins onto chromatin in living cells requires replication to unload. J Cell Biol 192, 29-41 (2011). 25. Braden, W. A., McClendon, A. K. & Knudsen, E. S. Cyclin-dependent kinase 4 / 6 activity is a critical determinant of pre-replication complex assembly. Oncogene 27, 7083-7093 (2008). 26. Noseda, M., Niessen, K., McLean, G., Chang, L. & Karsan, A. Notch-dependent cell cycle arrest is associated with downregulation of minichromosome maintenance proteins. Circ Res 97, 102- 104 (2005). 27. Madine, M. A. et al. The roles of the MCM, ORC, and Cdc6 proteins in determining the replication competence of chromatin in quiescent cells. J Struct Biol 129, 198-210 (2000). 28. Yuan, Z. et al. Structural mechanism of helicase loading onto replication origin DNA by ORC- Cdc6. Proceedings of the National Academy of Sciences 117, 888-17747 (2020). 29. Wang, B. et al. BET Bromodomain Blockade Mitigates Intimal Hyperplasia in Rat Carotid Arteries. EBioMedicine 2, 1650-1661 (2015). 30. Alexanian, M. et al. A transcriptional switch governs fibroblast activation in heart disease. Nature 595, 438-443 (2021). 31. Nicholls, S. J. et al. Apabetalone and hospitalization for heart failure in patients following an acute coronary syndrome: a prespecified analysis of the BETonMACE study. Cardiovasc Diabetol 20, 13 (2021). 32. Ding, N. et al. BRD4 is a novel therapeutic target for liver fibrosis. Proc Natl Acad Sci U S A 112, 15713-15718 (2015). PATENT Attorney Docket NO.: 51792-002WO2 33. Song, S. et al. Inhibition of BRD4 attenuates transverse aortic constriction- and TGF-beta- induced endothelial-mesenchymal transition and cardiac fibrosis. J Mol 900 Cell Cardiol 127, 83-96 (2019). 34. Sun, Y., Xie, Y., Du, L., Sun, J. & Liu, Z. Inhibition of BRD4 attenuates cardiomyocyte apoptosis via NF-kappaB pathway in a rat model of myocardial infarction. Cardiovasc Ther 36, (2018). 35. Wang, X. et al. Bromodomain-containing protein 4 contributes to renal fibrosis through the induction of epithelial-mesenchymal transition. Exp Cell Res 383, 906-111507 (2019). 36. Li, Z., Guo, J., Wu, Y. & Zhou, Q. The BET bromodomain inhibitor JQ1 activates HIV latency through antagonizing Brd4 inhibition of Tat-transactivation. Nucleic Acids Res 41, 277-287 (2013).37. Banerjee, C. et al. BET bromodomain inhibition as a novel strategy for reactivation of HIV-1. J Leukoc Biol 92, 1147-1154 (2012). 38. Bhadury, J. et al. BET and HDAC inhibitors induce similar genes and biological effects and synergize to kill in Myc-induced murine lymphoma. Proc Natl Acad Sci U S A 111, E2721-30 (2014). 39. Mota de Sá, P., Richard, A. J. & Stephens, J. M. Bromodomain and Extraterminal Inhibition by JQ1 Produces Divergent Transcriptional Regulation of Suppressors of Cytokine Signaling Genes in Adipocytes. Endocrinology 161, (2020). 40. Muhar, M. et al. SLAM-seq defines direct gene-regulatory functions of the BRD4- MYC axis. Science (1979) 360, 800-805 (2018). 41. Winter, G. E. et al. BET Bromodomain Proteins Function as Master Transcription Elongation Factors Independent of CDK9 Recruitment. Mol Cell 67, 5-18.e19 (2017). 42. Chavkin, N. W. et al. Endothelial cell cycle state determines propensity for arterial-venous fate. Nat Commun 13, 5891 (2022). 43. Yang, Y. et al. Cellular and molecular mechanism regulating blood flow recovery in acute versus gradual femoral artery occlusion are distinct in the mouse. J Vasc Surg 48, 1546-1558 (2008). 44. Lamalice, L., le Boeuf, F. & Huot, J. Endothelial cell migration during angiogenesis. Circ Res 100, 782-794 (2007). 45. Siekmann, A. F. & Lawson, N. D. Notch signalling limits angiogenic cell behaviour in developing zebrafish arteries. Nature 445, 781-784 (2007). 46. Wei, Y. et al. Nrf2 acts cell-autonomously in endothelium to regulate tip cell formation and vascular branching. Proc Natl Acad Sci U S A 110, E3910-8 (2013). 47. Jenkyn-Bedford, M. et al. A conserved mechanism for regulating replisome disassembly in eukaryotes. Nature 600, 743-747 (2021). 48. Seo, Y. S. & Kang, Y. H. The Human Replicative Helicase, the CMG Complex, as a Target for PATENT Attorney Docket NO.: 51792-002WO2 Anti-cancer Therapy. Front Mol Biosci 5, 26 (2018). 49. Kalogeropoulou, A., Lygerou, Z. & Taraviras, S. Cortical Development and Brain Malformations: Insights From the Differential Regulation of Early Events of DNA Replication. Front Cell Dev Biol 7, (2019). 50. Yan, T. et al. FAM222A encodes a protein which accumulates in plaques in Alzheimer’s disease. Nat Commun 11, 411 (2020). Example 3. Modulating FAM222A as a Method for Treating Disease We have identified FAM222A as a specific, novel, and unique gene / transcribed protein thatexerts fundamental effects in the endothelium, as well as in other settings, with findings that include:1) basal expression of FMA222A occurs in the endothelium; 2) FAM222A mRNA expression and protein levels further increased after BET inhibition and, specifically, after BRD4 inhibition / silencing; 3) FAM222A plays an essential role in allowing for and promoting core aspects of endothelial function, including endothelial cell (EC) migration and proliferation (e.g., both were significantly repressed after siRNA silencing of FAM222A); 4) FAM222A plays an essential role in angiogenesis, with coordinated modulation of pro-angiogenic VEGF (which is blocked by siFAM222A, FAM222A ASOs, and other FAM222A inhibitors) and anti-angiogenic NOTCH pathways (which increased by siFAM222A, FAM222A ASOs, and other FAM222A inhibitors); 5) FAM222A plays an essential role in cell cycling (e.g., siFAM222A and other FAM222A inhibitors blocked cell cycling by causing G1-S phase cell cycle arrest); 6) FAM222A plays an essential role during retinal development (FIG.49) through its expression in endothelial cells (FIG.50) and other cells types that are involved with the pathogenesis of retinopathy; 7) FAM222A expression is modulated by known angiogenic stimuli (e.g., VEGF, hypoxia, and flow) or mediators (e.g., EGR1, E2F1, and HIF1A) that are implicated during retinopathy (FIG.50), such as oxygen induced retinopathy in a well-established model of human retinopathy of prematurity (ROP), and was highly upregulated (FIG.51); 8) silencing or otherwise inhibiting FAM222A decreases ROP; 9) FAM222A plays an essential role in controlling cytoskeleton integrity and cytoskeletal remodeling during angiogenesis (FIG.53 and FIG.54). Silencing or inhibiting FAM222A decreases vascular leakage, as seen in retinopathy; 10) silencing and otherwise inhibiting FAM222A blocks angiogenic responses to hindlimb ischemia; 11) FAM222A exerts its transcriptional effects by binding to key nuclear transcription proteins including MCM, MEIS1, MEIS2 (FIGs.28-40); 12) a series of distinct BET inhibitor compounds increased FAM222A (FIG.29A and FIG.29B); 13) BRD4 is presented on FAM222A’s promoter and is essential for expression. BRD4 degradation, as seen with agents that degrade BRD4, decreased FAM222A levels (FIG.29C); 14) FAM222A plays an essential role in cancer by inhibiting cell cycling. P53, a tumor suppressor protein that is often found to be mutated in cancer resulting in uncontrolled cells proliferation represses FAM222A levels (FIG.55). Silencing or otherwise inhibiting FAM222A decreased ki67 expressing endothelial cells, a known prognostic marker in cancer indicating decreased cell proliferation and cell cycling activity; 15) inhibiting or silencing FAM222A decreased cell proliferation (FIG.57) and cell cycle target expression (FIG.58 and FIG.59) independently of hypoxia, a hallmark of cancer development and metastasis; PATENT Attorney Docket NO.: 51792-002WO2 16) blocking or inhibiting FAM222A decreased HIF1A translation and activation under hypoxia conditions (FIG.60), which is a known mediator of cancer survival, promoting chemoresistance, and metastasis; 17) FAM222A proteins levels are decreased in patients with pulmonary arterial hypertension (PAH) (FIG.61). FAM222A levels are also greatly increased by BMP9, a selective therapeutic agent approved for use in PAH; 18) blocking or inhibiting FAM222A with siFAM222A or FAM222A ASOs worsened PAH in rat (FIG.47) and mouse models (FIG.48). Targeting endothelial lung-enriched FAM222A with ASOs (FIG.68) promoted pulmonary hypertension in rats (FIG.69); 19) loss of FAM222A in the lungs or endothelial lung cells of mice worsened pulmonary hypertension in mice (FIG.70); 20) FAM222A protein levels were decreased in patients with atherosclerosis (FIG.62);21) SNPs associated with cardiovascular disease, including coronary artery disease, were found to bein close proximity to the FAM222A gene (FIG.63); 22) FAM222A is essential for maintaining endothelial identity, with loss of FAM222A causing ECs to undergo endothelial-to-mesenchymal transition (EndMT; FIG.64), with increased mesenchymal and profibrotic markers. EndMT markers were increased in the presence of proEndMT stimulation (FIG.65); 23) FAM222A plays an essential role in maintaining vascular integrity by inhibiting vascular leakage and endothelial barrier dysfunction in diseases associated with EndMT pathophysiology, including atherosclerosis and pulmonary hypertension (FIG.66); 24) FAM222A binds to and interacts directly with specific nuclear proteins, namely mini-chromosomal maintenance (MCM) proteins MCM3, MCM4, MCM7 as well as Myeloid Ectopic Viral Integration Site 2 homolog (MEIS2), MEIS1, and PBX homeobox genes (e.g., PBX1, PBX2, and PBX3), all of which are considered therapeutic targets (FIG.28). FAM222A’s interaction with MEIS2 has been established in a reciprocal strategy (FIGs.30-33), is mediated by the MEINOX protein domain of MEIS2 (FIG.36). Further, FAM222A’s interaction with MEIS2 is independent of DNA binding (FIG.37), of additional stimulation (FIG.39), and of poly ADP-ribose polymerase (PARP) activity (FIG.38). Silencing MEIS2 or MEIS1 modulates FAM222A-mediated EndMT responses (FIGs.41-46). FAM222A interacts also with PBX3 (FIG.35 and FIG.40), which has an established role in cancer, development, and cell-cycle regulation; 25) FAM222A exhibited anti-inflammatory properties. Silencing FAM222A increased endothelial inflammatory and DNA damage responses, while overexpressing FAM222A inhibited them (FIG.67). Taken together, these findings establish that: • Decreasing FAM222A levels, through silencing (e.g., by siRNA, ASO, or any other inhibitory nucleic acid molecule described here), FAM222A inhibition, genetic deletion via genetic tools (e.g., CRISPR), or with BET / BRD4 degrading agents would result in blocking angiogenesis, which is useful in conditions associated with angiogenesis, namely proliferative retinopathies, including retinopathy of prematurity, diabetic retinopathy, angiogenic tumors, cancers, malformations, and decreasing myeloproliferative or myelofibrosis (e.g., Pelabresib, an oral potent pan-BET inhibitor, downregulates the expression of genes involved in the heterogeneous pathology of MF and has been explored in patients with MF in the Phase II MANIFEST study) PATENT Attorney Docket NO.: 51792-002WO2 • FAM222A silencing, inhibition, or deletion can be used as a method for blocking cell cycling as a treatment for arresting cancer growth, treating vascular cancers / tumors, treating AV malformations, or treating other vascular structures (e.g., hemangiomas). • Increasing FAM222A levels through overexpression constructs, small molecules that increase FAM222A levels (e.g., BET and BRD4 specific inhibitors) would be useful in clinical disordersthat benefit from increased angiogenesis, blood supply, hypoxia, including chronic ischemic conditions (e.g., coronary disease, cerebrovascular disease, peripheral arterial disease), and pulmonary artery disease. • Increasing FAM222A levels would also be expected to benefit from conditions in which preserving or restoring endothelial identity would be helpful, including avoidance of fibrosis and scar formation that occurs with loss of endothelial function, including cardiomyopathies, cardiac pressure overload, atrial fibrosis associated with arrhythmias, excess scar formation, and other clinical conditions associated with endothelial-to-mesenchymal transition (EndoMT or EndMT). • Given reports of FAM222A being associated with Alzheimer's Disease, targeting FAM222A, whether in the endothelium or elsewhere, can be expected to offer potential benefit in Alzheimer's Disease and other central nervous system conditions. Advantageously, FAM222A has never been reported as a determinant of endothelial identity and function, which includes its being necessary for endothelial migration, proliferation, VEGF induced responses, including angiogenesis. Silencing / inhibiting FAM222A blocks all of these responses. The need for FAM222A in maintaining cell cycling as well as our finding that silencing FAM222A worsened retinopathy, peripheral arterial disease, and pulmonary artery hypertension are new and to our knowledge are not previously reported. As described above, we have strong in vitro and in vivo evidence that using both loss of function (e.g., silencing by siRNA, ASO, or other inhibitory nucleic acid molecules described herein) and overexpression models. We have also developed cell specific knock out mouse models. We have demonstrated the effects of specific BET inhibitors and BET modulators, including more specific BRD4 targeting agents for increasing or decreasing FAM222A levels. FAM222A inhibitors, e.g., siRNA, ASOs, therapeutic antibodies, and small molecules that decrease FAM222A or inhibit its activity, can be used to treat retinopathies and cancer, such as vascular cancers / tumors. FAM222A activators, e.g., overexpression constructs and BET / BRD4 degraders, can be used to treat chronic ischemic conditions that would benefit from angiogenesis.Other EmbodimentsAll publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each independent publication or patent application was specifically and individually indicated to be incorporated by reference. While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover PATENT Attorney Docket NO.: 51792-002WO2 any variations, uses, or adaptations following, in general, the principles and including such departures from the invention that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth, and follows in the scope of the claims. Other embodiments are within the claims.

Claims

PATENT Attorney Docket NO.: 51792-002WO2 CLAIMS 1. A method of treating retinopathy in a subject comprising administering to the subject an effective amount of a FAM222A (family with sequence similarity 222 member A) inhibitor.

2. The method of claim 1, wherein the retinopathy is selected from the group consisting of: diabetic retinopathy, retinopathy of prematurity (ROP), hypertensive retinopathy, and central serous retinopathy.

3. A method of treating cancer in a subject having a tumor, the method comprising administering to the subject an effective amount of a FAM222A inhibitor.

4. The method of claim 3, wherein the tumor is an angiogenic tumor.

5. The method of claim 3 or 4, wherein the FAM222A inhibitor reduces tumor angiogenesis.

6. The method of claim 3, wherein the tumor is a non-angiogenic tumor.

7. The method of any one of claims 3-6, wherein the tumor is chemoresistant and / or radioresistant.

8. The method of any one of claims 3-7, wherein the tumor comprises a mesenchymal cell.

9. The method of claim 8, wherein the mesenchymal cell is a migratory mesenchymal cell and treating the cancer reduces metastasis of the migratory mesenchymal cell.

10. The method of any one of claims 3-9, wherein the cancer is selected from the group consisting of breast cancer, colorectal cancer (CRC), esophageal cancer, gastric cancer ovarian cancer, prostate cancer, renal cell carcinoma (RCC), and non-small lung cancer (NSCLC).

11. The method of any one of claims 1-10, wherein the FAM222A inhibitor reduces angiogenesis of an endothelial cell in the subject.

12. The method of claims 1-11, wherein the FAM222A inhibitor reduces endothelial cell proliferation, endothelial cell migration, and / or endothelial-to-mesenchymal transition (EndMT).

13. The method of any one of claims 1-12, where the FAM222A inhibitor is an inhibitory nucleic acid molecule.PATENT Attorney Docket NO.: 51792-002WO2 14. The method of claim 13, wherein the inhibitory nucleic acid molecule is an anti-sense oligonucleotide (ASO), a small interfering RNA (siRNA), a short hairpin RNA (shRNA), a double- stranded RNA (dsRNA), or a microRNA (miRNA).

15. The method of claim 14, wherein the inhibitory nucleic acid molecule is an ASO.

16. The method of claim 15, wherein the ASO comprises 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 10-15.

17. The method of claim 13, wherein the inhibitory nucleic acid molecule is an siRNA.

18. The method of claim 17, wherein the siRNA comprises: (a) a sense strand comprising 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 16, 18, 20, 22, 24, 26, 28, and 30; and (b) an antisense strand comprising 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 17, 19, 21, 23, 25, 27, 29, and 31.

19. The method of any one of claims 13-18, wherein the inhibitory nucleic acid molecule comprises a non-natural or modified nucleoside or nucleotide.

20. The method of claim 19, wherein the wherein the non-natural or modified nucleoside or nucleotide comprises a 2′-O-methyl (2′-O-Me) modified nucleoside, a phosphorothioate (PS) bond between nucleosides, and a 2′-fluoro (2′-F) modified nucleoside.

21. The method of any one of claims 13-20, wherein the inhibitory nucleic acid molecule comprises a delivery vehicle.

22. The method of claim 21, wherein the delivery vehicle is a lipid nanoparticle (LNP).

23. The method of any one of claims 1-12, wherein the FAM222A inhibitor is a bromodomain- containing protein 4 (BRD4) degrader.

24. The method of any one of claims 1-23, wherein the method further comprises administering a second therapeutic agent to the subject.

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