APOM fusion constructs and methods of use in treating inflammatory diseases

Abstract

Described herein are ApoA1 and ApoM fusion proteins and methods of use thereof for treatment of vascular and inflammatory disorders. Also described herein are high-yield protocols to produce active ApoA1-ApoM fusion proteins from bacteria.

Description

[0001]APOM FUSION CONSTRUCTS AND METHODS OF USE IN TREATING INFLAMMATORY DISEASES RELATED APPLICATIONS This Application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No.63 / 554372, filed on February 16, 2024, the entire contents of which are incorporated herein by reference. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING The contents of the electronic sequence listing (C123370285W000-SEQ-ACZ.xml; Size: 77,351 bytes; and Date of Creation: February 14, 2025) are herein incorporated by reference in its entirety. FEDERALLY SPONSORED RESEARCH This invention was made with government support under Grant Number HL135821, awarded by the National Institutes of Health. The Government has certain rights in the invention. BACKGROUND OF INVENTION High-density lipoproteins (HDLs) are a group of small (~7-14 nm in diameter), spherical particles composed of proteins and lipids. The major proteins associated with HDL are apolipoprotein (apo) A1 and ApoA2, which are arranged radially around a phospholipid bilayer to form a discoidal structure. These nascent HDL particles sequester and incorporate cholesterol and phospholipid molecules from cells and other lipoproteins, in a process termed reverse cholesterol transport, to inhibit oxidation, inflammation, activation of the endothelium, and platelet aggregation. SUMMARY OF INVENTION Vascular endothelial dysfunction, which can be induced by metabolic stress, inflammatory and autoimmune states, is a major driver of chronic diseases, such as atherosclerosis, stroke, heart failure, rheumatoid arthritis, and systemic lupus erythematosus. Restoration of endothelial cell function attenuates inflammation-induced tissue damage, thrombosis, and chronic disease progression. Circulating high-density lipoprotein (HDL) particles regulate cholesterol homeostasis and endothelial function. However, therapeutic strategies based on such particles are currently unable to counter endothelial dysfunction. 12124565.1 To overcome this issue, the inventors have designed and characterized ApoA1-ApoM (A1M) fusion proteins that form spherical, nano-sized lipoprotein particles, chaperones multiple bioactive lipids (S1P and PGI2 analogs), protects the endothelium, and suppresses platelet aggregation and inflammatory responses in vitro and in vivo. Importantly, the inventors have surprisingly found that A1M fusion proteins disclosed herein do not need to be loaded with lipids prior to administration in order to achieve therapeutic effect. The inventors additionally developed high-yield protocols to produce active A1M fusion proteins from bacteria. Accordingly, aspects of this disclosure relate to a fusion protein comprising Apolipoprotein A1 (ApoA1) and Apolipoprotein M (ApoM), and methods of use thereof. ApoA1 and ApoM are HDL-associated proteins that can act as chaperones for biologically active lipids, such as S1P and prostacyclin (PGI2), to protect vascular endothelial cells and inhibit thromboinflammatory responses. In some embodiments, ApoA1 comprises an amino acid sequence that is 90% identical to any one of SEQ ID NOs: 1, 3, or 5 and ApoM comprises an amino acid sequence that is 90% identical to any one of SEQ ID NOs: 2 or 4. In some embodiments, ApoA1 comprises the amino acid sequence of any one of SEQ ID NOs: 1, 3, or 5. In some embodiments, ApoM comprises the amino acid sequence of any one of SEQ ID NOs: 2 or 4. In some embodiments, ApoA1 is fused to the N-terminus of ApoM. In some embodiments, ApoA1 is fused to the C-terminus of ApoM. In some embodiments, ApoA1 and ApoM are fused via a linker. In some embodiments, the linker is a peptide linker. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 7. In some embodiments, the fusion protein further comprises a signal peptide sequence. In some embodiments, the signal peptide sequence comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, the signal peptide is fused to the N-terminus of ApoA1. In some embodiments, the fusion protein does not comprise a signal peptide sequence. In some embodiments, the fusion protein further comprises a translation start site sequence. In some embodiments, the translation start site sequence comprises the amino acid sequence of SEQ ID NO: 15. In some embodiments, the translation start site sequence is fused to the N-terminus of ApoA1. In some embodiments, the fusion protein comprises an amino acid sequence that is 90% identical to any one of SEQ ID NOs: 8-10. In some embodiments, the fusion protein comprises the amino acid sequence of any one of SEQ ID NOs: 8-10. In some aspects, the present disclosure provides a nucleic acid molecule comprising a polynucleotide sequence encoding a fusion protein as described herein. In some embodiments, the polynucleotide sequence comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 11, 17, or 18, optionally wherein the polynucleotide sequence comprises the 12124565.1 nucleotide sequence of SEQ ID NO: 11, 17, or 18. In some embodiments, the polynucleotide sequence is operably linked to a promoter. In some embodiments, the polynucleotide sequence is operably linked to a translation start site sequence. In some embodiments, the translation start site sequence comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 16, optionally wherein the translation start site sequences comprises the nucleotide sequence of SEQ ID NO: 16. In some embodiments, this disclosure provides a construct comprises a nucleic acid molecule as described herein. In some embodiments, the construct is a plasmid or a vector. In some embodiments, the vector is a viral vector. In some aspects, the present disclosure provides a cell comprising a fusion protein as described herein, a nucleic acid sequence as described herein, or a construct as described herein. In some embodiments, the cell is a prokaryotic cell. In some embodiments, the prokaryotic cell is a bacterial cell. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the eukaryotic cell is a human cell. In some aspects, the present disclosure provides a lipoprotein comprising a fusion protein as described herein and a lipid. In some embodiments, the lipid is a S1P receptor agonist or antagonist, or a prostaglandin agonist or antagonist. In some embodiments, the lipid is selected from the group consisting of a prostaglandin, sphingosine 1-phosphate (S1P), a leukotriene, and phosphatidyl choline. In some embodiments, the phosphatidyl choline is 1,2-dimyristoyl-sn- glycero-3-phosphocholine (DMPC) or 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG). In some embodiments, the lipid is Iloprost. In some embodiments, the lipid is sphingosine-1- phosphate. In some embodiments, the lipid is 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG). In some embodiments, the ratio of DMPC:DMPG is 7:3 molar ratio. In some embodiments, the lipoprotein is non- covalently bound to the lipid. In some embodiments, the lipoprotein is covalently bound to the lipid. In some embodiments, the fusion protein comprises at least 60 mol% of the lipid. In some embodiments, the fusion protein comprises less than 1 mol% of the lipid. In some embodiments, the lipoprotein is incorporated into a nanoparticle. In some embodiments, the nanoparticle is a nanodisk. In some embodiments, the nanoparticle is 70% unlipidated fusion protein and 30% lipidated fusion protein. In some embodiments, the nanoparticle is an HDL-like nanoparticle. In some embodiments, the HDL-like nanoparticle is between 8-12 nm in diameter. 12124565.1 In some aspects, the present disclosure provides a pharmaceutical composition comprising a fusion protein as described herein, a lipoprotein as described herein, a nanoparticle as described herein, or a nanodisk as described herein. In some aspects, the present disclosure provides a method of treating a subject having a disease or disorder associated with vascular endothelial dysfunction, comprising administering a fusion protein as described herein, a lipoprotein as described herein, or a pharmaceutical composition as described herein. In some embodiments, the disease or disorder is thrombosis, or thrombotic inflammation. In some embodiments, the disease or disorder is cardiovascular disease, metabolic disorder, autoimmune disease, inflammatory disease, infectious disease, ocular disorder, or cancer. In some embodiments, the cardiovascular disease is cerebrovascular disease, hypercholesterolemia, atherosclerosis, stroke, heart failure, peripheral artery disease, acute liver failure, or acute kidney failure. In some embodiments, the metabolic disorder is diabetes, diabetic nephropathy, or diabetic retinopathy. In some embodiments, the autoimmune disease is rheumatoid arthritis, systemic lupus erythematosus, or an autoimmune syndrome. In some embodiments, the inflammatory disease is an acute inflammatory disease, a chronic inflammatory disease, acute respiratory distress syndrome, or sepsis. In some embodiments, the infectious disease is a respiratory virus infection. In some embodiments, the ocular disorder is retinal vascular disease or age-related macular degeneration. In some aspects, the present disclosure provides a method of reducing inflammation in a subject, comprising administering a fusion protein as described herein, a lipoprotein as described herein, or a pharmaceutical composition as described herein. Other aspects of the present disclosure provide a method of reducing systemic inflammation in a subject, the method comprising administering a fusion protein as described herein, a lipoprotein as described herein, or a pharmaceutical composition as described herein. In some embodiments, the method further comprises lipidating the fusion protein or the lipoprotein in vitro. In some embodiments, the fusion protein or lipoprotein is lipidated in vitro with 1,2- dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dimyristoyl-sn-glycero-3- phosphoglycerol (DMPG). In some embodiments, the ratio of DMPC:DMPG is a 7:3 molar ratio. In some embodiments, lipidation of the fusion protein or the lipoprotein in vitro produces HDL-like nanoparticles. In some embodiments, the HDL-like nanoparticles are between 8-12 nm in diameter. In some embodiments, the method does not comprise lipidating the fusion protein or lipoprotein in vitro. In some embodiments, the method comprises administering a therapeutically effective amount of the fusion protein, the lipoprotein, or the pharmaceutical composition. In some 12124565.1 embodiments, the method comprises administering the fusion protein, the lipoprotein, or the pharmaceutical composition intravenously. In some embodiments, the inflammation is associated with TNFalpha-induced NF- kappaB activation. In some embodiments, the inflammation is associated with cardiovascular disease, metabolic disorder, autoimmune disease, inflammatory disease, infectious disease, ocular disorder, or cancer. In some embodiments, the cardiovascular disease is cerebrovascular disease, hypercholesterolemia, atherosclerosis, stroke, heart failure, peripheral artery disease, acute liver failure, or acute kidney failure. In some embodiments, the metabolic disorder is diabetes, diabetic nephropathy, or diabetic retinopathy. In some embodiments, the autoimmune disease is rheumatoid arthritis, systemic lupus erythematosus, or an autoimmune syndrome. In some embodiments, the inflammatory disease is an acute inflammatory disease, a chronic inflammatory disease, acute respiratory distress syndrome, or sepsis. In some embodiments, the infectious disease is a respiratory virus infection. In some embodiments, the ocular disorder is retinal vascular disease or age-related macular degeneration. In some embodiments, the acute inflammatory disease is peritonitis. In some embodiments, the method as described herein further comprises administrating an anti-thrombin agent. In some embodiments, the anti-thrombin agent is Angiopoietin-1 (Ang- 1) or Activated Protein C (APC). In some aspects, the present disclosure provides a method of purifying an ApoA1-ApoM fusion protein from a cell. In some embodiments, the method comprises: (i) obtaining cells as provided above; (ii) lysing the cells; (iii) obtaining inclusion bodies from the lysed cells; (iv) denaturing the inclusion bodies to release the ApoA1-ApoM fusion protein; and, (v) refolding the ApoA1-ApoM fusion protein. In some embodiments, step (ii) is performed using polytron disruption. In some embodiments, step (iv) is performed with a chaotropic reagent. In some embodiments, step (v) is performed by step-wise dialysis. In some embodiments, step (v) is performed without arginine. BRIEF DESCRIPTION OF DRAWINGS The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. 12124565.1 FIGs.1A-1B show the amino acid (FIG.1A) and nucleotide (FIG.1B) sequences of an exemplary A1M fusion protein containing a native signal peptide (MKAVVLAVALVFLTGSQAWH) (SEQ ID NO: 72) and amino acids 1-264 of the open reading frame of murine ApoA1, followed by a flexible linker region (GGGGS) (SEQ ID NO: 31), amino acids 21-190 of murine ApoM, a 6X His-Tag (HHHHHH) (SEQ ID NO: 32), and a stop codon (STOP). FIGs.2A-2E show production, purification, and characterization of S1P binding by an exemplary A1M fusion protein. FIG.2A shows purified A1M (4 μg) from CHO-S cell conditioned media separated by reducing 10% SDS-PAGE and stained with Coomassie brilliant blue. FIG.2B shows S1P content of A1M-S1P analyzed by electrospray ionization-MS / MS. The data shows the mean ± standard deviation; n=5 biological replicates per group; **** p < 0.0001 by unpaired t-test. FIG.2C shows a representative FPLC elution profile (OD 280 nm) of 200 μl mouse plasma, purified recombinant A1M, and lipidated A1M with S1P loading (A1M- S1P). FPLC elution of plasma standards: vLDL / LDL (21-26 mL), HDL (28-31 mL), and soluble protein (31-35 mL) fractions. Elution profiles are representative of 3 independent experiments. FIG.2D shows a representative negative-stain transmission EM image of lipidated A1M-S1P complex with some particles highlighted in circles (top) and 2D averages with potential ApoM densities indicated by arrows (bottom). Box dimension of each 2D average is 215 Å. Images are representative of 3 independent experiments. FIG.2E shows a schematic model of A1M-S1P. FIGs.3A-3C show molecular dynamics simulation study of A1M-S1P complex. FIG. 3A shows a side-view of the A1M-S1P complex with and without the phospholipids, highlighting the S1P binding pocket in both the ApoM-S1P and ApoA1-fused systems. Detail of the binding pocket in A1M-S1P and ApoM-S1P are shown in the right panel. FIG.3B shows projection of protein atoms along the top two dominant eigenvectors (eigenvector 1 - PC1 and eigenvector 2 – PC2). Permanence times: cluster 1 – 9.6%, cluster 2 – 52.9%, cluster 3 – 18.7%, cluster 4 – 12.2%, cluster 5 – 6.5%. FIG.3C shows the distribution of the proportion of variance between the PCs. FIG.4 shows the ratio of solvent accessible surface area for the interfacial residues. Ratio of the solvent accessible surface area (SASA) (ratioSASA = A1M / S1PSASA / ApoM / S1PSASA) of the ApoM binding pocket residues was calculated for both monomer 1 (light grey) and monomer 2 (dark grey). Boxes highlight some key interfacial residues. FIGs.5A-5F show A1M-S1P activation of S1PRs in G-protein dissociation and β- arrestin coupling assays. FIG.5A shows temporal analysis of S1P-induced S1PR1 activation in cells treated with 100 nM of S1P complexed with various chaperones through a NanoBiT G- 12124565.1 protein dissociation assay (n=3 biological replicates per group). FIG.5B shows dose-response analysis of S1PR1-dependent Gαi activation by bovine serum albumin-S1P BSA-S1P), ApoM- Fc-S1P, or A1M-S1P by NanoBiT assay assessing G-protein dissociation (n=33 biological replicates per group). FIG.5C shows temporal analysis of S1P-induced S1PR1 activation in cells treated with 100 nM of S1P complexed with various chaperones through a NanoBit β- arrestin association assay (n=3 biological replicates per group). FIG.5D shows dose analysis of S1PR1-β-arrestin coupling induced by BSA-S1P, ApoM-Fc-S1P, or A1M-S1P by NanoBiT assay (n= 3 biological replicates per group). FIG.5E shows dose analysis of S1PR2-β-arrestin coupling induced by BSA-S1P, ApoM-Fc-S1P, or A1M-S1P by NanoBiT assay (n= 3 biological replicates per group). FIG.5F shows dose analysis of S1PR3-β-arrestin coupling induced by BSA-S1P, ApoM-Fc-S1P, or A1M-S1P by NanoBiT assay (N = 3 biological replicates per group). Data represent mean ± standard deviation. The bar graphs show the responses at 1 μM S1P. ** p < 0.01; *** p < 0.001; **** p < 0.0001 by ordinary one-way ANOVA with Tukey’s multiple comparisons test. FIGs.6A-6F show A1M-S1P maintenance of endothelial barrier function in vitro, rescue of thrombin-induced endothelial barrier breakdown by ApoM-Fc-S1P and Ang-1, and suppression of thrombin-induced barrier degradation. FIG.6A show the results of TEER analysis to measure barrier function performed on HUVECs treated with A1M-S1P, ApoM-Fc- S1P or chaperone only (n=3 biological replicates per group). FIG.6B show the results of TEER analysis to measure barrier function performed on HUVECs treated with Ang-1 (300 ng / mL), A1M-S1P (30 nM) or both (n=3 biological replicates per group). FIG.6C shows the results of TEER analysis to determine barrier function performed on HUVECs treated for 2 hours using ApoM-S1P (0, 3, 10, 30, and 100 nM S1P) in combination with thrombin (1U / ML) (n=3 biological replicates per group). FIG.6D shows the results of ) TEER analysis to determine barrier function performed on HUVECs treated for 2 hours with thrombin (1 U / ml) and ApoM- Fc-S1P (200 nM), angiopoietin (Ang-1, 300 ng / ml) or both (n=3 biological replicates per group). FIG.6E shows the results of TEER analysis to measure barrier function performed on HUVECs treated with APC (5 μg / mL) alone or with A1M-S1P (30 nM) (n=3 biological replicates per group). FIG.6F shows the results of TEER analysis to measure barrier function performed on HUVECs treated with APC (5 μg / mL) alone or with ApoM-Fc-S1P (30 nM) (n-3 biological replicates per group). For FIGs.6E-6F, thrombin was added for an additional 2 hours after 1 hour of pretreatment. Data are the mean ± standard deviation. *** p < 0.001; *** p< 0.0001; **** p < 0.0001 by ordinary one-way ANOVA with Dunnett’s multiple comparisons test (for FIGs.6C-6D) or with Tukey’s multiple comparisons test (for FIGs.6E-6F). 12124565.1 FIGs.7A-7E show production and characterization of A1M-iloprost and demonstrates endothelial barrier function induced by iloprost, HDl-S1P, and AUY954. FIG.7A shows results of a CREB-assay, in which cells expressing a CREB-luciferase reporter with prostacyclin receptor (IP) were stimulated with vehicle or A1M-iloprost for 8 hours, and cell lysates were assayed for luciferase activity (n≥3 biological replicates per group).0 nM was used as a reference control and was normalized to 1. Data are presented as means ± standard deviation * p < 0.05; ** p < 0.01; *** p < 0.001; **** < 0.0001 by ordinary one-way ANOVA with Dunnett’s multiple comparisons test. FIG.7B shows the results of TEER analysis to measure barrier function performed on HUVECs treated with A1M-S1P (8 μg / mL with 100 nM S1P), A1M-iloprost (25 μg / mL with 200 nM iloprost), or both (n=4 biological replicates per group). Data are presented as means ± standard deviation. **** p < 0.0001 by ordinary one-way ANOVA with Tukey’s multiple comparisons test. FIG.7C shows the results of TEER analysis to measure barrier function performed on HUVECs treated with HDL-S1P (100 nM S1P) alone or in combination with iloprost (200 nM) (n=4 biological replicates per group). Data are presented as means ± standard deviation. **** p < 0.0001 by ordinary one-way ANOVA with Tukey’s multiple comparisons test. FIG.7D shows the results of TEER analysis to measure barrier function performed on HUVECs treated with the S1PR1 agonist AUY954 (1 μM) alone or in combination with iloprost (200 nM) (n=4 biological replicates per group). Data are presented as means ± standard deviation. **** p < 0.0001 by ordinary one-way ANOVA with Tukey’s multiple comparisons test. FIG.7E shows the results of a study in which human platelets were assayed for aggregation in response to the thrombin receptor mimetic peptide SFLLRN in the presence of vehicle control, A1M-iloprost (10 nM), A1M-S1P (200 nM), or in combination. Representative data from 3 biological replicates per group. FIGs.8A-8B show iloprost and ApoA1-iloprost inhibit SFLLRN-induced human platelet aggregation. FIG.8A shows comparison of pretreatments with free iloprost and ApoA1- iloprost on dose-response curves for platelet aggregation by the PAR-1 agonist SFLLRM peptide (2 μM). ApoA1-iloprost was lipidated using the same method as A1M-iloprost and A1M-S1P. FIG.8B shows the IC50of iloprost and ApoA1-iloprost determined from 6 biological replicates per group. Data are presented as means ± standard deviation. FIGs.9A-9C show A1M attenuation of TNFα-dependent inflammation. FIG.9A shows HMEC-1 cells expressing an NF-κB-luciferase reporter were assayed for TNFα-induced NF-κB reporter activity in the presence of ApoA1, A1M, A1M-S1P, and ApoM-Fc-S1P (n=3 biological replicates per group). Data are presented as means + standard deviation. ** p < 0.01; *** p < 0.001; **** p < 0.0001 by ordinary two-way ANOVA with Tukey’s multiple 12124565.1 comparisons test. TNFα is the reference group. FIG.9B shows an ICAM-1 immunoblot assay of lysates from HUVECs starved for 1 h, pre-treated for 10 minutes with ApoM-Fc-S1P (100 nM), iloprost (200 nM), both ApoM-Fc-S1P and iloprost, A1M (200 µg / mL), A1M-S1P (200 µg / mL), or A1M-iloprost (200 µg / mL) and induced with TNFα (10 ng / mL) for 5 hours. Quantification of immunoblots was analyzed from 3 biological replicates per group. Data are presented as means ± standard deviation. * p < 0.05; ** p < 0.01; **** p < 0.0001 by ANOVA with post-hoc Holm- Sidak’s multiple comparisons test. FIG.9C shows cholesterol efflux in response to human HDL (hHDL), human ApoA1 protein (hApoA1), and bacterial A1M (bA1M) in PMA-induced THP-1 cells (N ≥ 5 independent experiments). Data are presented as means + standard deviation. * p < 0.05; *** p < 0.001; **** p < 0.0001 by one-way ANOVA with Dunnett’s multiple comparisons test. FIGs.10A-10B show A1M-S1P suppression of inflammation in a murine peritonitis model. FIG.10A shows total isolated neutrophils of mice treated intraperitoneally with thioglycolate and either PBS, A1M (0.2 mg / animal), or lipidated A1M-S1P (0.2 mg / animal). Peritoneal cells were collected at 4 h and analyzed by flow cytometry. N = 4-5 mice per group. Data are presented as means ± standard deviation. * p < 0.05 by one-way ANOVA with Dunnett’s multiple comparisons test. Vehicle was the reference group. FIG.10B shows cytokine array analysis of l=peritoneal lavage supernatant. Statistical tests were done on analytes with changes in abundance after normalization (control was set at 100%). N = 3 mice per group. Data are presented as means ± standard deviation. * p < 0.05; *** p < 0.001; **** p < 0.0001 by ANOVA with Sidak’s multiple comparisons test. FIGs.11A-11I show the production, purification, and characterization of bacterial recombinant ApoA1-ApoM (bA1M) fusion protein. The nucleotide (FIG.11A) and amino acid (FIG.11B) sequence of an exemplary bacterial A1M fusion (bA1M) protein contains a pET15b- derived derived translation start site sequence (MGSSSSGLVPRGSHM) (SEQ ID NO: 33) embedded with a 6X histidine tag (HHHHHH) (SEQ ID NO: 32) and amino acids 25-264 of the open reading frame of murine ApoA1, followed by a flexible linker region (GGGGS) (SEQ ID NO: 31), amino acids 21-190 of murine ApoM, and a stop codon (STOP). FIG.11C show purified bA1M (4 μg / lane) separated by reducing 10% SDS-PAGE and stained with Coomassie brilliant blue. FIG.11D show representative FPLC elution profiles (OD 280 nm) of purified bA1M (right curve), and lipidated bA1M with S1P loading (bA1M-S1P, left curve). Elution profiles are representative of 3 independent experiments. FIG.11E shows titration analysis of bA1M-S1P-dependent enhancement of barrier function in HUVECs (8 μg / ml A1M contains ~ 100 nM S1P). N = 4 biological replicates per group. Data are the mean ± standard deviation. 12124565.1 **** p < 0.0001 by ordinary one-way ANOVA with Dunnett’s multiple comparisons test. FIG. 11F shows western blot analysis of 0.5 μl of plasma from 4 mice before and after bA1M injection (80 mg / kg) at 24 hours post-injection. ApoM antibody was applied to detect injected A1M and endogenous ApoM in plasma. FIG.11G shows FPLC-derived HDL fraction analyzed by Western blot with ApoM antibody. FIG.11H shows analysis of plasma for S1P content by electrospray ionization-MS / MS. FIG.11I shows analysis of FPLC-derived HDL fraction for S1P content by electrospray ionization-MS / MS. For FIGs.11H-11I, each data point represents data from an individual mouse. ** p < 0.01, **** p < 0.0001 by unpaired t-test. FIGs.12A-12E show suppression of the systemic inflammatory response induced by LPS by the ApoA1 moiety of bA1M. FIGs.12A-12C show the murine sepsis score (FIG.12A), body temperature (FIG.12B), and plasma IL-6 (FIG.12C) of mice injected intravenously with 40 mg / Kg of bA1M or PBS (vehicle) for 1 hour to allow for A1M lipidation in vivo before being injected intraperitoneally with 10 mg / Kg of LPS or saline. Groups were saline (N ≥ 6 mice), LPS alone (N ≥ 14 mice), and LPS with A1M (N ≥ 10 mice). Mice were assessed at 12 hours post-LPS injection. Data are presented as means ± standard deviation. *** p < 0.001, **** p < 0.0001 by one-way ANOVA with Tukey’s multiple comparisons test. FIG.12D shows Western blot analysis of 0.5 μl of plasma for ApoM from mice injected with LPS alone (10 mg / Kg) or LPS and bA1M (40 mg / Kg) at 12 hours post-injection. Each lane represents an individual mouse. FIG.12E shows plasma S1P content in mice injected with saline, LPS alone, or LPS and bA1M at 12 hours post-injection were analyzed by LC-MS / MS (n=4 mice per group). Data are presented as means ± standard deviation. *** p < 0.001 by one-way ANOVA with Tukey’s multiple comparisons test. DETAILED DESCRIPTION OF INVENTION The present disclosure relates to recombinant fusion proteins of Apolipoprotein A1 (ApoA1) and Apolipoprotein M (ApoM), methods of improving production capacity of ApoA1- ApoM (A1M) fusion proteins, and methods of use thereof. Additional aspects of this disclosure relates to methods of using unlipidated A1M fusion proteins to achieve a therapeutic effect, and high-yield protocols to produce active A1M fusion proteins from bacteria. ApoA1 and ApoM are high-density lipoprotein (HDL)-associated proteins that can act as chaperones for multiple biologically active lipids. Apolipoprotein A1 (ApoA1) is a 28-kDa protein encoded in humans by the APOA1 gene that is the major structural protein of HDL. ApoA1 sequences of this disclosure have been designed for bacterial cell expression or eukaryotic cell expression. In some embodiments, 12124565.1 ApoA1 comprises an amino acid sequence that is at least 70% identical to any one of SEQ ID NOs: 1, 3, or 5. For example, ApoA1 may comprise an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identical to any one of SEQ ID NOs: 1, 3, or 5. In some embodiments, ApoA1 comprises an amino acid sequence that is 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%, or 99% identical to any one of SEQ ID NOs: 1, 3, or 5. In some embodiments, ApoA1 is at least 90% identical to any one of SEQ ID NOs: 1, 3, or 5. In some embodiments, ApoA1 comprises the amino acid sequence of any one of SEQ ID NOs: 1, 3, or 5. In some embodiments, ApoA1 consists of the amino acid sequence of any one of SEQ ID NOs: 1, 3, or 5. Apolipoprotein M (ApoM) is a 26-kDa protein encoded in humans by the APOM gene that is mainly associated with high-density lipoprotein (HDL) in mammalian (e.g., human) plasma, with a small proportion present in triglyceride-rich lipoproteins (TGRLP) and low- density lipoproteins (LDL). It belongs to lipocalin protein superfamily. ApoM is only expressed in liver and in kidney and small amounts are found in fetal liver and kidney. Expression of native ApoM could be regulated by platelet activating factor (PAF), transforming growth factors (TGF), insulin-like growth factor (IGF) and leptin in vivo and / or in vitro. The ApoM may be from a human or a murine, such as a mouse. The amino acid sequences of wild-type human and mouse ApoM, and nucleotide sequences encoding such are provided in Table 1. The ApoM moiety acts as a chaperone for the biologically active sphingolipid, Sphingosine-1-Phosphate (S1P). ApoM sequences of this disclosure have been designed for bacterial cell expression or eukaryotic cell expression. In some embodiments, ApoM comprises an amino acid sequence that is at least 70% identical to any one of SEQ ID NOs: 2 or 4. For example, ApoM may comprise an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identical to any one of SEQ ID NOs: 2 or 4. In some embodiments, ApoM comprises an amino acid sequence that is 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%, or 99% identical to any one of SEQ ID NOs: 2 or 4. In some embodiments, ApoM comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs: 2 or 4. In some embodiments, ApoM comprises the amino acid sequence of any one of SEQ ID NOs: 2 or 4. In some embodiments, ApoM consists of the amino acid sequence of any one of SEQ ID NOs: 2 or 4. 12124565.1 A1M Fusion Proteins Aspects of this disclosure relate to a fusion protein comprising ApoA1 and ApoM (A1M). A “fusion protein” is a polypeptide comprising protein domains from at least two different proteins. One protein may be located at the amino-terminal (N-terminal) portion of the fusion protein or at the carboxy-terminal (C-terminal) portion of the fusion protein, thus forming an “amino-terminal fusion protein” or a “carboxy-terminal fusion protein”, respectively. A fusion protein may comprise different domains, for example, an ApoM domain and ApoA1 domain. In some embodiments, ApoA1 is fused to the N-terminus of ApoM. In some embodiments, ApoA1 is fused to the C-terminus of ApoM. Linkers The different domains of a fusion protein may be fused by end-to-end conjoining of the domains or via a linker. In some embodiments, ApoA1 and ApoM are fused via a linker. A “linker” is a chemical group or a molecule linking two molecules or moieties, e.g., two domains of a fusion protein. Typically, the linker is positioned between, or flanked by, two groups, molecules, domains, or other moieties and connected to each one via a covalent bond. In some embodiments, the linker is a flexible linker, a rigid linker, or a cleavable linker. The linker may be as simple as a covalent bond, or it may be a polymeric linker many atoms in length. In some embodiments, the linker is a polypeptide or based on amino acids. In some embodiments, the linker is not peptide-like. In some embodiments, the linker is a covalent bond (e.g., a carbon- carbon bond, disulfide bond, carbon-heteroatom bond, etc.). In some embodiments, the linker is a carbon-nitrogen bond of an amide linkage. In some embodiments, the linker is a cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic or heteroaliphatic linker. In some embodiments, the linker is polymeric (e.g., polyethylene, polyethylene glycol, polyamide, polyester, etc.). In some embodiments, the linker comprises a monomer, dimer, or polymer of aminoalkanoic acid. In some embodiments, the linker comprises an aminoalkanoic acid (e.g., glycine, ethanoic acid, alanine, beta-alanine, 3-aminopropanoic acid, 4-aminobutanoic acid, 5-pentanoic acid, etc.). In some embodiments, the linker comprises a monomer, dimer, or polymer of aminohexanoic acid (Ahx). In some embodiments, the linker is based on a carbocyclic moiety (e.g., cyclopentane, cyclohexane). In other embodiments, the linker comprises a polyethylene glycol moiety (PEG). In other embodiments, the linker comprises amino acids. In some embodiments, the linker comprises a peptide. In some embodiments, the linker comprises an aryl or heteroaryl moiety. In some embodiments, the linker is based on a phenyl ring. The linker may include functionalized moieties to facilitate attachment of a 12124565.1 nucleophile (e.g., thiol, amino) from the peptide to the linker. Any electrophile may be used as part of the linker. Exemplary electrophiles include, but are not limited to, activated esters, activated amides, Michael acceptors, alkyl halides, aryl halides, acyl halides, and isothiocyanates. Methods of choosing a suitable linker are known in the art, and may be chosen based on the desired function. For example, a linker may be chosen to increase stability and / or folding of a fusion protein, to increase expression of a fusion protein, to improve the biological activity of a fusion protein, or to enable targeting of a fusion protein. In some embodiments, the linker is an amino acid or a plurality of amino acids (e.g., a peptide or protein). In some embodiments, ApoA1 and ApoM are fused via a peptide linker. In some embodiments, the linker is a bond (e.g., a covalent bond), an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker is 1-100 amino acids in length, for example, 1, 2, 3, 4, 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, 30-35, 35-40, 40-45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-110, 110-120, 120-130, 130-140, 140-150, or 150-200 amino acids in length. Longer or shorter linkers are also contemplated. In some embodiments, the linker comprises the amino acid sequence of any one of SEQ ID NOs: 19-31 or a combination of any of these, wherein n is independently an integer between 1 and 30, and wherein X is any amino acid. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, the linker comprises SGSETPGTSESATPES (SEQ ID NO: 26), and SGGS (SEQ ID NO: 19). In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 7. Signal Peptides Fusion proteins of the present disclosure may further comprise a signal peptide. A “signal peptide” is a short peptide (usually between 16-30 amino acids in length) present at the N-terminus. A signal peptide can be a native signal peptide (e.g., the naturally occurring signal sequence found at the N-terminus of a protein in its native, unmodified form within an organism) or a non-native signal peptide (e.g., a signal peptide that has been modified to improve protein production, secretion, or other desired characteristics). In some embodiments, A1M further comprises a signal peptide sequence. In some embodiments, the signal peptide sequence comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, a signal peptide sequence is fused to the N-terminus of ApoA1. In some embodiments, the signal peptide sequence is fused to the N-terminus of ApoM. In some embodiments, A1M does not comprise a signal peptide sequence. 12124565.1 Translation Start Sites A fusion protein of the present disclosure may further comprise a translation start site sequence. A “translation start site” is a sequence that signals the beginning of protein translation and serves as the initiation point for the assembly of the ribosome and synthesis of the polypeptide chain. The translation start site serves as the initiation point for translation machinery, influences the reading frame, can impact the efficiency of translation initiation, protein synthesis, overall yield, and may improve expression system compatibility. Translation start site sequences may include the canonical Kozak motif (ACCAUGG) or the Shine-Dalgarno motif (AGGAGGU). Translation start site sequences may also be chosen based on the desired protein expression vector. For example, pET vector-derived translation start sites (e.g., pET15b- derived translation start sites) may include a T7 promoter sequence and a Shine-Dalgarno sequence. In some embodiments, A1M further comprises a translation start site sequence. In some embodiments, the translation start site sequence comprises the amino acid sequence of SEQ ID NO: 15. In some embodiments, the translation start site sequence is fused to the N- terminus of ApoA1. In some embodiments, the translation start site sequence is fused to the N- terminus of ApoM. In some embodiments, A1M does not comprise a translation start site sequence. Thus, in some embodiments, the A1M fusion protein comprises, from N-terminus to C- terminus: ApoA1, a linker, and ApoM. In some embodiments, an A1M fusion protein comprises, from N-terminus to C-terminus: a signal peptide, ApoA1, a linker, and ApoM. In some embodiments, the A1M fusion protein comprises, from N-terminus to C-terminus: a translation start site, ApoA1, a linker, and ApoM. In some embodiments, the A1M fusion protein comprises, from N-terminus to C-terminus: a signal peptide, ApoA1, a linker, ApoM, and an affinity tag. In some embodiments, the A1M fusion protein comprises, from N-terminus to C-terminus, a translation start site, an affinity tag, ApoA1, a linker, and ApoM. In some embodiments, the A1M fusion protein comprises, from N-terminus to C- terminus, the signal peptide as set forth in SEQ ID NO: 6, ApoA1 as set forth in SEQ ID NO: 5, the linker as set forth in SEQ ID NO: 7, and ApoM as set forth in SEQ ID NO: 4. In some embodiments, the A1M fusion protein comprises, from N-terminus to C-terminus, the signal peptide as set forth in SEQ ID NO: 6, ApoA1 as set forth in SEQ ID NO: 5, the linker as set forth in SEQ ID NO: 7, ApoM as set forth in SEQ ID NO: 4, and a 6x-His tag (HHHHHH). In some embodiments, the A1M fusion protein comprises the amino acid sequence of SEQ ID NO: 8. 12124565.1 In some embodiments, the A1M fusion protein comprises, from N-terminus to C- terminus, the translation start site as set forth in SEQ ID NO: 15, ApoA1 as set forth in SEQ ID NO: 3, the linker as set forth in SEQ ID NO: 7, and the ApoM as set forth in SEQ ID NO: 4. In some embodiments, the A1M fusion protein comprises, from N-terminus to C-terminus, the translation start site as set forth in SEQ ID NO: 15, a 7x-His tag (HHHHHHH) (SEQ ID NO: 34), ApoA1 as set forth in SEQ ID NO: 3, the linker as set forth in SEQ ID NO: 7, and the ApoM as set forth in SEQ ID NO: 4. In some embodiments, the A1M fusion protein comprises the amino acid sequence of SEQ ID NO: 9. In some embodiments, the A1M fusion protein comprises, from N-terminus to C- terminus, the translation start site as set forth in SEQ ID NO: 15, a 7x-His tag (HHHHHH) (SEQ ID NO: 34), ApoA1 as set forth in SEQ ID NO: 1, the linker as set forth in SEQ ID NO: 7, and the ApoM as set forth in SEQ ID NO: 2. In some embodiments, the A1M fusion protein comprises, from N-terminus to C-terminus, the translation start site as set forth in SEQ ID NO: 15, ApoA1 as set forth in SEQ ID NO: 1, the linker as set forth in SEQ ID NO: 7, and the ApoM as set forth in SEQ ID NO: 2. In some embodiments, the A1M fusion protein comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, A1M comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of any one of SEQ ID NOs: 8-10. In some embodiments, A1M comprises an amino acid sequence that is 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%, or 99% identical to any one of SEQ ID NOs: 8-10. In some embodiments, A1M comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 8-10. In some embodiments, A1M comprises the amino acid sequence of any one of SEQ ID NOs: 8-10. In some embodiments, A1M comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, A1M comprises the amino acid sequence of SEQ ID NO: 9. In some embodiments, A1M comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, A1M consists of the amino acid sequence of any one of SEQ ID NOs: 8-10. A1M Lipoproteins In some embodiments, this disclosure provides a lipoprotein comprising a fusion protein (e.g., an A1M fusion protein) and a therapeutic. In some embodiments, the therapeutic is a lipid. In some embodiments, the therapeutic interacts with a lipid receptor. In some embodiments, the 12124565.1 therapeutic is selected from the group consisting of S1P receptor agonists or antagonists; or prostaglandin agonists or antagonists; or combination thereof. S1P receptor agonists and antagonists are well known in the art and are described in Park SJ et al. Biomolecules & therapeutics 25.1 (2017): 80. In some embodiments, the S1P receptor antagonist is MT-1303 or JTE-013. In some embodiments, the S1P receptor agonist is selected from the group consisting of SEW2871, KRP-203, Siponimod (BAF312), AUY954, Ponesimod (ACT-128800), Ceralifimod (ONO-4641), GSK2018682, Ozanimod (RPC1063), CS-0777, and Fingolimod (FTY720, Gilenya). In some embodiments, the therapeutic inhibits production of prostaglandins. In some embodiments, the therapeutic is a non-steroidal anti-inflammatory drug that inhibits prostaglandin production. Prostaglandin receptor agonists and antagonists are well known in the art and are described in Sharif NA et al. British journal of pharmacology 176.8 (2019): 1059-1078. In some embodiments, the prostaglandin receptor agonist is selected from the group consisting of Cloprostenol, Fluprostenol (travoprost acid), 16-Phenoxy-ω-tetranor-PGF2α, 17-Phenyl-ω- trinor-PGF2α (bimatoprost acid), 13,14-Dihydro-17-phenyl-ω-trinor-PGF2α (latanoprost-free acid) (PhXA85), AFP-172 (tafluprost acid), and AL-12182 acid (AL-12180). In some embodiments, the prostaglandin receptor antagonist is selected from the group consisting of PGF2α dimethylamide, PGF2α dimethylamine, Phloretin, Glibenclamide, tolbutamide, AL- 8810, AL-3138, AS604872, THG-113.31, PDC113.824, AL-8810, and AGN 211377. In some embodiments, the lipoprotein comprises a fusion protein (e.g., an A1M fusion protein) and a lipid. In some embodiments, the lipid is selected from the group consisting of prostaglandin, sphingosine 1-phosphate (S1P), a leukotriene, and phosphatidyl choline. In some embodiments, the prostaglandin is Iloprost. In some embodiments, the prostaglandin is selected from the group consisting of prostaglandin E2 (PGE2), prostacyclin (PGI2), prostaglandin D2 (PGD2), and prostaglandin F2α (PGF2α). In some embodiments, the phosphatidyl choline is 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or 1,2-dimyristoyl-sn-glycero-3- phosphoglycerol (DMPG). In some embodiments, the phosphatidyl choline is 1,2- Dipalmitoylphosphatidylcholinee (DPPC) or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). In some embodiments, the lipid is S1P. In some embodiments, the lipid is 1,2- dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or 1,2-dimyristoyl-sn-glycero-3- phosphoglycerol (DMPG). In some embodiments, the lipid binds to ApoA1 of the fusion protein. In some embodiments, the lipoprotein is non-covalently bound to the lipid. In some embodiments, the lipoprotein is covalently bound to the lipid. 12124565.1 In some embodiments, a fusion protein (e.g., an A1M fusion protein) comprises at least 60 mol% of a lipid (e.g., S1P or Iloprost). In some embodiments, the fusion protein (e.g., an A1M fusion protein) comprises about 60 mol%, about 61 mol%, about 62 mol%, about 63 mol%, about 64 mol%, about 65 mol%, about 66 mol%, about 67 mol%, about 68 mol%, about 69 mol%, or about 70 mol% of a lipid (e.g., S1P or Iloprost). In some embodiments, the fusion protein (e.g., an A1M fusion protein) comprises at least 60 mol%, at least 65 mol%, at least 70 mol%, at least 75 mol%, at least 80 mol%, at least 85 mol%, at least 90 mol%, at least 95 mol%, at least 99 mol%, or 100 mol% of a lipid (e.g., S1P or Iloprost). In some embodiments, the fusion protein (e.g., an A1M fusion protein) comprises less than 5 mol% of a lipid (e.g., S1P). In some embodiments, the fusion protein (e.g., an A1M fusion protein) comprises less than 1 mol% of a lipid (e.g., S1P). In some embodiments, the fusion protein (e.g., an A1M fusion protein) comprises about 1 mol%, 0.9 mol%, 0.8 mol%, 0.7 mol%, 0.6 mol%, 0.5 mol%, 0.4 mol%, 0.3 mol%, 0.2 mol%, 0.1 mol%, or 0 mol% of a lipid (e.g., S1P). Methods of profiling lipoproteins are known and may include techniques such as ultracentrifugation, liquid chromatography (e.g., fast protein liquid chromatography (FPLC), ultra performance liquid chromatography (UPLC)), mass spectrometry (e.g., electrospray ionization tandem mass spectrometry (ESI-MS), matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS)), nuclear magnetic resonance (NMR), or combinations thereof. Nanoparticles In some embodiments, the lipoprotein as described herein is incorporated into a nanoparticle. In some embodiments, the fusion protein as described herein is incorporated into a nanoparticle. In some embodiments, the nanoparticle is a liposome. In some embodiments, the nanoparticle is a nanodisk. A nanodisk is a discoidal particle comprising a lipid bilayer and proteins that form disc-like or spherical structure, where the proteins encircle the lipid bilayer. In some embodiments, the lipoprotein incorporated into a nanoparticle (e.g., a nanodisk) is bound to a phospholipid (e.g., lipidated lipoprotein). In some embodiments, the fusion protein incorporated into a nanoparticle is bound to a phospholipid (e.g., lipidated fusion protein). In some embodiments, the phospholipid is phosphatidyl choline. In some embodiments, the phosphatidyl choline is 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2- dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG). In some embodiments, the ratio of DMPC:DMPG is 7:3 molar ratio. In some embodiments, the nanoparticle comprises at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 12124565.1 80%, at least 90%, at least 95%, or at least 99% unlipidated fusion protein. In some embodiments, the nanoparticle comprises 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%- 60%, 60%-70%, 70%-80%, 80%-90%, or 90%-100% of unlipidated fusion protein compared to lipidated fusion protein. In some embodiments, the nanoparticle comprises a ratio of lipidated fusion protein to unlipidated fusion protein of 1:10, 1:7, 1:5, 1:3, 1:2, 1:1, 2:1, 3:1, 5:1, 7:1, or 10:1. In some embodiments, the nanoparticle comprises 3 lipidated fusion proteins for every 7 unlipidated fusion proteins. In some embodiments, the nanoparticle comprises 30% lipidated fusion protein and 70% unlipidated fusion protein. In some embodiments, the nanoparticle comprises a fusion protein bound to a therapeutic as described herein. In some embodiments, the nanoparticle comprises a therapeutic as described herein. In some embodiments, lipidation of a fusion protein or a lipoprotein occurs in vitro (e.g. prior to administration to a subject). In some embodiments, lipidation of a fusion protein or a lipoprotein does not occur in vitro. In some embodiments, lipidation of a fusion protein or a lipoprotein occurs in vivo (e.g., after administration to a subject). In some embodiments, lipidation of a fusion protein of a lipoprotein in vitro produces HDL-like nanoparticles. In some embodiments, the nanoparticle is an HDL-like nanoparticle. An HDL-like nanoparticle is a non- naturally occurring (e.g., synthetic) nanoparticle that mimics one or more features (e.g., composition, surface properties, size) of naturally-occurring HDLs. For example, the structural diversity of HDL-like nanoparticles can vary in terms of the composition of nanoparticle core (e.g., cholesterol esters, inorganic scaffolds), the shape (e.g., discoidal, spherical), the protein associated with the nanoparticle (e.g., ApoA1), the phospholipids (e.g., DOPC, DMPG, DMPC), and the lipid layer surrounding the nanoparticle (e.g., monolayer, bilayer). In addition, HDL-like nanoparticles can be used for drug delivery by, for instance, modifying the mechanism of drug loading (e.g., covalent attachment, encapsulation, integration in lipid layer). In some embodiments, the HDL-like nanoparticle is between 6-14 nm, between 7-13 nm, between 8-12 nm, between 8-13 nm, between 9-13 nm, between 9-12 nm, between 7-9 nm, between 7-8 nm, or between 8-9 nm in diameter. In some embodiments, the HDL-like nanoparticle is around 7 nm, 7.5 nm, 8 nm, 8.5 nm, 9 nm, 9.5 nm, 10 nm, 10.5 nm, 11 nm, 11.5 nm, 12 nm, or 12.5 nm in diameter. In some embodiments, the HDL-like nanoparticle is between 8-12 nm in diameter. Modified Fusion Proteins In some embodiments, the fusion protein described herein comprises a modification. Proteins or polypeptides comprising modifications have additional features other than amino acid contents. As used herein, a “modification” or "derivative" of a protein or polypeptide (e.g., 12124565.1 a fusion protein described herein) produces a modified or derivatized polypeptide, which is a form of a given peptide that is chemically modified relative to the reference peptide, the modification including, but not limited to, oligomerization or polymerization, modifications of amino acid residues or peptide backbone, cross-linking, cyclization, conjugation, PEGylation, glycosylation, acetylation, phosphorylation, acylation, carboxylation, lipidation, thioglycolic acid amidation, alkylation, methylation, polyglycylation, glycosylation, polysialylation, adenylylation, PEGylation, fusion to additional heterologous amino acid sequences, or other modifications that alter the stability, solubility, or other properties of the peptide while substantially retaining the activity of the polypeptides described herein. It is to be understood that a fusion protein comprising such modifications, are cross-linked, cyclized, conjugated, acylated, carboxylated, lipidated, acetylated, thioglycolic acid amidated, alkylated, methylated, polyglycylated, glycosylated, polysialylated, phosphorylated, adenylylated, PEGylated, or combination thereof. In some embodiments, a modified fusion protein of the present disclosure may contain non-amino acid elements, such as polyethylene glycols, lipids, poly- or mono- saccharide, and phosphates. A fusion protein of the present disclosure may comprise the modifications disclosed herein at the C-terminus (e.g., C-terminal amidation), N-terminus (e.g., N-terminal acetylation). Terminal modifications are useful, and are well known, to reduce susceptibility to proteinase digestion, and therefore serve to prolong half-life of the polypeptides in solutions, particularly biological fluids where proteases may be present. In some embodiments, the fusion proteins described herein are further modified within the sequence, such as, modification by terminal-NH2 acylation, e.g., acetylation, or thioglycolic acid amidation, by terminal-carboxylamidation, e.g., with ammonia, methylamine, and the like terminal modifications. Terminal modifications are useful, to reduce susceptibility by proteinase digestion, and therefore can serve to prolong half-life of the polypeptides in solution, particularly in biological fluids where proteases may be present. Amino terminus modifications include methylation (e.g., --NHCH3 or --N(CH3)2), acetylation (e.g., with acetic acid or a halogenated derivative thereof such as a-chloroacetic acid, a-bromoacetic acid, or a-iodoacetic acid), adding a benzyloxycarbonyl (Cbz) group, or blocking the amino terminus with any blocking group containing a carboxylate functionality defined by RCOO-- or sulfonyl functionality defined by R--SO2--, where R is selected from the group consisting of alkyl, aryl, heteroaryl, alkyl aryl, and the like, and similar groups. One can also incorporate a desamino acid at the N-terminus (so that there is no N-terminal amino group) to decrease susceptibility to proteases or to restrict the 12124565.1 conformation of the polypeptide. In certain embodiments, the N-terminus is acetylated with acetic acid or acetic anhydride. Carboxy terminus modifications include replacing the free acid with a carboxamide group or forming a cyclic lactam at the carboxy terminus to introduce structural constraints. One can also cyclize the peptides described herein, or incorporate a desamino or descarboxy residue at the termini of the peptide, so that there is no terminal amino or carboxyl group, to decrease susceptibility to proteases or to restrict the conformation of the peptide. Methods of circular peptide synthesis are known in the art, for example, in U.S. Patent Application No. 20090035814; Muralidharan and Muir, 2006, Nat Methods, 3:429-38; and Lockless and Muir, 2009, Proc Natl Acad Sci U S A. Jun 18, Epub. C-terminal functional groups of the peptides described herein include amide, amide lower alkyl, amide di(lower alkyl), lower alkoxy, hydroxy, and carboxy, and the lower ester derivatives thereof, and the pharmaceutically acceptable salts thereof. In some embodiments, the fusion proteins described herein are phosphorylated. One can also readily modify peptides by phosphorylation, and other methods (e.g., as described in Hruby, et al. (1990) Biochem J.268:249-262). In some embodiments, one can also replace the naturally occurring side chains of the genetically encoded amino acids (or the stereoisomeric D amino acids) with other side chains, for instance with groups such as alkyl, lower (C1-6) alkyl, cyclic 4-, 5-, 6-, to 7-membered alkyl, amide, amide lower alkyl amide di(lower alkyl), lower alkoxy, hydroxy, carboxy and the lower ester derivatives thereof, and with 4-, 5-, 6-, to 7-membered heterocycles. For example, proline analogues in which the ring size of the proline residue is changed from 5 members to 4, 6, or 7 members can be employed. Cyclic groups can be saturated or unsaturated, and if unsaturated, can be aromatic or non-aromatic. Heterocyclic groups preferably contain one or more nitrogen, oxygen, and / or sulfur heteroatoms. Examples of such groups include the furazanyl, furyl, imidazolidinyl, imidazolyl, imidazolinyl, isothiazolyl, isoxazolyl, morpholinyl (e.g. morpholino), oxazolyl, piperazinyl (e.g., 1-piperazinyl), piperidyl (e.g., 1-piperidyl, piperidino), pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolidinyl (e.g., 1- pyrrolidinyl), pyrrolinyl, pyrrolyl, thiadiazolyl, thiazolyl, thienyl, thiomorpholinyl (e.g., thiomorpholino), and triazolyl groups. These heterocyclic groups can be substituted or unsubstituted. Where a group is substituted, the substituent can be alkyl, alkoxy, halogen, oxygen, or substituted or unsubstituted phenyl. In some embodiments, the fusion proteins described herein may be attached to one or more polymer moieties. In some embodiments, these polymers are covalently attached to the 12124565.1 fusion proteins of the disclosure. In some embodiments, for therapeutic use of the end product preparation, the polymer is pharmaceutically acceptable. One skilled in the art will be able to select the desired polymer based on such considerations as whether the polymer-peptide conjugate will be used therapeutically, and if so, the desired dosage, circulation time, resistance to proteolysis, and other considerations. Suitable polymers include, for example, polyethylene glycol (PEG), polyvinyl pyrrolidone, polyvinyl alcohol, polyamino acids, divinylether maleic anhydride, N-(2- Hydroxypropyl)-methacrylamide, dextran, dextran derivatives including dextran sulfate, polypropylene glycol, polyoxyethylated polyol, heparin, heparin fragments, polysaccharides, cellulose and cellulose derivatives, including methylcellulose and carboxymethyl cellulose, starch and starch derivatives, polyalkylene glycol and derivatives thereof, copolymers of polyalkylene glycols and derivatives thereof, polyvinyl ethyl ethers, and α,β-Poly[(2- hydroxyethyl)-DL-aspartamide, and the like, or mixtures thereof. Such a polymer may or may not have its own biological activity. The polymers can be covalently or non-covalently conjugated to the fusion protein. Methods of conjugation for increasing serum half-life and for radiotherapy are known in the art, for example, in U.S. Pat. Nos.: 5,180,816, 6,423,685, 6,884,780, and 7,022,673. In some embodiments, the fusion protein described herein may be attached to one or more water soluble polymer moieties. The water soluble polymer may be, for example, polyethylene glycol (PEG), copolymers of ethylene glycol / propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), poly(n-vinyl-pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, and polyoxyethylated polyols. A preferred water soluble polymer is PEG. The polymer may be of any molecular weight, and may be branched or unbranched. The average molecular weight of the reactant PEG is preferably between about 3,000 and about 50,000 Daltons (the term "about" indicating that in preparations of PEG, some molecules will weigh more, and some less, than the stated molecular weight). More preferably, the PEG has a molecular weight of from about 10 kDa to about 40 kDa, and even more preferably, the PEG has a molecular weight from 15 to 30 kDa. Other sizes may be used, depending on the desired therapeutic profile (e.g., duration of sustained release desired; effects, if any, on biological activity; ease in handling; degree or lack of antigenicity; and other effects of PEG on a therapeutic peptide known to one skilled in the art). 12124565.1 The number of polymer molecules attached may vary; for example, one, two, three, or more water-soluble polymers may be attached to a peptide of the disclosure. The multiple attached polymers may be the same or different chemical moieties (e.g., PEGs of different molecular weight). In certain embodiments, PEG may be attached to at least one terminus (N-terminus or C- terminus) of the fusion protein described herein. In some embodiments, PEG may be attached to a linker moiety of the fusion protein. In some embodiments, the linker contains more than one reactive amine capable of being derivatized with a suitably activated PEG species. PEGylation is routinely achieved by incubation of a reactive derivative of PEG with the target macromolecule. The covalent attachment of PEG to a drug or therapeutic protein can "mask" the agent from the host's immune system (reduced immunogenicity and antigenicity), and increase the hydrodynamic size (size in solution) of the agent which prolongs its circulatory time by reducing renal clearance. PEGylation can also provide water solubility to hydrophobic drugs and proteins. PEGylation, by increasing the molecular weight of a molecule, can impart several significant pharmacological advantages over the unmodified form, such as: improved drug solubility, reduced dosage frequency, without diminished efficacy with potentially reduced toxicity, extended circulating life, increased drug stability, and enhanced protection from proteolytic degradation. In addition, PEGylated drugs have wider opportunities for new delivery formats and dosing regimens. Methods of PEGylating molecules, proteins and peptides are well known in the art, e.g., as described in U. S. Patent No.5,766,897; 7,610,156; 7,256,258 and the International Application No. WO / 1998 / 032466. Encompassed herein are conjugates of a fusion protein described herein. The fusion proteins can be conjugated to other polymers in addition to polyethylene glycol (PEG). The polymer may or may not have its own biological activity. Further examples of polymer conjugation include but are not limited to polymers such as polyvinyl pyrrolidone, polyvinyl alcohol, polyamino acids, divinylether maleic anhydride, N-(2-Hydroxypropyl)-methacrylamide, dextran, dextran derivatives including dextran sulfate, polypropylene glycol, polyoxyethylated polyol, heparin, heparin fragments, polysaccharides, cellulose and cellulose derivatives, including methylcellulose and carboxymethyl cellulose, starch and starch derivatives, polyalkylene glycol and derivatives thereof, copolymers of polyalkylene glycols and derivatives thereof, polyvinyl ethyl ethers, and α,β-Poly[(2-hydroxyethyl)-DL-aspartamide, and the like, or mixtures thereof. Conjugation to a polymer can improve serum half-life, among other effects. A variety of chelating agents can be used to conjugate the peptides described herein. These chelating agents include but are not limited to ethylenediaminetetraacetic acid (EDTA), 12124565.1 diethylenetriaminopentaacetic acid (DTPA), ethyleneglycol-0,0′-bis(2-aminoethyl)-N,N,N′,N′- tetraacetic acid (EGTA), N,N′-bis(hydroxybenzyl)ethylenediamine-N,N′-diacetic acid (HBED), triethylenetetraminehexaacetic acid (TTHA), 1,4,7,10-tetra-azacyclododecane-N,N′,N″,N′″- tetraacetic acid (DOTA), 1,4,7,10-tetraazacyclotridecane- 1,4,7,10-tetraacetic acid (TITRA), 1,4,8,11-tetraazacyclotetradecane-N,N′,N″,N′″-tetraacetic acid (TETA), and 1,4,8,11- tetraazacyclotetradecane (TETRA). Methods of conjugation are well known in the art, for example, P. E. Thorpe, et. al, 1978, Nature 271, 752 – 755; Harokopakis E., et. al., 1995, Journal of Immunological Methods, 185:31-42; S. F. Atkinson, et. al., 2001, J. Biol. Chem., 276:27930- 27935; and U. S Pat. Nos.:5,601,825, 5,180,816, 6,423,685, 6,706,252, 6,884,780, and 7,022,673. Other methods for stabilizing peptides known in the art may be used with the methods and compositions described herein. For example, using D-amino acids, using reduced amide bonds for the peptide backbone, and using non-peptide bonds to link the side chains, including, but not limited to, pyrrolinone and sugar mimetics can each provide stabilization. The design and synthesis of sugar scaffold peptide mimetics are described by Hirschmann et al. (J. Med. Chem., 1996, 36, 2441-2448). Further, pyrrolinone-based peptide mimetics present the peptide pharmacophore on a stable background that has improved bioavailability characteristics (see, for example, Smith et al., J. Am. Chem. Soc.2000, 122, 11037-1103). All combinations of the different modifications and derivatizations are envisioned for the fusion protein described herein. Modifications, derivatives and methods of reprivatizing polypeptides are described in Published International Application WO 2010 / 014616. Methods of Producing Fusion Proteins Other aspects of this disclosure provide methods of producing a fusion protein described herein. The fusion protein will generally be produced by expression from recombinant nucleic acids in appropriate cells (e.g., bacterial cells or eukaryotic cells) and isolated. To produce the fusion protein, nucleic acids encoding the fusion protein may be introduced to a cell (e.g., a bacterial cell or a eukaryotic cell). The cells may be cultured under conditions that allow the fusion protein to express from the nucleic acids encoding the fusion protein. Fusion proteins comprising a signal peptide can be secreted, e.g., into the culturing media and can subsequently be recovered. The fusion protein may be isolated using any methods of purifying a protein known in the art, or by methods described below. Nucleic Acids Encoding Fusion Proteins 12124565.1 The nucleic acids encoding the fusion proteins described herein may be obtained, and the nucleotide sequence of the nucleic acids determined, by any method known in the art. Non- limiting exemplary nucleotide sequences encoding the fusion protein or variants described herein are provided in Table 2, e.g., SEQ ID NOs: 11, 17, or 18. In some embodiments, the nucleic acid sequence encoding an A1M fusion protein is at least 70% (e.g., at least 70%, 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%) identical to SEQ ID NO: 11, 17, or 18. In some embodiments, the nucleic acid sequence encoding an A1M fusion protein is at least 95% identical to any one of SEQ ID NOs: 11, 17, or 18. In some embodiments, the nucleic acid sequence encoding an A1M fusion protein comprises SEQ ID NO: 11, 17, or 18. In some embodiments, the nucleic acid sequence encoding an A1M fusion protein consists of SEQ ID NO: 11, 17, or 18. One skilled in the art is able to identify the nucleotide sequence encoding the fusion protein from the amino acid sequence of the fusion protein. The nucleic acids encoding the fusion protein of the present disclosure, may be DNA or RNA, double-stranded or single stranded. In some embodiments, the nucleotide sequence encoding the fusion protein may be codon optimized to adapt to different expression systems (e.g., for mammalian expression or for bacterial expression). In some embodiments, the nucleic acid is comprised within a nucleic acid construct. In some embodiments, the construct is a plasmid or a vector such as an expression vector. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a viral expression vector. In some embodiments, the vector comprises a promoter operably linked to the nucleic acid. In some embodiments, the vector comprises a translation start site sequence operably linked to the nucleic acid. In some embodiments, the translation start site sequence comprises a nucleotide sequence that is at least 70% (e.g., at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%) identical to SEQ ID NO: 6. In some embodiments, the translation start site sequence comprises the nucleotide sequence of SEQ ID NO: 6. In some embodiments, the translation start site sequence consists of the nucleotide sequence of SEQ ID NO: 6. In some embodiments, the vector comprises an affinity tag for protein purification. Promoters A variety of promoters can be used for expression of a fusion protein described herein, including, but not limited to, cytomegalovirus (CMV) intermediate early promoter, a viral LTR such as the Rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR, the simian virus 40 (SV40) early promoter, E. coli lac UV5 promoter, and the herpes simplex tk virus promoter. Regulatable 12124565.1 promoters can also be used, such as those using the lac repressor from E. coli as a transcription modulator to regulate transcription from lac operator-bearing mammalian cell promoters [Brown, M. et al., Cell, 49:603-612 (1987)], those using the tetracycline repressor (tetR) [Gossen, M., and Bujard, H., Proc. Natl. Acad. Sci. USA 89:5547-5551 (1992); Yao, F. et al., Human Gene Therapy, 9:1939-1950 (1998); Shockelt, P., et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526 (1995)]. Other systems include FK506 dimer, VP16 or p65 using astradiol, RU486, diphenol murislerone, or rapamycin. Inducible systems are available from Invitrogen, Clontech and Ariad. Regulatable promoters that include a repressor with the operon can be used. In one embodiment, the lac repressor from Escherichia coli can function as a transcriptional modulator to regulate transcription from lac operator-bearing mammalian cell promoters [M. Brown et al., Cell, 49:603-612 (1987)]; Gossen and Bujard (1992); [M. Gossen et al., Natl. Acad. Sci. USA, 89:5547-5551 (1992)] combined the tetracycline repressor (tetR) with the transcription activator (VP 16) to create a tetR-mammalian cell transcription activator fusion protein, tTa (tetR-VP 16), with the tetO-bearing minimal promoter derived from the human cytomegalovirus (hCMV) major immediate-early promoter to create a tetR-tet operator system to control gene expression in mammalian cells. In one embodiment, a tetracycline inducible switch is used (Yao et al., Human Gene Therapy; Gossen et al., Natl. Acad. Sci. USA, 89:5547-5551 (1992); Shockett et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526 (1995)). Affinity Tags Affinity tags are short peptide sequences (typically between 6-30 amino acids in length) that are fused to a protein of interest (e.g., an A1M fusion protein) to facilitate the purification and detection of the protein of interest. An affinity tag may be fused to the N-terminus or C- terminus of a protein of interest (e.g., an A1M fusion protein). In some embodiments, an affinity tag is fused to the N-terminus of an A1M fusion protein described herein. In some embodiments, an affinity tag is fused to the C-terminus of an A1M fusion protein described herein. Non- limiting examples of affinity tags include albumin-binding protein, alkaline phosphatase, AU1 epitope (DTYRYI) (SEQ ID NO: 35), AU5 epitope (TDFYLK) (SEQ ID NO: 36), bacteriophage T7 epitope (MASMTGGQQMG) (SEQ ID NO: 37), bacteriophage V5 epitope (GKPIPNPLLGLDST) (SEQ ID NO: 38), Biotin-carboxy carrier protein, bluetongue virus tag (QYPALT) (SEQ ID NO; 39), calmodulin binding peptide, chloramphenicol acetyl transferase, cellulose binding domain, chitin binding domain, choline-binding domain, dihydrofolate reductase, E2 epitope (SSTSSDFRDR) (SEQ ID NO: 40), FLAG epitope (DYKDDDK) (SEQ 12124565.1 ID NO: 41), Galactose-binding protein, green fluorescent protein, Glu-Glu tag (EYMPME (SEQ ID NO: 42) or EFMPME (SEQ ID NO: 43)), glutathione S-transferase, human influenza hemagglutinin, HaloTag®, histidine affinity tag (KDHLIHNVHKEFHAHAHNK) (SEQ ID NO: 44), horseradish peroxidase, HSV epitope (QPELAPED) (SEQ ID NO: 45), ketosteroid isomerase, KT3 epitope (KPPTPPPEPET) (SEQ ID NO: 46), LacZ, luciferase, maltose-binding protein, Myc epitope (CEQKLISEEDL) (SEQ ID NO: 47), NusA, PDZ domain, PDZ ligand, polyarginine (RRRRR (SEQ ID NO: 48) or RRRRRR (SEQ ID NO: 49)), polyaspartate (Dn, where n = 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 (SEQ ID NO: 50)), polycysteine (CCCC) (SEQ ID NO: 51), polyhistidine (Hn, where n = 2, 3, 4, 5, 6, 7, 8, 9, or 10) (SEQ ID NO: 52), polyphenylalanine (FFFFFFFFFFF) (SEQ ID NO: 53), protein C, S1-tag (NANNPDWDF) (SEQ ID NO: 54), S-tag (KETAAAKFERQHMDS) (SEQ ID NO: 55), streptavidin-binding peptide, Staphylococcal protein A, Staphylococcal protein G, Strep-tag (WSHPQFEK (SEQ ID NO: 56) or AWAHPQPGG (SEQ ID NO: 57)), streptavidin, small ubiquitin-like modifier, T7 epitope, thioredoxin, TrpE, ubiquitin, universal (HTTPHH) (SEQ ID NO: 58), VSV-6 (YTDIEMNRLGK) (SEQ ID NO: 59), and combinations thereof. In some embodiments, an 6x- His tag (HHHHHH) (SEQ ID NO: 32) is fused to the N-terminus of an A1M fusion protein described herein. In some embodiments, a 7x-His tag (HHHHHHH) (SEQ ID NO: 34) is fused to the N-terminus of an A1M fusion protein described herein. In some embodiments, an 6x-His tag (HHHHHH) (SEQ ID NO: 32) is fused to the C-terminus of an A1M fusion protein described herein. In some embodiments, a 7x-His tag (HHHHHHH) (SEQ ID NO: 34) is fused to the C-terminus of an A1M fusion protein described herein. Thus, in some embodiments, A1M comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of any one of SEQ ID NOs: 12-14, wherein X is any affinity tag described herein (e.g., a 6x-His tag or a 7x-His tag) . In some embodiments, A1M comprises an amino acid sequence that is 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%, or 99% identical to any one of SEQ ID NOs: 12-14, wherein X is any affinity tag described herein (e.g., a 6x-His tag or a 7x-His tag). In some embodiments, A1M comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 12-14, wherein X is any affinity tag described herein (e.g., a 6x-His tag or a 7x-His tag). In some embodiments, A1M comprises the amino acid sequence of any one of SEQ ID NOs: 12-14, wherein X is any affinity tag described herein (e.g., a 6x-His tag or a 7x- His tag). In some embodiments, A1M comprises the amino acid sequence of SEQ ID NO: 12, wherein X is any affinity tag described herein (e.g., a 6x-His tag or a 7x-His tag). In some 12124565.1 embodiments, A1M comprises the amino acid sequence of SEQ ID NO: 13, wherein X is any affinity tag described herein (e.g., a 6x-His tag or a 7x-His tag). In some embodiments, A1M comprises the amino acid sequence of SEQ ID NO: 14, wherein X is any affinity tag described herein (e.g., a 6x-His tag or a 7x-His tag). In some embodiments, A1M consists of the amino acid sequence of any one of SEQ ID NOs: 12-14, wherein X is any affinity tag described herein (e.g., a 6x-His tag or a 7x-His tag). In some embodiments, a vector described herein may further comprise a protease cleavage site. A protease cleavage site refers to a specific amino acid sequence within a protein that is recognized and cleaved by a protease enzyme. In recombinant proteins, a protease cleavage site is often situated between an affinity tag and the protein of interest (e.g., an A1M fusion protein) and facilitates the removal of the affinity tag after purification. Non-limiting examples of protease cleavage sites include thrombin (LVPRGS) (SEQ ID NO: 60), factor Xa, PreScission, enterokinase, genenase I, and TEV. Additionally, the vector can contain, for example, some or all of the following: a selectable marker gene, such as the neomycin gene for selection of stable or transient transfectants in mammalian cells; enhancer / promoter sequences from the immediate early gene of human CMV for high levels of transcription; transcription termination and RNA processing signals from SV40 for mRNA stability; SV40 polyoma origins of replication and ColE1 for proper episomal replication; internal ribosome binding sites (IRESes), versatile multiple cloning sites; and T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA. Suitable vectors and methods for producing vectors containing transgenes are well known and available in the art. An expression vector comprising the nucleic acid can be transferred to a host cell by conventional techniques (e.g., electroporation, liposomal transfection, and calcium phosphate precipitation) and the transfected cells are then cultured by conventional techniques to produce the fusion proteins described herein. In some embodiments, the expression of the fusion proteins described herein is regulated by a constitutive, an inducible or a tissue-specific promoter. Cells Comprising a Fusion Protein The host cells used to express the fusion proteins described herein may be either prokaryotic cells, like bacterial cells (e.g., Escherichia coli cells) or eukaryotic cells (e.g., mammalian cells, such as Chinese hamster ovary (CHO) cells). A variety of host-expression 12124565.1 vector systems may be utilized to express the fusion proteins described herein. Such host- expression systems represent vehicles by which the coding sequences of the isolated fusion proteins described herein may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express the fusion proteins described herein in situ. These include, but are not limited to, microorganisms such as bacteria (e.g., E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing coding sequences for the fusion proteins described herein; yeast (e.g., Saccharomyces pichia) transformed with recombinant yeast expression vectors containing sequences encoding the fusion proteins described herein; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing the sequences encoding the fusion proteins described herein; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus (CaMV) and tobacco mosaic virus (TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing sequences encoding the fusion proteins described herein; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 293T, 3T3 cells, lymphocytic cells (see U.S. Pat. No.5,807,715), Per C.6 cells (human retinal cells developed by Crucell) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter). In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the fusion proteins being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of fusion proteins described herein, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Rüther et al. (1983) “Easy Identification Of cDNA Clones,” EMBO J.2:1791-1794), in which the coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye et al. (1985) “Up-Promoter Mutations In The lpp Gene Of Escherichia Coli,” Nucleic Acids Res.13:3101-3110; Van Heeke et al. (1989) “Expression Of Human Asparagine Synthetase In Escherichia Coli,” J. Biol. Chem.24:5503-5509); and the like. pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to a matrix glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to 12124565.1 include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety. In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The coding sequence may be cloned individually into non-essential regions (e.g., the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (e.g., the polyhedrin promoter). In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, the coding sequence of interest may be ligated to an adenovirus transcription / translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable and capable of expressing the immunoglobulin molecule in infected hosts (e.g., see Logan et al. (1984) “Adenovirus Tripartite Leader Sequence Enhances Translation Of mRNAs Late After Infection,” Proc. Natl. Acad. Sci. USA 81:3655-3659). Specific initiation signals may also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bitter et al. (1987) “Expression And Secretion Vectors For Yeast,” Methods in Enzymol.153:516-544). In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Purification and modification of recombinant proteins is well known in the art such that the design of the polyprotein precursor could include a number of embodiments readily appreciated by a skilled worker. Any known proteases or peptidases known in the art can be used for the described modification of the precursor molecule, e.g., thrombin or factor Xa (Nagai et al. (1985) “Oxygen Binding Properties Of Human Mutant Hemoglobins Synthesized In Escherichia Coli,” Proc. Nat. Acad. Sci. USA 82:7252-7255, and reviewed in Jenny et al. (2003) “A Critical Review Of The Methods For Cleavage Of Fusion Proteins With Thrombin And Factor Xa,” Protein Expr. Purif.31:1-11), enterokinase (Collins-Racie et al. (1995) “Production Of Recombinant Bovine Enterokinase 12124565.1 Catalytic Subunit In Escherichia Coli Using The Novel Secretory Fusion Partner DsbA,” Biotechnology 13:982-987), furin, and AcTEV (Parks et al. (1994) “Release Of Proteins And Peptides From Fusion Proteins Using A Recombinant Plant Virus Proteinase,” Anal. Biochem. 216:413-417) and the Foot and Mouth Disease Virus Protease C3. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells include but are not limited to CHO, VERY, BHK, HeLa, COS, MDCK, 293, 293T, 3T3, WI38, BT483, Hs578T, HTB2, BT20 and T47D, CRL7030 and Hs578Bst. For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express fusion proteins described herein may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines which express the fusion proteins described herein. Such engineered cell lines may be particularly useful in screening and evaluation of fusion proteins that interact directly or indirectly with the fusion proteins described herein. A number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al. (1977) “Transfer Of Purified Herpes Virus Thymidine Kinase Gene To Cultured Mouse Cells,” Cell 11: 223-232), hypoxanthine-guanine phosphoribosyltransferase (Szybalska et al. (1992) “Use Of The HPRT Gene And The HAT Selection Technique In DNA-Mediated Transformation Of Mammalian Cells First Steps Toward Developing Hybridoma Techniques And Gene Therapy,” Bioessays 14: 495-500), and adenine phosphoribosyltransferase (Lowy et al. (1980) “Isolation Of Transforming DNA: Cloning The Hamster aprt Gene,” Cell 22: 817-823) genes can be employed in tk−, hgprt− or aprt− cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for 12124565.1 the following genes: dhfr, which confers resistance to methotrexate (Wigler et al. (1980) “Transformation Of Mammalian Cells With An Amplifiable Dominant-Acting Gene,” Proc. Natl. Acad. Sci. USA 77:3567-3570; O'Hare et al. (1981) “Transformation Of Mouse Fibroblasts To Methotrexate Resistance By A Recombinant Plasmid Expressing A Prokaryotic Dihydrofolate Reductase,” Proc. Natl. Acad. Sci. USA 78: 1527-1531); gpt, which confers resistance to mycophenolic acid (Mulligan et al. (1981) “Selection For Animal Cells That Express The Escherichia coli Gene Coding For Xanthine-Guanine Phosphoribosyltransferase,” Proc. Natl. Acad. Sci. USA 78: 2072-2076); neo, which confers resistance to the aminoglycoside G-418 (Tolstoshev (1993) “Gene Therapy, Concepts, Current Trials And Future Directions,” Ann. Rev. Pharmacol. Toxicol.32:573-596; Mulligan (1993) “The Basic Science Of Gene Therapy,” Science 260:926-932; and Morgan et al. (1993) “Human Gene Therapy,” Ann. Rev. Biochem.62:191-217) and hygro, which confers resistance to hygromycin (Santerre et al. (1984) “Expression Of Prokaryotic Genes For Hygromycin B And G418 Resistance As Dominant- Selection Markers In Mouse L Cells,” Gene 30:147-156). Methods commonly known in the art of recombinant DNA technology which can be used are described in Ausubel et al. (eds.), 1993, Current Protocols in Molecular Biology, John Wiley & Sons, NY; Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY; and in Chapters 12 and 13, Dracopoli et al. (eds), 1994, Current Protocols in Human Genetics, John Wiley & Sons, NY.; Colberre-Garapin et al. (1981) “A New Dominant Hybrid Selective Marker For Higher Eukaryotic Cells,” J. Mol. Biol.150:1-14. The expression levels of the fusion protein described herein can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol.3 (Academic Press, New York, 1987). When a marker in the vector system expressing a fusion protein described herein is amplifiable, increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the nucleotide sequence of a fusion protein described herein or a fusion protein described herein, production of the fusion protein will also increase (Crouse et al. (1983) “Expression And Amplification Of Engineered Mouse Dihydrofolate Reductase Minigenes,” Mol. Cell. Biol.3:257-266). Once a fusion protein described herein has been recombinantly expressed, it may be purified by any method known in the art for purification of polypeptides, polyproteins or antibodies (e.g., analogous to antibody purification schemes based on antigen selectivity) for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific 12124565.1 antigen, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of polypeptides or antibodies. In some embodiments, a fusion protein comprises an affinity tag (e.g., a 6x-His tag or a 7x-His tag) as described herein to facilitate isolation of the fusion protein by affinity chromatography. For the purpose of affinity purification, relevant matrices for affinity chromatography, such as glutathione-, amylase-, and nickel- or cobalt- conjugated resins are used. Many of such matrices are available in “kit” form, such as the Pharmacia GST purification system and the QIAexpressTM system (Qiagen) useful with (HIS6) fusion partners. Methods of Isolating and Purifying Fusion Proteins Aspects of this disclosure provide a method of isolating and purifying a fusion protein (e.g., an A1M fusion protein) from a cell (e.g., a bacterial cell or a eukaryotic cell). The steps required to isolate and purify a fusion protein from a cell may vary, in part, on the cellular complexity of the cell and the choice of affinity tag, but generally requires: (1) obtaining cells comprising a fusion protein; (2) lysing the cells; and (3) purifying the fusion protein. Cell comprising a fusion protein (e.g., cells comprising a nucleic acid molecule encoding the fusion protein or cells comprising a vector comprising said nucleic acid molecule) may be induced to express the fusion protein. Methods for inducing fusion protein expression are known in the art to vary based on the specific cell system used. For example, inducing fusion protein expression in prokaryotic cells (e.g., E. coli cells) may be performed by adding an inducer molecule (e.g., IPTG for lac promoter-based systems). Other inducers are contemplated, including, but not limited to, tetracycline for Tet-On and Tet-Off systems, arabinose for arabinose promoter-based systems, lactose for lac promoter-based systems, and tamoxifen in Cre recombinase systems. In some embodiments, fusion protein expression may result in the formation of inclusion bodies. “Inclusion bodies” are insoluble protein aggregates that may contain misfolded protein and result from high-level protein expression (e.g., from strong promoters on high copy number plasmids). Once the fusion protein has been expressed in a cell, the cell can be lysed and the fusion protein can be purified from the cell debris using techniques known in the art. For example, cell lysis can be performed using physical methods (e.g., freeze-thaw, mechanical disruption, such as polytron disruption or mechanical grinding, sonication) or chemical methods (e.g., a detergent) and the fusion protein can be purified using chromatography (e.g., affinity chromatography, ion exchange chromatography, size exclusion chromatography, liquid chromatography), centrifugation, precipitation (e.g., acid precipitation, salting out), filtration, buffer exchange 12124565.1 (e.g., dialysis). In some embodiments, multiple purification steps using one or more techniques may be required to obtain purified protein. One of ordinary skill can be readily determine the appropriate protocol required for protein expression, lysis, and purification without undue experimentation based on the protein of interest, cell system of choice (e.g., promoter system, affinity tag), and downstream applications. Where inclusion bodies are obtained, additional steps to solubilize and refold the fusion protein may be required prior to purification. These may include isolation of the inclusion body from the cell lysate (e.g., by centrifugation), one or more wash steps to remove contaminants and cellular debris, solubilization to disrupt protein aggregates (e.g., using denaturing agents, and / or reducing agents), and refolding. Solubilization of inclusion bodies is the process of releasing a protein of interest (e.g., an A1M fusion protein) from insoluble aggregates by disrupting the hydrophobic interactions that hold the protein aggregates together to allow the protein of interest to unfold and solubilize. This may be achieved, in some instances, by the use of denaturing agents such as chaotropic agents and / or reducing agents. A chaotropic agent is a substance that interferes with the hydration shell around proteins, leading to the disruption of hydrogen bonds and hydrophobic interactions and destabilization of the native protein structure. Non-limiting examples of chaotropic agents include urea, guanidine and guanidine salts (e.g., guanidine hydrochloride), formamide, thiocyanate salts (e.g., ammonium thiocyanate), and ionic salts (e.g., sodium iodide). A reducing agent is a substance that donates electrons to break disulfide bonds (e.g., covalent bonds formed between cysteine residues) and converts them into thiol groups, leading to destabilization of the native protein structure. Non-limiting examples of reducing agents include β-mercaptoethanol, dithiothreitol (DTT), Tris(20carboxyethyl)phosphine, and sodium borohydride. Once solubilized, the denaturing agents can then be gradually removed by one or more dilution or dialysis steps to promote protein folding. For example, step-wise dialysis may be used to incrementally lower the concentration of denaturants using 2 or more dialysis steps. In some embodiments, step-wise dialysis may include 2, 3, 4, 5, 6, 7, 8, 9 or 10 dialysis steps. In some embodiments, each dialysis step reduces the concentration of denaturants by at least 20% (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%) relative to the previous dialysis step. In some embodiments, one or more dialysis steps may require use of a redox buffer. A redox buffer is a pair of chemical species in solution that can undergo oxidation-reduction reactions to maintain a constant ratio of oxidized and reduced forms of a molecule in a solution, thereby stabilizing the redox state of the system and facilitating proper folding and protein 12124565.1 stability, particularly for proteins containing disulfide bonds. Thus, in some embodiments, redox buffers may be used in one or more dialysis steps to promote disulfide bond formation during refolding. Non-limiting examples of redox buffers include glutathione (GSH) and oxidized glutathione (GSSG) (SEQ ID NO: 73), cysteine and cystine, NAD+ and NADH, NADP+ and NADPH, and ferredoxin and ferredoxin oxidoreductase. In some embodiments, arginine is not added to a redox buffer. Additional dialysis steps for desalting, removing small molecules (e.g., detergents, reducing agents, or chaotropic agents), exchanging buffer components, and protein concentration prior to purification are contemplated depending on the downstream assay or application. For example, where proteins are expressed in Gram-negative bacteria (e.g., E. coli), additional dialysis steps (e.g., using lysine cellulose) may be required to remove endotoxins (e.g., lipopolysaccharides) which may interfere with downstream applications. Thus, in some embodiments, this disclosure provides a method of purifying a fusion protein (e.g., an A1M fusion protein) from a cell described herein, comprising: (i) obtaining the cells; (ii) lysing the cells; (iii) obtaining inclusion bodies from the lysed cells; (iv) denaturing the inclusion bodies to release the fusion protein; and (v) refolding the fusion protein. In some embodiments, step (ii) is performed using polytron disruption. In some embodiments, step (iv) is performed with a chaotropic reagent. In some embodiments, step (v) is performed by step-wise dialysis. In some embodiments, step (v) is performed without arginine. In some embodiments, step (v) is performed with a redox buffer. Methods of Treatment Further provided herein are methods of using the fusion proteins, lipoproteins, nanoparticles, nanodisks, etc. (e.g., in therapeutic applications). For example, in some embodiments, a fusion protein described herein is formulated in a pharmaceutical composition. Methods of treating a disease or disorder using a fusion protein described herein, a lipoprotein described herein, or a pharmaceutical composition described herein are also provided. In some embodiments, the method using the fusion protein described herein comprises contacting the fusion protein with S1P. Contacting the fusion protein described herein with S1P results in the formation of a complex between the fusion protein and S1P. In some embodiments, such contacting is carried out in a cell. In some embodiments, a composition comprising a fusion protein (e.g. a fusion protein, fusion protein bound to a therapeutic, lipoprotein, nanoparticle, or nanodisk) as described herein is used in methods of treating a disease or disorder associated with reduced level of sphingosine- 12124565.1 1-phosphate (S1P), the method comprising administering to a subject in need thereof a therapeutically effective amount of a composition comprising the fusion protein. Such fusion protein binds to S1P and activates a S1P receptor, triggering downstream signaling pathway. In some embodiments, the S1P receptor is S1P1. It is demonstrated herein that the fusion protein- S1P complex specifically activates the S1P1 receptor, compared to other types of S1P receptors, e.g., S1P2 or S1P3. In some embodiments, the S1P receptor (e.g., S1P1) is vascular, i.e., found on the surface of an endothelial cell in a blood vessel. A disease or disorder “associated with reduced level of S1P” refers to an abnormal condition where the level or activity of S1P, or S1P-triggered signaling pathway is reduced in a subject that has the disease or disorder, compared to a healthy subject. In some embodiments, the level or activity of S1P, or S1P-triggered signaling pathway is reduced by at least 20% in a subject that has the disease or disorder, compared to a healthy subject. For example, the level or activity of S1P, or S1P-triggered signaling pathway may be reduced by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% in a subject that has the disease or disorder, compared to a healthy subject. In some embodiments, the level or activity of S1P, or S1P-triggered signaling pathway is reduced by 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% in a subject that has the disease or disorder, compared to a healthy subject. Administering a fusion protein or a composition comprising such fusion protein described herein to a subject having a disease or disorder associated with reduced level of S1P increased S1P signaling, e.g., by activating S1P receptor such as S1P1. In some embodiments, the S1P signaling is increases by at least 20%, in the presence of the fusion protein, compared to without the fusion protein. For example, the S1P signaling may be increased by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2-fold, at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 1000-fold, or more, in the presence of the fusion protein, compared to without the fusion protein. In some embodiments, the S1P signaling is increased by 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 1000-fold, or more, in the presence of the fusion protein, compared to without the fusion protein. In some embodiments, the diseases or disorders associated with reduced level of S1P include, without limitation: infection, sepsis, diabetes, cardiovascular diseases, retinal vascular diseases, peripheral vascular diseases, metabolic syndromes, and respiratory diseases. In some embodiments, the diseases or disorders associated with reduced level of S1P include, without limitation: primary and / or secondary resistant hypertension, neurogenic 12124565.1 hypertension, gestational hypertension, diabetic hypertension, hypertension of chronic kidney disease, cardiac and non-cardiac reperfusion injury, ischemic injury, stroke, pulmonary edema, myocardial infarction, acute coronary syndrome, angina, atherosclerosis, and age-related macular degeneration. A disease or disorder “associated with reduced level of prostaglandin” refers to an abnormal condition where the level or activity of prostaglandin, or prostaglandin-triggered signaling pathway is reduced in a subject that has the disease or disorder, compared to a healthy subject. In some embodiments, the level or activity of prostaglandin, or prostaglandin-triggered signaling pathway is reduced by at least 20% in a subject that has the disease or disorder, compared to a healthy subject. For example, the level or activity of prostaglandin, or prostaglandin-triggered signaling pathway may be reduced by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% in a subject that has the disease or disorder, compared to a healthy subject. In some embodiments, the level or activity of prostaglandin, or prostaglandin-triggered signaling pathway is reduced by 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% in a subject that has the disease or disorder, compared to a healthy subject. Administering a fusion protein or a composition comprising such fusion protein described herein to a subject having a disease or disorder associated with reduced level of prostaglandin increased prostaglandin signaling, e.g., by activating prostaglandin receptor. In some embodiments, the prostaglandin signaling is increased by at least 20%, in the presence of the fusion protein, compared to without the fusion protein. For example, the prostaglandin signaling may be increased by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2-fold, at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 1000-fold, or more, in the presence of the fusion protein, compared to without the fusion protein. In some embodiments, the prostaglandin signaling is increased by 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 1000-fold, or more, in the presence of the fusion protein, compared to without the fusion protein. In some embodiments, a fusion protein or a composition comprising such fusion protein described herein is used in methods of treating a subject having a disease or disorder associated with vascular endothelial dysfunction. In some embodiments, the subject has poor blood circulation. In some embodiments, the subject has pulmonary hypertension. In some embodiments, the subject has thrombosis. In some embodiments, a fusion protein or a composition comprising a fusion protein is used in methods of treating a subject having a 12124565.1 disease or disorder associated thrombotic inflammation. In some embodiments, the subject has a cardiovascular disease, an autoimmune disease, an inflammatory disease, an infectious disease, an ocular disorder, or cancer. For example, a fusion protein or a composition comprising a fusion protein can be used to enhance cancer therapy, e.g., by vascular normalization. In some embodiments, a fusion protein or a composition comprising a fusion protein is used in methods of treating a subject having a cardiovascular disease (e.g., hypercholesterolemia, atherosclerosis, stroke, heart failure, peripheral artery disease, acute liver failure, and / or acute kidney failure). In some embodiments, a fusion protein or a composition comprising a fusion protein is used in methods of treating a subject having a metabolic disorder (e.g., diabetes, diabetic nephropathy, and / or diabetic retinopathy). In some embodiments, a fusion protein or a composition comprising a fusion protein is used in methods of treating a subject having an autoimmune disease (e.g., rheumatoid arthritis, systemic lupus erythematosus, and / or an autoimmune syndrome). In some embodiments, a fusion protein or a composition comprising a fusion protein is used in methods of treating a subject having an inflammatory disease (e.g., an acute inflammatory disease, a chronic inflammatory disease, acute respiratory distress syndrome, and / or sepsis). In some embodiments, a fusion protein or a composition comprising a fusion protein is used in methods of treating a subject having a infectious disease (e.g., a respiratory virus infection) or an ocular disorder (e.g., retinal vascular disease or age-related macular degeneration. In some embodiments, a fusion protein or a composition comprising such fusion protein described herein is used in methods of reducing inflammation (e.g., systemic and / or local inflammation) in a subject. In some embodiments, a fusion protein or a composition comprising such fusion protein described herein is used in methods of reducing systemic inflammation in a subject. In some embodiments, the inflammation is associated with TNFα-induced NF-κβ activation. Thus, in some embodiments, a fusion protein or a composition comprising such fusion protein described herein is used to treat TNFα-induced NF-κβ activation. In some embodiments, a fusion protein or a composition comprising such fusion protein described herein is used to decrease TNFα-induced NF-κβ activation. In some embodiments, the inflammation is associated with a cardiovascular disease, an autoimmune disease, an inflammatory disease, an infectious disease, an ocular disorder, or cancer. For example, a fusion protein or a composition comprising a fusion protein can be used to enhance cancer therapy, e.g., by vascular normalization. In some embodiments, a fusion protein or a composition comprising a fusion protein is used in methods of treating a subject having a cardiovascular disease (e.g., hypercholesterolemia, atherosclerosis, stroke, heart failure, peripheral artery disease, acute liver 12124565.1 failure, and / or acute kidney failure). In some embodiments, a fusion protein or a composition comprising a fusion protein is used in methods of treating a subject having a metabolic disorder (e.g., diabetes, diabetic nephropathy, and / or diabetic retinopathy). In some embodiments, a fusion protein or a composition comprising a fusion protein is used in methods of treating a subject having an autoimmune disease (e.g., rheumatoid arthritis, systemic lupus erythematosus, and / or an autoimmune syndrome). In some embodiments, a fusion protein or a composition comprising a fusion protein is used in methods of treating a subject having an inflammatory disease (e.g., an acute inflammatory disease, such as peritonitis, a chronic inflammatory disease, acute respiratory distress syndrome, and / or sepsis). In some embodiments, a fusion protein or a composition comprising a fusion protein is used in methods of treating a subject having a infectious disease (e.g., a respiratory virus infection) or an ocular disorder (e.g., retinal vascular disease or age-related macular degeneration. In some embodiments, a fusion protein or a composition comprising a fusion protein is combined with an anti-thrombin agent to treat a disease described herein. In some embodiments, the anti-thrombin agent is Angiopoietin1 or Activated Protein C (APC). In some embodiments, S1P and / or Iloprost bound fusion protein nanoparticles inhibited formyl peptide- stimulated oxidative burst. In some embodiments, Iloprost bound A1M inhibits platelet aggregation. Pharmaceutical Compositions A “pharmaceutical composition”, as used herein, refers to the formulation of the fusion protein, composition comprising a fusion protein (e.g. a fusion protein bound to a therapeutic, a lipoprotein, a nanoparticle, or a nanodisk) described herein in combination with a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical compositions further comprise an additional therapeutic agent as described herein. The pharmaceutical composition can further comprise additional agents (e.g. for specific delivery, increasing half- life, or other therapeutic agents). Pharmaceutically-acceptable carriers refers to a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the fusion protein from one site (e.g., the delivery site) of the body, to another site (e.g., organ, tissue, or portion of the body). A pharmaceutically acceptable carrier is “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the tissue of the subject 9e.g., physiologically compatible, 12124565.1 sterile, physiologic pH, etc.). Some examples of materials which can serve as pharmaceutically- acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and / or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; (22) C2-C12 alcohols, such as ethanol; and (23) other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservative and antioxidants can also be present in the formulation. The terms such as “excipient”, “carrier”, “pharmaceutically acceptable carrier” or the like are used interchangeably herein. In some embodiments, a pharmaceutical composition comprising a fusion protein of the present disclosure is administered by injection, by means of a catheter, by means of a suppository, or by means of an implant, the implant being of a porous, non-porous, or gelatinous material, including a membrane, such as a sialastic membrane, or a fiber. Typically, when administering the composition, materials to which the fusion protein of the disclosure does not absorb are used. In some embodiments, a pharmaceutical composition comprising a fusion protein of the present disclosure is delivered in a controlled release system. In one embodiment, a pump may be used (see, e.g., Langer, 1990, Science 249:1527-1533; Sefton, 1989, CRC Crit. Ref. Biomed. Eng.14:201; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med. 321:574). In another embodiment, polymeric materials can be used. (See, e.g., Medical Applications of Controlled Release (Langer and Wise eds., CRC Press, Boca Raton, Fla., 1974); Controlled Drug Bioavailability, Drug Product Design and Performance (Smolen and Ball eds., Wiley, New York, 1984); Ranger and Peppas, 1983, Macromol. Sci. Rev. Macromol. Chem. 23:61. See also Levy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol.25:351; 12124565.1 Howard et al., 1989, J. Neurosurg.71:105.) Other controlled release systems are discussed, for example, in Langer, supra. A fusion protein of the present disclosure can be administered as pharmaceutical compositions comprising a therapeutically effective amount of a binding agent and one or more pharmaceutically compatible ingredients. In some embodiments, the pharmaceutical composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous or subcutaneous administration to a subject, e.g., a human being. Typically, compositions for administration by injection are solutions in sterile isotonic aqueous buffer. Where necessary, the pharmaceutical can also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the pharmaceutical is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the pharmaceutical is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration. A pharmaceutical composition for systemic administration may be a liquid, e.g., sterile saline, lactated Ringer's or Hank’s solution. In addition, the pharmaceutical composition can be in solid forms and re-dissolved or suspended immediately prior to use. Lyophilized forms are also contemplated. The pharmaceutical composition can be contained within a lipid particle or vesicle, such as the nanodisk described herein, which is also suitable for parenteral administration. The particles can be of any suitable structure, such as unilamellar or plurilamellar, so long as compositions are contained therein. The fusion proteins of the present disclosure can be entrapped in 'stabilized plasmid-lipid particles' (SPLP) containing the fusogenic lipid dioleoylphosphatidylethanolamine (DOPE), low levels (5-10 mol%) of cationic lipid, and stabilized by a polyethyleneglycol (PEG) coating (Zhang Y. P. et al., Gene Ther.1999, 6:1438- 47). Positively charged lipids such as N-[1-(2,3-dioleoyloxi)propyl]-N,N,N-trimethyl- amoniummethylsulfate, or "DOTAP," are particularly preferred for such particles and vesicles. The preparation of such lipid particles is well known. See, e.g., U.S. Patent Nos.4,880,635; 4,906,477; 4,911,928; 4,917,951; 4,920,016; and 4,921,757. The pharmaceutical compositions of the present disclosure may be administered or packaged as a unit dose, for example. The term "unit dose" when used in reference to a 12124565.1 pharmaceutical composition of the present disclosure refers to physically discrete units suitable as unitary dosage for the subject, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent; i.e., carrier, or vehicle. In some embodiments, the pharmaceutical composition can be provided as a pharmaceutical kit comprising (a) a container containing a fusion protein of the disclosure in lyophilized form and (b) a second container containing a pharmaceutically acceptable diluent (e.g., sterile water) for injection. The pharmaceutically acceptable diluent can be used for reconstitution or dilution of the lyophilized fusion protein of the disclosure. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. In another aspect, an article of manufacture containing materials useful for the treatment of the diseases described herein is included. In some embodiments, the article of manufacture comprises a container and a label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers may be formed from a variety of materials such as glass or plastic. In some embodiments, the container holds a composition that is effective for treating a disease described herein and may have a sterile access port. For example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle. The active agent in the composition is an fusion protein of the disclosure. In some embodiments, the label on or associated with the container indicates that the composition is used for treating the disease of choice. The article of manufacture may further comprise a second container comprising a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer's solution, or dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. The terms “treatment” or “to treat” refer to both therapeutic and prophylactic treatments. If the subject is in need of treatment of a conditions (e.g. pathogenic inflammation) then “treating the condition” refers to ameliorating, reducing or eliminating one or more symptoms associated with the disease or preventing any further progression of the disease. If the subject in need of treatment is one who is at risk of a disease (e.g. pathogenic inflammation), then treating the subject refers to reducing the risk of the subject having the disease or preventing the subject from developing the disease. 12124565.1 A subject shall mean a human or vertebrate animal or mammal including but not limited to a rodent, e.g., a rat or a mouse, dog, cat, horse, cow, pig, sheep, goat, turkey, chicken, and primate, e.g., monkey. The methods of the present disclosure are useful for treating a subject in need thereof. A subject in need thereof can be a subject who has a risk of developing a disease or disorder associated with reduced S1P or vascular endothelial dysfunction, or a subject who has such a disease or disorder. Pharmaceutically compositions that may be used in accordance with the present disclosure may be directly administered to the subject or may be administered to a subject in need thereof in a therapeutically effective amount. The term “therapeutically effective amount” refers to the amount necessary or sufficient to realize a desired biologic effect. For example, a therapeutically effective amount of a cancer-target liposome associated with the present disclosure may be that amount sufficient to ameliorate one or more symptoms of the disease or disorder. Combined with the teachings provided herein, by choosing among the various active compounds and weighing factors such as potency, relative bioavailability, patient body weight, severity of adverse side-effects and preferred mode of administration, an effective prophylactic or therapeutic treatment regimen can be planned which does not cause substantial toxicity and yet is entirely effective to treat the particular subject. The effective amount for any particular application can vary depending on such factors as the disease or condition being treated, the particular pharmaceutically compositions being administered the size of the subject, or the severity of the disease or condition. One of ordinary skill in the art can empirically determine the effective amount of a particular therapeutic compound associated with the present disclosure without necessitating undue experimentation. Subject doses of the fusion protein described herein for delivery typically range from about 0.1 µg to 10 mg per administration, which depending on the application could be given daily, weekly, or monthly and any other amount of time there between. In some embodiments, a single dose is administered during the critical consolidation or reconsolidation period. The formulations of the present disclosure are administered in pharmaceutically acceptable solutions, which may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, and optionally other therapeutic ingredients. For use in therapy, an effective amount of the fusion protein of the present disclosure can be administered to a subject by any mode that delivers the fusion protein to the desired location, e.g., mucosal, injection, systemic, etc.. Administering the pharmaceutical composition of the present disclosure may be accomplished by any means known to the skilled artisan. In some 12124565.1 embodiments, the fusion protein is administered subcutaneously, intracutaneously, intravenously, intramuscularly, intraarticularly, intraarterially, intrasynovially, intrasternally, intrathecally, intralesionally, or intracranially. Some of the embodiments, advantages, features, and uses of the technology disclosed herein will be more fully understood from the Examples below. The Examples are intended to illustrate some of the benefits of the present disclosure and to describe particular embodiments, but are not intended to exemplify the full scope of the disclosure and, accordingly, do not limit the scope of the disclosure. EXAMPLES Example 1. Designer high-density lipoprotein particles enhance endothelial barrier function and suppress inflammation. Abstract High-density lipoprotein (HDL) nanoparticles promote endothelial cell (EC) function and suppress inflammation, but their utility in treating EC dysfunction has not been fully explored. This Example describe a fusion protein named ApoA1-ApoM (A1M) consisting of apolipoprotein A1 (ApoA1), the principal structural protein of HDL that forms lipid nanoparticles, and ApoM, a chaperone for the bioactive lipid sphingosine 1-phosphate (S1P). A1M forms HDL-like particles, binds S1P, and is signaling competent. Molecular dynamic simulations showed that the S1P-bound ApoM moiety in A1M efficiently activated the EC surface receptors. Treatment of human umbilical vein endothelial cells (HUVECs) with A1M- S1P stimulated barrier function either alone or cooperatively with other barrier-enhancing molecules, including the stable prostacyclin analog iloprost, and suppressed cytokine-induced inflammation. A1M-S1P injection into mice during sterile inflammation suppressed neutrophil influx and inflammatory mediator secretion. Moreover, systemic A1M administration led to a sustained increase in circulating HDL-bound S1P and suppressed inflammation in a murine model of LPS-induced endotoxemia. This Example suggests that A1M administration may enhance vascular endothelial barrier function, suppress cytokine storm, and promote resilience of the vascular endothelium. Introduction Vascular endothelial dysfunction, which is induced by metabolic stress (such as diabetes or hypercholesterolemia), inflammatory and autoimmune states, is a major driver of chronic diseases, such as atherosclerosis, stroke, heart failure, rheumatoid arthritis, and systemic lupus 12124565.1 erythematosus (Pober and Sessa 2007). Restoration of endothelial cell function attenuates inflammation-induced tissue damage, thrombosis, and chronic disease progression (Kiseleva et al.2018). However, therapeutic strategies to counter endothelial dysfunction are not available at present. Circulating high-density lipoprotein (HDL) particles are important for cholesterol homeostasis and endothelial function. The major structural protein of HDL is apolipoprotein A1 (ApoA1), an amphipathic polypeptide that becomes lipidated after interaction with ABCA1 / ABCG1 transporters and membranes (Yvan-Charvet et al.2010). Nascent discoidal HDL particles transport cholesterol from tissues to the liver, a process termed reverse cholesterol transport (Ouimet et al.2019). Although reverse cholesterol transport was originally thought to be solely responsible for the cardiovascular protective effects of HDL, the ability to attenuate endothelial dysfunction may be equally important (Remaley et al.2014; Rohatgi et al.2021). HDL is associated with numerous proteins and lipids that confer cytoprotective and anti- inflammatory properties under normal conditions (Heinecke 2010; Shao and Heinecke 2018). Among the HDL-associated proteins, the major structural protein ApoA1 decreases cytokine and endotoxin-induced NF-κB activation in myeloid cells (macrophages and neutrophils) (Suzuki et al.2010) by interfering with inflammatory receptor signal transduction (Fotakis et al.2019). ApoA1 also enhances endothelial-derived nitric oxide (NO) secretion, which promotes blood flow (Mineo and Shaul 2013). In addition, HDL suppresses thrombosis by enhancing the activity of prostacyclin (PGI2) and attenuation of tissue factor expression (Mineo et al.2006; Morishita et al.1990). Apolipoprotein M (ApoM), a member of the lipocalin family of proteins associated with a subpopulation of HDL particles, has been shown to be a key physiological chaperone for sphingosine 1-phosphate (S1P), a high affinity ligand for G-protein coupled S1P receptors (S1PRs) (Christoffersen et al.2011). The S1P / S1PR1 axis is critical for vascular development, vascular barrier function, NO synthesis, and endothelial survival (Proia and Hla 2015). HDL- bound S1P attenuates cytokine-induced NF-κB activation and adhesion molecule expression by a mechanism that involves S1PR1 (Galvani et al.2015). This signaling axis suppresses lymphopoiesis in the bone marrow (Blaho et al.2015), endothelial injury in the lung (Cao et al. 2020) and liver fibrosis and promotes hepatocyte regeneration (Ding et al.2016). In addition, S1PR1 signaling enables trans-endothelial passage of HDL particles into tissue parenchymal spaces (Velagapudi et al.2021). These studies suggest that HDL-bound S1P activation of endothelial S1PR1 may be therapeutically tractable to suppress endothelial dysfunction and suppress pathologic inflammation. 12124565.1 S1P bound to ApoM as well as other carrier proteins, such as albumin and ApoA4 can activate the S1PRs (Obinata et al.2019). However, mice that are deficient for ApoM exhibit increased vascular leak in lung microvessels (Christoffersen et al.2011; Christensen et al.2016) and penetrating arterioles of the brain (Mathiesen Janiurek et al.2019), suggesting that the ability of ApoM-bound S1P to promote vascular barrier function is physiologically critical. Solution binding as well as molecular dynamics (MD) simulation studies indicate that S1P dissociation from ApoM requires free energy to overcome the electrostatic interactions between the anionic head group of S1P and the cationic amino acid residues that form the lid of ApoM that blocks the of the ligand binding pocket (Zhang et al.2016). Thus, S1P transfer from ApoM to S1PRs may be fundamentally different from albumin-S1P / S1PR activation. This may explain the sustained and Gαi-biased signaling property of HDL-bound S1P (Galvani et al.2015). An ApoM-Fc fusion protein (ApoM-Fc-S1P) that enhances endothelial barrier function, suppresses ischemia-reperfusion injury of the heart and the brain, and immune complex- mediated and acid-induced lung injury has previously been developed, but was unable to mimic the endothelial protective effects of HDL-S1P (Cao et al.2020; Swendeman et al.2017; Burg et al.2018). This Example shows the design and characterization of an ApoA1-ApoM (A1M) fusion protein which forms spherical nano-sized lipoprotein particles, chaperones multiple bioactive lipids (S1P and PGI2 analogs), protects the endothelium, and suppresses platelet aggregation and inflammatory responses in vitro and in vivo. Results Design, expression, and characterization of ApoA1-ApoM-S1P (A1M-S1P) nanoparticles An ApoA1-ApoM (A1M) fusion protein was designed and constructed with a flexible linker domain (GGGGS) and 6X-Histidine tag at the carboxyl-terminus (FIGs.1A-1B) (Trinh et al.2004). The A1M protein was expressed and purified from conditioned media from stably transfected CHO-S cells (FIG.2A). A1M was loaded with S1P as described previously. Briefly, purified A1M was incubated with S1P for 24 hours, lipidated, and purified by FPLC gel filtration chromatography (Swendeman et al.2017). Unloaded and loaded A1M proteins had ~0.5 mol% and ~65 mol% of S1P, respectively (FIG.2B). To prepare reconstituted HDL-like particles, A1M-S1P was lipidated with a mixture of phospholipids dimyristoyl phosphatidylcholine and dimyristoyl phosphatidyl glycerol (DMPC / DMPG) as described for ApoA1 (Swendeman et al.2017; Oda et al.2006). FPLC elution profiles of the samples were compared to a reference of fractionated mouse plasma (FIG.2C). Lipidated A1M-S1P eluted near the HDL fractions, suggesting conversion of 12124565.1 nascent A1M to HDL-like particles. Lipidated A1M-S1P particles were further analyzed by negative staining and analyzed by transmission electron microscopy. The majority of the particles were ~ 8-12 nm in diameter with various shapes (FIG.2D, top), in line with the conformational heterogeneity of A1M due to the flexible linker between ApoA1 and ApoM. Several two-dimensional (2D) averages showed two side lobes connecting to a central disc- shaped density (FIG.2D, bottom), which is consistent with the flexibility of the linker domain that connects ApoA1 and ApoM domains (FIG.2E). These studies suggested that lipidated A1M-S1P complex forms nanometer-sized lipoprotein particles that stably bind to S1P. Molecular dynamic simulation study of the A1M-S1P complex Next, molecular dynamic (MD) simulation analyses of lipidated A1M-S1P were performed using the known protein structure of ApoM-S1P complex (PDB: 2YG21) (Christoffersen et al.2011) and the double belt structure of ApoA1 (Jurrus et al.2018; Pourmousa et al.2018). The HDL-like particle contained POPC:unesterified cholesterol:ApoA1 at a ratio of 160:24:2 in an antiparallel arrangement with LL5 / 5 registry (Segrest et al.1999) (FIG.3A). To understand the structural stability of the lipidated A1M-S1P versus ApoM-S1P complexes at near physiological conditions, the root mean square deviation (RMSD) was calculated for all backbone residues. The time evolution of the RMSD of the conformation of ApoM-S1P reached a plateau around 2.3 ± 0.3 Å (ApoM-S1P interface: 1.7 ± 0.3 Å), suggesting the stability of this complex. However, the overall system of A1M-S1P exhibited a much higher degree of conformational variability with RMSD values of 18.4 ± 4.6 Å likely due to the presence of a flexible linker that connects ApoA1 and ApoM domains. Nevertheless, the ApoM- S1P moiety in the A1M-S1P particle displayed an RMSD of 2.4 ± 0.2 Å and 2.2 ± 0.3 Å in monomers A and B, respectively. Both S1P interfaces remained highly stable (RMSD – monomer A ApoM-S1P Interface: 1.5 ± 0.4 Å; monomer B ApoM-S1P Interface: 1.9 ± 0.3 Å). To better evaluate the conformational diversity, principal component analysis (PCA) of sampled conformations in the A1M particle was performed to allow the reduction of high-dimensional structural space and to understand main collective motions. The top two dominant eigenvectors were found to be responsible for over 40% of the dominant collective variances in the structure set. The ensemble of structures was clustered by similarity into 5 representative clusters (FIG. 3B). The resulting eigenvalue contribution of PCA (FIG.3C) showed that the first seven principal components were responsible for 86.4% of the total mean-square displacement of atom positional fluctuations. This capacity of rapid interconversions of conformations agreed with the electron microscopy data (FIG.2D). 12124565.1 Although large-scale conformational changes of the fused A1M protein were observed, the overall accessibility of the S1P binding interface was similar in both A1M-S1P and ApoM- S1P complexes. The ratio between the solvent accessible surface area (SASA) of interfacial residues on both A1M-S1P and ApoM-S1P was stable (close to 1, FIG.4). Furthermore, the network of hydrogen bonds established between ApoM and S1P was also stably maintained in both complexes (Table 1). The more accessible conformational space explored by the fused protein led to greater movement of the polar S1P groups and replacement of the Arg143 contribution within this network by Arg98. Hydrogen network involvement in the S1P coupling is the main energetic barrier for S1P dissociation from the complex (Zhang et al.2016). Overall, analysis of the MD simulation data pointed to a stable binding of S1P to ApoM in both ApoM- S1P and lipidated A1M-S1P complexes. Further, the conformational diversity of ApoA1 and ApoM joined by a flexible linker in the A1M fusion protein may enable efficient action at cell surface S1P receptors. Table 1. Hydrogen bonds between S1P and five interfacial ApoM residues. Occupancy times are listed. Values higher than 100% means that more than 1 hydrogen bond can be established with the studied residue. A1M-S1P preferentially activates S1PR1 as a Gαi-biased agonist The ability of the A1M-S1P complex to activate S1P receptors was studied using the real-time NanoBiT system that monitors receptor-induced heterotrimeric G protein dissociation and receptor-β-arrestin association (Hisano et al.2019). Temporal and dose-response analysis revealed A1M-S1P induced rapid and potent S1PR1-Gαi dissociation compared to ApoM-S1P and bovine serum albumin (BSA)-S1P, suggesting ApoM chaperone function was not impaired by fusion to ApoA1 or nanoparticle formation of the A1M-S1P particle (FIGs.5A-5B). However, β-arrestin coupling to S1PR1 in response to A1M-S1P and ApoM-Fc-S1P was slower than BSA-S1P (FIG.5C). Moreover, over a 4-log range of ligand concentrations, similar S1PR1-dependent activation of β-arrestin was observed for A1M-S1P, BSA-S1P, and ApoM- 12124565.1 Fc-S1P (FIG.5D). These data suggested that A1M-S1P, similar to ApoM-Fc-S1P, is a Gαi- biased activator of S1PR1. The ability of various chaperones to activate S1PR2 (FIG.5E) and S1PR3 (FIG.5F) was also observed by conducting β-arrestin coupling assays. Greater maximum stimulation of these receptors was observed with BSA-S1P compared to either A1M- S1P and ApoM-Fc-S1P, suggesting that ApoM-bound S1P activates S1PR1 preferentially when compared to S1PR2 and S1PR3. A1M-S1P stimulates endothelial barrier function To determine the ability of lipidated A1M-S1P particles to regulate endothelial cell barrier function, trans-endothelial electrical resistance (TEER) analysis was performed using HUVECs (Swendeman et al.2017). A dose-response study of A1M-S1P was performed (FIG. 6A) which showed similar potency of ApoM-Fc- and A1M-bound S1P. EC50 values of A1M- S1P and ApoM-Fc-S1P are 1.5 µg / mL and 2.2 µg / mL, respectively, which corresponded to ~ 21 and 29 nM S1P. Unloaded A1M and ApoM-Fc had no barrier stimulatory activity. This result suggests the equivalent functionality of ApoM as an S1P chaperone whether it is fused to the Fc or ApoA1 domains. Angiopoietin-1 (Ang-1) is a polypeptide that stimulates endothelial barrier function and attenuates vascular leak during inflammation and thrombosis (Jeansson et al.2011; Parikh 2017). A1M-S1P and Ang-1 enhanced endothelial barrier function in an additive manner (FIG. 6B). Thrombin, a protease produced during blood clotting, activates G protein-coupled protease- activated receptors (PARs) to disrupt endothelial barrier function. S1P suppressed thrombin- induced barrier breach by activating S1PRs (Garcia et al.2017). It was found that ApoM-Fc-S1P treated EC blocked thrombin-induced barrier degradation in a dose-dependent manner (FIG. 6C). In addition, ApoM-Fc-S1P and Ang-1 cooperated to suppress thrombin-induced barrier breach (FIG.6D). In contrast to thrombin, activated protein C (APC) interacts with endothelial cell surface receptors such as thrombomodulin and the endothelial protein C receptor (EPCR) to enhance EC barrier function (Finigan et al.2005). Sub-optimal concentrations of APC (5 μg / mL) and S1P (30 nM) were combined either complexed with A1M-S1P (FIG.6E) or ApoM- Fc-S1P (FIG.6F) to test if these combinations blocked thrombin-induced HUVEC barrier breach. Combination of suboptimal APC and A1M-S1P or ApoM-Fc-S1P blocked thrombin- induced barrier breach ~50% in a synergistic manner (FIGs.6E-6F). Together these data reveal that A1M-S1P, Ang-1, and APC cooperate to protect the endothelial barrier. 12124565.1 A1M binds to the stable prostacyclin analog iloprost, which cooperates with S1P to enhance barrier function Endothelial cell derived PGI2 acts on the prostacyclin receptor (IP) to activate the Gαs / adenylate cyclase / cAMP pathway to suppress platelet aggregation, vascular smooth muscle dilation and inflammatory responses (Pluchart et al.2017). IP activators also suppress inflammation-induced vascular leak by activating the cAMP / EPAC / Rap1 pathway (Birukova et al.2007). Moreover, the lability of PGI2 due to autohydrolysis is inhibited by HDL association (Morishita et al.1990). Therefore, A1M supplemented with iloprost, a stable PGI2 analog, was evaluated to determine if it would possess EC protective effects (Skuballa et al.2983). It was found that lipidated A1M-iloprost induced an IP / cAMP-responsive CREB luciferase reporter activity (FIG.7A).6 nM of iloprost showed an equivalent response to 1 μg / mL of A1M- iloprost. These data suggest that A1M-bound iloprost is biologically active and activates the IP receptor potently. Because the cAMP / EPAC / Rap1 pathway stimulates endothelial barrier function, A1M- iloprost was evaluated for its ability to stimulate endothelial barrier function to a similar degree as low dose A1M-S1P (30 nM S1P) (FIG.7B). Although effective as a single agent, combining A1M-iloprost and A1M-S1P provided additive barrier protection (FIG.7B). Similarly, free iloprost (200 nM) cooperated with HDL-S1P (FIG.7C) and the S1PR1-selective agonist AUY954 (FIG.7D) to enhance endothelial barrier function in an additive manner. Prostacyclin signaling through the IP receptor inhibits platelet aggregation, thereby inhibiting thrombosis (Moncada et al.1977). A1M-iloprost potently inhibited platelet aggregation induced by the thrombin receptor activating peptide SFLLRN (SEQ ID NO: 61) (FIG.7E). In contrast, A1M-S1P did not inhibit platelet aggregation to the same degree. Dose- response studies indicated an EC50of ~ 11 nM for free iloprost and ~9 nM for ApoA1- associated-iloprost (FIGs.8A-8B), suggesting a complete retention of potency when iloprost is associated with HDL-like nanodiscs. These data suggest that A1M supplemented with iloprost can function both as an anti-thrombotic as well as an endothelial barrier protective agent. A1M attenuates TNFα-induced NF-κB activity and ICAM-1 expression Intercellular adhesion molecule 1 (ICAM-1) is a cell surface glycoprotein and an adhesion receptor that regulates leukocyte recruitment from circulation to sites of inflammation (Bui et al.2020). HDL-bound S1P attenuates TNFα-induced ICAM-1 expression (Galvani et al. 2015; Cockerill et al.1995). In contrast, albumin-bound S1P did not suppress ICAM-1 expression, suggesting that S1PR1 agonism alone is not sufficient to suppress cytokine 12124565.1 inflammatory responses. The ApoA1 moiety of HDL has been suggested to engage endothelial cells to induce NO release (Yuhanna et al.2001) and suppress TLR4- and TNFα-induced activation of the proinflammatory transcription factor NF-κB in myeloid cells (Fotakis et al. 2019). This study showed that ApoA1, A1M, and lipidated A1M-S1P inhibited TNFα-induced NF-κB B activity ~25% in a luciferase-based NF-κB transcriptional reporter assay (FIG.9A). Moreover, A1M lipoprotein particles, regardless of whether the bound lipid mediator was S1P or iloprost, suppressed the TNF-α-induced increase in ICAM-1 expression in HUVECs (FIG.9B). In contrast, ApoM-Fc-S1P did not inhibit NF-κB activity or ICAM-1 up-regulation in response to TNF-α. Moreover, A1M-S1P induced cholesterol efflux in PMA-treated THP-1 cells (FIG. 9C), suggesting that similar to HDL particles, A1M-S1P can efflux cholesterol from cells by interacting with ATP-binding cassette transporters ABCA1 and ABCG1. This property of A1M likely promotes suppression of ICAM-1 expression. A1M suppresses inflammation in vivo Next, A1M-S1P was evaluated for its impact on the inflammatory responses in a murine sterile inflammation model. Thioglycolate injection into the peritoneum induces rapid neutrophil extravasation, resulting in acute inflammation (peritonitis) (Michaud et al.2006). It was found that the intraperitoneal injection of lipidated A1M-S1P particle, but not non-S1P loaded A1M, markedly suppressed neutrophil influx into the peritoneum (FIG.10A). Furthermore, peritoneal lavage fluid in A1M-S1P treated animals contained reduced amounts of inflammatory factors, including complement C5a, G-CSF, soluble ICAM-1, IL-6, IL-16, M-CSF and CCL2 (FIG.10B). These data provide evidence for an anti-inflammatory and EC barrier protective functions of A1M-S1P in vivo. To determine if systemic administration of nascent A1M suppresses the inflammatory response, intravenously injected recombinant A1M was evaluated for its ability to acquire S1P and become lipidated into HDL particles in vivo. Bacterial A1M fusion from E.coli (bA1M) was constructed and purified (FIGs.11A-11C). Nascent A1M was loaded with S1P and lipidated (bA1M-S1P) (FIG.11D). Similar to CHO cell-derived A1M, bA1M-S1P induced barrier function in HUVECs in a dose-dependent manner (FIG.11E). When bA1M was injected intravenously into mice, it was rapidly lipidated and appeared in the HDL fraction in plasma within 24 hours (FIGs.11F-11G). In addition, S1P content of plasma and HDL fractions were increased by 2.5 and 4.5-fold, respectively (FIGs.11H-11I), suggesting that intravenously injected nascent A1M protein picked up endogenous S1P and was lipidated into HDL particles. 12124565.1 Systemic A1M administration was evaluated for its influence on LPS-induced systemic inflammatory response in mice. C57Bl / 6 mice were dosed intravenously with bA1M before being intraperitoneally injected with LPS. Although A1M treatment did not change the murine sepsis score (FIG.12A), the LPS-induced decrease in body temperature was partially reversed by bA1M treatment (FIG.12B). Similarly, plasma IL-6 levels were suppressed by bA1M (FIG. 12C). A1M-injected mice contained significant levels of circulating A1M and 2.13-fold more plasma S1P (FIGs.12D-12E). These data suggest that the ApoA1 moiety of A1M suppressed systemic inflammatory responses induced by LPS in vivo. Discussion Vascular endothelial dysfunction during infections, trauma, metabolic diseases and aging has been identified as a primary event that drives chronic pathology. Endothelial injury drives vascular leak and the subsequent activation of the innate immune response. Circulating HDL particles have vascular endothelial protective properties (Rohatgi et al.2021; Mineo and Shaul 2013). Specifically, ApoA1, the principal structural protein of HDL that carries out reverse cholesterol efflux, attenuates cytokine driven inflammatory responses (Robert et al.2021). In addition, HDL binds to and stabilizes endothelial-derived prostacyclin, which inhibits platelet activation and thrombosis, blocks neutrophil adherence to the endothelium, and enhances endothelial barrier function (Birukova et al.2013). It was previously found that the HDL- associated protein ApoM is the physiological chaperone for S1P, which enhances endothelial barrier function (Christoffersen et al.2011; Christensen et al.2016) and protects from multiple organ injuries. The vascular endothelial protective properties of HDL were used to create a recombinant A1M fusion protein. A1M had similar biochemical properties to HDL particles while retaining stable S1P binding, which is intrinsic to ApoM. Transmission electron microscopy analysis suggested that the A1M-S1P complex exhibited conformational heterogeneity of 10-12 nm size HDL-like particles. It is likely that the flexible linker between the ApoA1 and ApoM components enables variable orientations of those components in the A1M-S1P complex. Only 5% of endogenous plasma-borne HDL particles from human and mice contain ApoM (Christoffersen et al.2011). The ApoM content of HDL was needed for S1P association. Given that plasma ApoM levels are decreased by pathological conditions such as sepsis, diabetes, and viral infections, as well as normal aging (Cao et al.2020; Bisgaard and Christoffersen 2019; Frej et al.2016; Marfia et al.2021; Winkler et al.2021), A1M designer HDL particles that contain 12124565.1 ~20x more ApoM than endogenous HDL may be able to replenish ApoM-S1P during pathological conditions. MD simulation analysis of the ApoM-S1P and A1M-S1P complexes led to several insights that either added to or concurred with the experimental data. As previously reported, the ApoM-S1P complex is stable, with the ligand S1P buried in the internal binding pocket of the lipocalin fold of ApoM (Christoffersen et al.2011; Zhang et al.2016; Swendeman et al.2017). The exterior lid of the ApoM moiety, which contains charged and polar residues, are thought to restrict dissociation of S1P from ApoM. The MD simulation study showed that A1M-S1P complex maintains the stable S1P binding property of the ApoM protein. However, the flexible linker between ApoA1 and ApoM enables high conformational flexibility of the ApoM-S1P complex in the A1M molecule. These studies suggest that A1M-S1P should allow both ApoA1 and ApoM portions of this fusion protein to function independently to regulate cellular functions. It is speculated that the ApoA1 interaction with its receptors on endothelial cells, such as scavenger receptor-B1 (Lee et al.2017), would dock A1M-S1P complex at the endothelial cell surface. Conformational flexibility of the linker and the stability of the ApoM-S1P complex would then allow productive interaction with the S1P receptors on cell surface to induce intracellular signals. HDL particles that contain ApoM exhibit anti-inflammatory properties of ApoA1 as well as the barrier protecting properties of S1P. Even though A1M-S1P can activate both Gαi and β- arrestin coupling, it shows a marked Gαi-biased signaling property. Although β-arrestin activation normally leads to termination of S1PR1 signaling, Gαi-biased A1M-S1P signaling is expected to activate EC protective pathways in a sustained manner, which was confirmed by TEER analysis. Additionally, it was previously shown that A1M-S1P activates endothelial S1PR1, S1PR2, and S1PR3, but preferentially activated the barrier protective S1PR1 rather than the barrier disruptive S1PR2 (Swendeman et al.2017). Both A1M and A1M-S1P were anti- inflammatory, reducing both TNFα-induced NF-κB activation in a reporter assay and inhibiting downstream inflammatory ICAM-1 expression in endothelium. It was observed that other S1P chaperones ApoM-Fc and albumin had no inhibitory activity, suggesting that the ApoA1 moiety is the primary inhibitor of TNFR signaling and inflammatory responses. Lipidated ApoA1 or A1M can bind to the stable PGI2 analog iloprost, consistent with the previous findings that PGI2 association with HDL or ApoA1 prolongs the half-life of this unstable lipid mediator by suppressing autohydrolysis (Pirich et al.1993; Yui et al.1988). This Example demonstrated that A1M / iloprost potently activated PKA-dependent CREB-signaling, promoted endothelial barrier function and completely inhibited human platelet aggregation. 12124565.1 This Example also shows that A1M-bound iloprost cooperated with A1M-S1P to stimulate EC barrier function, suggesting that the IP / Gαs / cAMP / EPAC / Rap1 and S1PR1 / Gαi / Rac1 pathways converge to assemble EC cell-cell junctions and enhance barrier function. Free iloprost has been previously suggested to be a protective factor in EC barrier integrity protection under endotoxic conditions (Birukova et al.2015). Indeed, this Example demonstrated the in vivo functionality of A1M-S1P in suppressing neutrophil infiltration into the peritoneum and inflammatory mediator secretion in a sterile peritonitis model of inflammation. The suppression of neutrophil influx by A1M-S1P but not A1M alone suggests that the ability of A1M-S1P to suppress inflammation- induced vascular leak is critical in the recruitment of neutrophils into the peritoneum. In the LPS model of endotoxemia, A1M suppressed cytokine storm in an ApoA1-dependent manner. These results suggest that A1M could be used not only to enhance endothelial barrier function but also to suppress inflammatory cytokine storm induction in vivo, which occurs in several pathological states. Inflammatory activation of the endothelium leads to thrombin formation, which not only disrupts the endothelial barrier, but also induces thrombosis (Mineo and Shaul 2013; Schmaier et al.2021). This is counteracted by the activated protein C (APC) pathway, which preserves the EC barrier and suppresses thrombin formation by the formation of APC and its cell surface functions. This Example demonstrated that A1M-S1P blocked thrombin-induced barrier disruption and enhanced the barrier-protective activity of APC. Moreover, it was shown that A1M-iloprost inhibited thrombin-driven platelet aggregation in vitro. Thus, A1M-bound S1P and iloprost would be predicted to suppress pathologic vascular permeability and thrombosis. Together, these results argue that recombinant A1M-bound S1P and prostacyclin provide a three-pronged approach to recapitulate the endothelial protective effects of HDL by enhancing endothelial barrier function through S1PR1 agonism, inhibiting inflammatory cytokine signaling with ApoA1, and dampening platelet activation and thrombotic responses by prostanoid receptor signaling. In contrast to small molecule S1PR functional antagonists, S1P chaperone-based receptor activation does not lead to inhibition of lymphocyte egress (Swendeman et al.2017). Therefore, it is suggested that A1M administration would not lead to lymphopenia and immune suppression. This approach has potential therapeutic utility in inflammatory and thrombotic states in which endothelial damage is driven by infectious pathogens, vascular reperfusion injury after trauma or in organ transplantation, as well as more chronic vasculopathies associated with atherosclerosis, diabetes, and autoimmunity. Materials and Methods 12124565.1 Creation of the A1M fusion constructs The A1M fusion was constructed using plasmids for murine ApoA1 (MR203500) and murine ApoM (MR201811) obtained from OriGene. The cDNA for ApoA1 was amplified to include the endogenous Kozak sequence and ORF of ApoA1 and to replace the stop codon with a codon for glycine. The cDNA for ApoM was amplified by sequential PCR to include a linker sequence encoding one copy of a five amino acid sequence linker (GGGGS; SEQ ID NO: 31) added to the 5’-end of ApoM, to remove the signal peptide (amino acids 1-20) and to insert a glycine codon and a 6X histidine sequence before the stop codon. The two PCR products were linked after NOT1 digestion and the final fusion PCR product was cloned into the pCDH-puro (Invitrogen) expression vector. Primers used for ApoA1 cloning were: 5’- TTTTCTAGAGAGGAGATCTGCCGCCGCGATCG-3’ (forward) (SEQ ID NO: 62) and 5’-TTTGCGGCCGCGTACGTCTGGGCAGTCAGAG-3’ (reverse) (SEQ ID NO: 63). Primers used for ApoM cloning were: 5’-GGTGGAGGTGGATCTAATCAGTGCCCTGAGCACAGT-3’ (forward) (SEQ ID NO: 64) and 5’GATGGTGATGTCCCTTGCTGGACAGCGGGCAGG-3’ (reverse) (SEQ ID NO: 65) or 5’-TTTGCGGCCGCTTGGTGGAGGTGGATCTAATCAGTG-3’ (forward) (SEQ ID NO: 66) and 5’-TTTGGATCCTCAGTGATGGTGATGGTGATGTCCCTTGCTGG-3’ (reverse) (SEQ ID NO: 67). Expression and purification of the A1M fusion protein from CHO-S cells The resulting pA1M plasmid was transfected into adherent CHO-S cells (A11364-01, Invitrogen) using polyethylenimine (PEI, Sigma). Positive transfectants were obtained by selection in Puromycin (30 µg / mL, GIBCO) for 4 days. Cells were tested for expression of the fusion protein by immunoblot analysis for ApoA1 (Abcam), ApoM (Abcam), and His-tag (Santa Cruz). The drug-selected recombinant CHO-S cells were adapted to serum-free suspension culture using CD FortiCHO medium (Thermo Fisher). For large-scale cultures, cells were suspended at 3-5 x 105cells per mL and maintained in culture to 2-4 x 106cells / mL in a spinner flask (Corning). To collect secreted A1M protein from the conditioned medium, cells were removed from culture by centrifugation at 800 x g for 10 minutes at 4°C. The culture supernatant was further clarified by ultracentrifugation at >100,000 x g for 30 minutes at 4°C. 12124565.1 The resulting supernatant was incubated with Ni-Sepharose beads (HisPur Ni-NTA resin, Thermo Fisher) at a final concentration of 2 mL of packed beads per 500 mL of culture overnight at 4°C. Next, beads were concentrated by centrifugation at 10,000 x g for 5 minutes at 4°C. Beads were washed with 50 volumes of His-wash buffer (20 mM Tris pH 8, 400 mM NaCl, 10 mM imidazole) until the flow through did not contain detectable protein. Protein was eluted from the column in the elution buffer (20 mM Tris pH 8, 400 mM NaCl, 300 mM imidazole). The resulting protein fractions were assayed using Bio-Rad protein reagent and were concentrated with Amicon Ultra-15 filters (Millipore Sigma). The final purified protein preparation was analyzed by SDS-PAGE and stained with Coomassie Brilliant Blue R-250 (Bio- Rad). Preparation of S1P, loading S1P onto A1M, mass spectrometry and electron microscope (EM) analysis S1P (1 mg) (Avanti Polar lipids) was resuspended in 13.4 mL of methanol and maintained at 37°C for 12 hours to achieve complete suspension. The solution was dispersed into 134 µl aliquots and dried under vacuum for 45 minutes to 1 hour at 37°C. Dried S1P was stored at -20°C until use. A method for loading S1P onto recombinant ApoM-Fc was previously described (Swendeman et al.2017). Essentially, purified protein was suspended in PBS at 1 mg / mL (~20 µM) and mixed with 160 µM S1P by gentle pipetting. The sample was subjected to 3 x 30 s sonication in a bath sonicator and allowed to incubate for >24 hours by nutation. The resulting product was subjected to FPLC to separate the free S1P from the A1M-S1P protein complex and the resulting protein fractions were concentrated using Amicon Ultra-15 filters. A1M-S1P was lipidated as below. S1P was loaded onto albumin as described (Lee et al.1998). Essentially, a 0.4% fatty acid free BSA (Sigma) solution in PBS was added to dried S1P and subjected to bath sonication for 3 cycles for 1 minute / cycle and maintained at 4°C for 24 hours. Mass spectroscopy-based quantitation of S1P was performed as described previously (Engelbrecht et al.2020). S1P-bound ApoM or A1M proteins or plasma from mice injected with these proteins was extracted in methanol (20:1 vol / vol methanol:analyte). For EM analysis, A1M samples were diluted to 0.02 mg / mL and 2.5 µL was applied to glow-discharged copper EM grids covered with a thin layer of carbon film. Grids were stained with 1.5% (w / v) uranyl formate, blotted, and allowed to air dry. Negatively stained EM grids were imaged on a Tecnai T12 electron microscope (Thermo Fisher Scientific) operated at 120 kV at a nominal magnification of 67,000 x using a 4k x 4k CCD camera (UltraScan 4000, Gatan), corresponding 12124565.1 to a pixel size of 1.68 Å. EM images were binned by two (3.36 Å per pixel), and particles were selected and subjected to two-dimensional classification (Ru et al.2015). Lipidation of A1M-S1P and A1M-iloprost A standard sonication and thermal cycling method for creating lipidated A1M was employed as described previously (Swendeman et al.2017; Oda et al.2006). A combination of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dimyristoyl-sn-glycero-3- phosphoglycerol (DMPG) (7:3 molar ratio) (Avanti) was resuspended in chloroform: methanol (95:5) and distributed into Eppendorf tubes and dried under vacuum. The lipid mixture was resuspended in PBS buffer, heated at 37°C for 5-10 minutes, and sonicated for 20 seconds to create a turbid lipid suspension. To generate lipidated A1M-S1P, S1P-loaded A1M was added to the lipid slurry (100:1 mol / mol lipid:protein). To generate A1M-iloprost, iloprost (Cayman Chemical) was dissolved in DMSO and added to the lipid solution at a final concentration of 1 mM. Purified A1M protein or human ApoA1 (Sigma) was resuspended in PBS and added to the lipid-iloprost slurry (100:1 mol / mol lipid:protein). Next, the solution was subjected to continuous bath sonication at 37°C for 30 min until the suspension exhibited clarification. The resulting lipidated solution was subjected to an FPLC purification (Swendeman et al.2017), and relevant fractions corresponding to recombinant HDL nanoparticles were collected and concentrated by Amicon Filters. TEER analysis of HUVEC To study endothelial barrier function, transendothelial electrical resistance (TEER) on primary human umbilical vein endothelial cells (HUVECs; PCS-100-013, ATCC) was performed using a 96-well ECIS system (96W10idf PET array, Applied BioPhysics), as described previously (Swendeman et al.2017). Essentially, HUVECs were maintained in HGM medium (M199 medium supplemented with 10% FCS, 1:100 penicillin-streptomycin, 8 mM glutamine, 2.5 U / mL heparin) and endothelial cell growth supplement (Hla and Neilson 1992) and on fibronectin (2 µg / mL in saline) coated plates. Wells were coated with fibronectin for 30 minutes at room temperature. HUVECs were harvested, resuspended in HGM medium at a cell density of 2.5-3 x 104cells / well, and allowed to adhere overnight. Prior to analysis, culture media was removed and replaced with M199 media supplemented with 1% FCS and penicillin- streptomycin / glutamine for 30 minutes. For stimulation studies, either iloprost (Cayman Biochemicals), ApoA1-iloprost, A1M-iloprost, A1M-S1P, or ApoM-Fc-S1P were added to cultures, and TEER studies were performed from 3-24 hours. To study thrombin-induced 12124565.1 endothelial barrier degradation, thrombin (Millipore Sigma) was used at a final concentration of 1 U / mL. Angiopoietin-1 (Ang-1, R&D systems) and activated protein C (APC, Enzyme Research laboratories) were used at a final concentration of 300 ng / mL and 5 μg / mL, respectively. For initial studies, Ang-1 was evaluated alone and in combination with ApoM-Fc- S1P. For thrombin studies, Ang-1, ApoM-Fc-S1P, and thrombin were co-added at the initiation of the study. For APC experiments, APC alone or in combination with A1M-S1P was added to the culture for 1 hour prior to the addition of thrombin, and TEER analysis was performed for an additional 2-8 hours. Data from TEER studies were analyzed using GraphPad Prism 7 (GraphPad Software, San Diego, CA). The endothelial barrier index (positive values) and endothelial barrier degradation (negative values) were measured by analyzing the area under the curve (AUC) of normalized TEER values for a given period of time. Nanobit analysis of S1P receptor activation The NanoBiT system employs split luciferase consisting of small and large fragments (SmBiT and LgBiT, respectively) and was previously utilized to monitor interactions of GPCRs with β-arrestin or specific Gα / βγ complexes in response to GPCR activation (Hisano et al. 2019). This system was used to characterize A1M nanoparticles containing S1P. Briefly, HEK293A cells (R70507, Invitrogen) maintained in DMEM (GIBCO) supplemented with 10% FCS and penicillin-streptomycin, were dispersed into 6 well plates and allowed to adhere overnight. For functional studies, cells were transfected with the appropriate combinations of reporter plasmids. S1PR1-SmBiT and LgBiT-β-arrestin fusion proteins or Gαi-SmBiT combined with Gβ1 and LgBiT-Gγ1 were employed as described previously (Hisano et al.2019). After 24 hrs, cells were harvested, resuspended with the luciferase substrate coelenterazine (Cayman Chem; 50 µM), dispersed into white opaque-bottom 96-well plates (Greiner) and maintained at room temperature for 2 hours to quench the background. S1P-containing samples were added by multi-channel pipetting and the plate was immediately analyzed for luminescence for 30 minutes in a SpectraMax L 96-well plate reader (Molecular Devices). Data was integrated as described (Hisano et al.2019). Inhibition of TNF-α-dependent NF-κB activation TNF-α-dependent NF-κB signaling was determined using an NFκB-Luciferase based reporter assay system (pGL4.32[luc2P / NF-κB-RE / Hygro]; Promega). A stable NFκB reporter cell line was created in human microvascular endothelial cells (HMEC-1; CRL-3243, ATCC). HMEC-1 cells were maintained in 10% FCS / penicillin-streptomycin MCDB media 12124565.1 supplemented with 2 mM L-glutamine (Sigma), EGF (2 ng / mL; R&D systems) and hydrocortisone (1 ng / mL; Sigma). The 5 x105reporter cells were distributed to 12 well plates and allowed to adhere for 24 hours. Media was replaced with MCDB media supplemented with 1% FCS and L-glutamine (2 mM). Cells were pre-treated with either media alone, purified human ApoA1 (Sigma), A1M, A1M-S1P, ApoM-Fc-S1P, or BSA-S1P for 10 minutes and 2 ng / mL of TNFα (R&D Systems). Samples were extracted using cell lysis buffers (Promega), luciferin substrate was added, and the plates were measured for luminescence for 8-15 minutes in a SpectraMax L 96-well plate reader (Molecular Devices). Inhibition of TNF-α-induced ICAM-1 expression in HUVECs 24 hours after plating, HUVECs were starved in M199 media supplemented with 1% FCS, 8 mM glutamine, and 1X penicillin-streptomycin. After 30 minutes, cells were pre-treated for 10 minutes with media, ApoM-Fc-S1P (100 nM S1P), iloprost (100 nM), A1M (200 μg / mL), A1M-iloprost (200 μg / mL with 100 nM Iloprost) or A1M-S1P (200 μg / mL with 100 nM S1P) or in combinations and subsequently treated with TNF-α (10 ng / mL). After 5 hours, cells were lysed with cell lysis buffer (TBS-T; 20 mM Tris-pH 8, 160 mM NaCl, 1% Triton, 1X protease inhibitor cocktail (Sigma)). Cell lysates were collected and centrifuged at 20,000 x g for 5 minutes at 4°C. The supernatant extracts were analyzed by 10% SDS-PAGE, transferred to nitrocellulose membrane (Bio-Rad), blocked in 5% milk, and probed with antibodies to ICAM-1 (G-5, 1:1,000, sc-8439, Santa Cruz Biotech) and actin (C-2, 1:5,000, sc-8432, Santa Cruz Biotech). Blots were developed with appropriate secondary antibodies linked to HRP and visualized by chemiluminescence (Immobilon Western, EMD Millipore) using a ChemiDoc Imaging (Bio-Rad) or Azure Imaging Systems (AZI600-1, Azure Biosystems). Iloprost-induced activation of the CREB–luciferase signaling The downstream cAMP-dependent CREB-luciferase reporter system (pGL4.29 [luc2P / CRE / Hygro]; Promega) was used to evaluate the activity of lipoprotein-bound Iloprost to activate the prostanoid receptor (IP). HEK293T (CRL-3216, ATCC) cells were maintained in DMEM supplemented with 10% FCS and penicillin-streptomycin. Cells were plated at 1 x 105cells / well in 96-well plates and allowed to adhere for 24 hours. Media was replaced, and cells were transfected using PEI (0.5 µg / well) with either 0.1 µg / well of reporter plasmid alone or co- transfected with pCDH-IP receptor (0.1 µg / well). After 24 hours, media was replaced, and cells were incubated with media containing either vehicle, iloprost, or A1M-iloprost by titration for 8 12124565.1 hours at 37°C. Samples were lysed, luciferin was added, and luminescence was measured for 30 minutes in a SpectraMax L 96-well plate reader (Molecular Devices). Inhibition of human platelet aggregation in vitro Human studies were approved by the institutional review board of the Beth Israel Deaconess Medical Center (PI: R. Flaumenhaft). Whole blood samples were drawn from healthy donors in the presence of 10% sodium citrate (Sigma, S577-50mL) and were spun at rcf of 168 xg using Beckman centrifuge (GS-6K) for 20 min. The top platelet-rich plasma layer was collected and rested in a 37ºC water bath for 30 min. The platelet-rich plasma layer was diluted with one-fifth volume of acid-citrate-dextrose buffer, and prostaglandin E1 (PGE1) was added to a final concentration of 0.15 µM. The mixture was spun in a conical-bottom tube at 2,000 g for 10 min, and the platelet pellet was collected and suspended in a pre-warmed HEPES-Tyrode- Glucose Buffer (HTG). The platelet concentration was determined and adjusted to ~2.5 x 105 / µL with HTG on a Hematology cell counter System (Drew Scientific, 850FS). Light transmission aggregometry was used to evaluate platelet responses to agonists and antagonists. In a 4-channel aggregometer (Platelet ionized calcium aggregometer, Chronolog Corp Model 660), platelets were stirred in a cuvette at 37°C. Human platelets were incubated with A1M-iloprost, ApoA1- iloprost, or A1M-S1P alone or in combination for ~10 min before adding the thrombin receptor (PAR-1) agonist SFLLRN peptide (2 µM). Data were analyzed using AGGRO / LINK software package Ver 5.2.5 and Microsoft Office Professional Plus 2013. Neutrophil Influx and cytokine content of peritoneal lavage fluid in thioglycolate-induced sterile peritonitis C57Bl / 6 mice were obtained from Jackson Labs (RRID: IMSR_JAX:000664, Bar Harbor, Maine). All in vivo experiments were performed according to an approved experimental protocol (1770) by IACUC at Boston Children’s Hospital. Mice were injected with 2 mL of 3% thioglycolate into the peritoneal cavity as described (Michaud et al.2006). One hour after thioglycolate induction, mice were intraperitoneally injected with either PBS (vehicle), or 200 µg of either A1M or A1M-S1P. After 4 hours, resident and infiltrating cells were isolated from peritoneal lavage by flushing with HBSS. Cells were washed, counted, and stained for flow cytometry using the neutrophil marker Ly6G (127608; BioLegend), gating for side-scatter and PE+ cells. Approximately 3.5 mL of peritoneal lavage fluid was collected from each mouse after thioglycolate induction (see above). After cell clarification by centrifugation at 300 x g for 5 12124565.1 min, 1 mL aliquots of fluid were prepared and immediately frozen in liquid N2. 500 μL of peritoneal fluid was analyzed for cytokine expression using the Proteome Profiler Mouse Cytokine array Kit, Panel A (ARY006; R&D Systems), following the manufacturer’s instructions. After development, blots were subjected to ImageJ analysis, and data were analyzed using GraphPad Prism 7 as above. In silico analyses of A1M-S1P and ApoM-S1P complexes Two protein-ligand systems were constructed: ApoM with S1P (ApoM-S1P) and ApoA1 fused to ApoM with S1P (A1M-S1P). The crystal structure with Protein Databank ID 2YG2 was used to construct ApoM-S1P (Christoffersen et al.2011). The APBS-PDB2PQR software suite was used to ensure protonation states (Jurrus et al.2018). All residues were included in their physiological protonation states (charged Glu, Asp, Lys, and Arg, all other residues neutral), except for His337, which was considered as double protonated / positively charged. His260, His277, and His386 were modeled as delta protonated and His317 as epsilon protonated. The substrate S1P was fully optimized, with a total charge of -1 (two negative charges on the phosphate and a positive ammonium group) using Open Babel (O’Boyle et al.2011) and ACPYPE (Sousa da Silva and Vranken 2012) (or AnteChamber PYthon Parser interfacE), a wrapper script around the ANTECHAMBER software (Wang et al.2006; Wang et al.2004). To construct the HDL complex, ApoA1 was fused to ApoM-S1P using the described linker. Due to its inherent high flexibility, ApoA1 exists in discoidal and spherical assemblies with varying lipid stoichiometries. Here, the double belt structure already modeled and stabilized by previously performed long molecular dynamics (MD) simulations was used (Jurrus et al.2018; Pourmousa et al.2018). Specifically, the double belt model for a nascent HDL of a disc containing 160:24:2 POPC:UC:ApoA1 (POPC: 1-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine; UC: unesterified cholesterol) in an antiparallel arrangement with LL5 / 5 registry was used (Segrest et al.1999). To perform the MD simulations, GROMACS 2019.4 (Pronk et al.2013; Tan et al.2022) with the CHARMM36 force field was used (Sippl 1993). To generate the final structures and input files for the MD simulations, CHARMM-GUI (Jo et al.2008; Jo et al.2014) was utilized using its Membrane / Bilayer builder. The ApoM-S1P and the A1M systems were composed of 38.082 and 408.528 atoms, respectively. The MD simulations were carried out in explicit solvent with the TIP3P water model, within the physiologic ionic concentration of 0.15 mM. To minimize electrostatic interactions between periodic images of the solute, a 10 Å separation was used between each edge of the box and the closest solute atom. The systems were subjected to an initial minimization using the steepest descent algorithm, 12124565.1 followed by various production replicas. Six replicas of 0.5 μs for ApoM-S1P and 6 replicas of 0.35 μs for the A1M-S1P system were run due to its high size (11 times bigger than ApoM- S1P). To ensure temperature coupling, a Nose-Hoover (Hoover 1985) thermostat was used with a time constant of 1 ps and to ensure pressure coupling, the semi-isotropic Parrinello–Rahman barostat was used (Aoki and Yonezawa 1992). In all MD simulations, the LINCS algorithm (Hess 2008) was used to constrain the bond lengths involving hydrogens. To treat long-range electrostatic interactions, periodic boundary conditions using fast smooth Particle-Mesh Ewald (Wang et al.2016) were used. To investigate the stability of the complexes, the RMSDs for all MD simulations were calculated. To highlight the main collective movements, the observed variation within the 3D atomic coordinates of the protein structures was reduced by identifying the significant PCs through PCA using the Bio3D package (Grant et al.2021) of the R programming language. The retrieved PCs were subsequently subjected to agglomerative hierarchical cluster analysis, another unsupervised technique, using the complete-linkage algorithm. Cluster analysis allowed grouping of the explored conformational space into subsets of similar structures. The binding interfaces were calculated using a 5 Å cut-off for the interatomic distance between protein residues and S1P. The sampled residues with permanency times higher than 40% of the simulation time for were chosen for further analysis. Because solvent accessible surface area (SASA) is a key factor for protein binding, it was assessed with FreeSASA within the vanddraabe R package (Patel et al.2014). The ratioSASA by residue was measured at the binding pocket (ratioSASA = A1M-S1PSASA / ApoM-S1PSASA). Hydrogen bonds were measured using the Visual Molecular Dynamics (VMD) package (Humphrey et al.1996). All figures / videos were generated using the PYMOL, Affinity Designer, iMovie, and R packages. Creation and characterization of the bacterial recombinant A1M (bA1M) fusion proteins The A1M fusion for bacterial expression contains the pET15b derived translation start site sequence with an embedded 6x histidine tag for purification followed by the murine ApoA1 amino acids 25-264 of the open reading frame, a flexible linker region (GGGGS; SEQ ID NO: XX), and amino acids 21-190 of murine ApoM with a stop codon. The bA1M fusion was expressed in bacteria using the pET15b prokaryotic expression vector (Novagen). The open reading frame from the pCDH-A1M-puro plasmid described herein was amplified by PCR using the following primers: 5’-TTTCATATGGATGAACCCCAGTCCCAATGGGACA-3’ (for ApoA1) (SEQ ID NO: 68) and 12124565.1 5’-TTTGGATCCTCACTTGCTGGACAGCGGGCAGGCCTCTT-3’ (for ApoM) (SEQ ID NO: 69). The resulting PCR products and the pET15b vector were digested using NdeI and BamHI (NEB), purified and ligated using standard protocols. Positive clones were transformed into the BL21(DE3) protein expression strain (NEB). Protein expression was performed using the standard IPTG induction protocols with several modifications. Overnight cultures were grown in terrific broth (TB) supplemented with 5 mM glucose and 100 μg / mL ampicillin. Cultures were diluted 1:30 in TB supplemented with 5 mM glucose and carbenicillin (Sigma) 100 μg / mL and maintained at 37˚C at 180 rpm until OD600 reached 0.9. Cultures were diluted with 2 volumes of TB / carbenicillin for 4.5-5.5 hours at 180 rpm, pelleted by centrifugation, and frozen at -80°C. Pellets were resuspended in 50 mM Tris buffer pH8, 100 mM NaCl, 10 mM benzamidine (Sigma) supplemented with lysozyme and DNaseI and rocked at room temperature for 1 hour. The suspension was subjected to polytron disruption and centrifuged at 100,000 x g for 30 minutes. The resulting pellet was thoroughly disrupted using the B-Per Reagent (ThermoScientific) supplemented with 10 mM benzamidine and centrifuged at 100,000 x g for 30 minutes. The final pellet was considered to be inclusion bodies. Inclusion bodies were disrupted in 6 M guanidine-HCl (Sigma) / 50 mM Tris pH8, rocked for 30 minutes, and centrifuged at 100,000 x g for 30 minutes. The resulting supernatant was mixed with HisPur Ni- NTA resin (ThermoFisher; 4 mL beads / L culture) for overnight bulk incubation at 4°C. The beads were washed with 50 volumes of wash buffer (PBS, 250 mM NaCl, 5 mM imidazole, 10 mM benzamidine) and protein was eluted as described herein. Eluted protein was refolded step- wise using a previously published dialysis protocol (Thomson et al.2012). The resulting protein was cleared of residual endotoxin using Pierce high-capacity endotoxin resin (ThermoFisher) according to the manufacturer’s protocol, concentrated with Amicon® Ultra-4 Centrifugal Filter Unit (Ultracel® - 10K) and protein concentration was determined using the Bio-Rad Protein Assay. Western blot analyses were performed on 0.5 μL of isolated plasma using protocols described herein using a rabbit monoclonal antibody specific for ApoM (EPR2904, ab91656, Abcam). Analysis of the effects of A1M on endotoxemia in vivo C57Bl / 6J mice were obtained from Jackson Labs (Bar Harbor, Maine). 12-week-old male mice were pre-treated for 1 hour with saline or 40 mg / Kg of purified bA1M by intravenous injection. Mice were administered LPS (E. coli O111:B4, L4130, Sigma-Aldrich) 10 mg / Kg by 12124565.1 intraperitoneal injection. Body temperature was measured at 0, 4, 8, and 12 hours post-injection using Temperature Controllers (TCAT-2DF, Physitemp). Clinical assessment was performed using the murine sepsis score (MSS) system based on observational characteristics (Shrum et al. 2014). At 12 hours, mice were sacrificed, and blood was collected by cardiac puncture and maintained in EDTA. Complete blood count (CBC) was performed on an aliquot of blood using the Hemavet 950 (Drew Scientific). Plasma was collected after centrifugation at 2,000 × g for 15 minutes at 4°C. Plasma was maintained at -80°C until analyzed by Western blot or ELISA. An IL-6 ELISA was performed on 10 μl of plasma using the mouse Duoset ELISA kit (R&D systems) and all data were derived by comparison to the supplied IL-6 standards. Cholesterol efflux assay The cholesterol efflux was performed with the Cholesterol Efflux Assay Kit (ab196985, Abcam) by tracing fluorescently labeled cholesterol. Briefly, 100 nM phorbol 12-myristate-13- acetate (PMA) for 72 h was used to differentiate THP-1 cells (TIB-202, ATCC) to macrophage- like phenotype. Cells were harvested and plated 1 × 105cells / well in a 96-well plate. After 4 h incubation, adherent cells were washed with serum-free medium and cultured in labeling medium in the dark for 1 h. After removing the labeling medium, cells were treated with the equilibration medium and incubated overnight. Cells were treated with cholesterol acceptors such as HDL and lipidated A1M particles for 5 h in the dark. The supernatant and cell lysates were collected and measured for fluorescence by plate reader at Ex / Em 485 / 523 nm. Cholesterol efflux from the labeled cells to HDL-like particles was calculated by dividing the fluorescence intensity (RFU) obtained for the supernatant by the sum of the fluorescence intensity of the supernatant and cell lysate of the same treatment. Statistical analysis Data are presented as means ± SD. Statistical analysis was performed using Prism software (GraphPad). Multiple comparison testing was performed according to the reference (Staffa and Surakowski 2020). P values < 0.05 were considered statistically significant. Table 2. Sequences 12124565.1 12124565.1 12124565.1 12124565.1 *In SEQ ID NOs: 20-25 and 27, n = 1, 2, 3, 4, or 5 12124565.1

Claims

CLAIMS What is claimed is:

1. A fusion protein comprising ApoA1 and ApoM, wherein ApoA1 comprises an amino acid sequence that is 90% identical to any one of SEQ ID NOs: 1, 3, or 5 and wherein ApoM comprises an amino acid sequence that is 90% identical to any one of SEQ ID NOs: 2 or 4.

2. The fusion protein of claim 1, wherein ApoA1 comprises the amino acid sequence of any one of SEQ ID NOs: 1, 3, or 5.

3. The fusion protein of claim 1 or claim 2, wherein ApoM comprises the amino acid sequence of any one of SEQ ID NOs: 2 or 4.

4. The fusion protein of any one of claims 1-3, wherein the ApoA1 is fused to the N- terminus of ApoM.

5. The fusion protein of any one of claims 1-3, wherein the ApoA1 is fused to the C- terminus of ApoM.

6. The fusion protein of any one of claims 1-5, wherein the ApoA1 and ApoM are fused via a linker, optionally wherein the linker is a peptide linker.

7. The fusion protein of claim 6, wherein the linker comprises the amino acid sequence of SEQ ID NO:

7.

8. The fusion protein of any one of claims 1-7, further comprising a signal peptide sequence.

9. The fusion protein of claim 8, wherein the signal peptide sequence comprises the amino acid sequence of SEQ ID NO:

6.

10. The fusion protein of claim 8 or claim 9, wherein the signal peptide is fused to the N- terminus of ApoA1. 12124565.

111. The fusion protein of any one of claims 1-7, wherein the fusion protein does not comprise a signal peptide sequence.

12. The fusion protein of any one of claims 1-11, further comprising a translation start site sequence.

13. The fusion protein of claim 12, wherein the translation start site sequence comprises the amino acid sequence of SEQ ID NO:

15.

14. The fusion protein of claim 12 or claim 13, wherein the translation start site sequence is fused to the N-terminus of ApoA1.

15. The fusion protein of any one of claims 1-14, comprising an amino acid sequence that is 90% identical to any one of SEQ ID NOs: 8-10.

16. The fusion protein of any one of claims 1-15, wherein the fusion protein comprises the amino acid sequence of any one of SEQ ID NOs: 8-10.

17. A nucleic acid molecule comprising a polynucleotide sequence encoding the fusion protein of any one of claims 1-16.

18. The nucleic acid molecule of claim 17, wherein the polynucleotide sequence comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 11, 17, or 18, optionally wherein the polynucleotide sequence comprises the nucleotide sequence of SEQ ID NO: 11, 17, or 18.

19. The nucleic acid molecule of claim 17 or claim 18, wherein the polynucleotide sequence is operably linked to a promoter.

20. The nucleic acid molecule of any one of claims 17-19, wherein the polynucleotide sequence is operably linked to a translation start site sequence.

21. The nucleic acid molecule of claim 20, wherein the translation start site sequence comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 16, optionally 12124565.1wherein the translation start site sequences comprises the nucleotide sequence of SEQ ID NO:

16.

22. A construct comprising the nucleic acid molecule of any one of claims 17-21.

23. The construct of claim 22, wherein the construct is a plasmid or a vector.

24. The construct of claim 23, wherein the vector is a viral vector.

25. A cell comprising the fusion protein of any one of claims 1-16, the nucleic acid sequence of any one of claims 17-21, or the construct of any one of claims 22-24.

26. The cell of claim 25, wherein the cell is a prokaryotic cell.

27. The cell of claim 26, wherein the prokaryotic cell is a bacterial cell.

28. The cell of claim 25, wherein the cell is a eukaryotic cell.

29. The cell of claim 28, wherein the eukaryotic cell is a human cell.

30. A lipoprotein comprising the fusion protein of any one of claims 1-16 and a lipid.

31. The lipoprotein of claim 30, wherein the lipid is a S1P receptor agonist or antagonist, or a prostaglandin agonist or antagonist.

32. The lipoprotein of claim 30 or claim 31, wherein the lipid is selected from the group consisting of a prostaglandin, sphingosine 1-phosphate (S1P), a leukotriene, and phosphatidyl choline.

33. The lipoprotein of claim 32, wherein the phosphatidyl choline is 1,2-dimyristoyl-sn- glycero-3-phosphocholine (DMPC) or 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG).

34. The lipoprotein of claim 30, wherein the lipid is Iloprost. 12124565.

135. The lipoprotein of claim 30, wherein the lipid is sphingosine-1-phosphate.

36. The lipoprotein of claim 30, wherein the lipid is 1,2-dimyristoyl-sn-glycero-3- phosphocholine (DMPC) and 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG).

37. The lipoprotein of claim 36, wherein the ratio of DMPC:DMPG is 7:3 molar ratio.

38. The lipoprotein of any one of claims 30-37, wherein the lipoprotein is non-covalently bound to the lipid.

39. The lipoprotein of any one of claims 30-37, wherein the lipoprotein is covalently bound to the lipid.

40. The lipoprotein of any one of claims 30-39, wherein the fusion protein comprises at least 60 mol% of the lipid.

41. The lipoprotein of any one of claims 30-39, wherein the fusion protein comprises less than 1 mol% of the lipid.

42. The lipoprotein of any one of claims 30-41, wherein the lipoprotein is incorporated into a nanoparticle.

43. The lipoprotein of claim 42, wherein the nanoparticle is a nanodisk.

44. The lipoprotein of claim 42 or claim 43, wherein the nanoparticle is 70% unlipidated fusion protein and 30% lipidated fusion protein.

45. The lipoprotein of any one of claims 42-44, wherein the nanoparticle is an HDL-like nanoparticle.

46. The lipoprotein of claim 45, wherein the HDL-like nanoparticle is between 8-12 nm in diameter. 12124565.

147. A pharmaceutical composition comprising the fusion protein of any one of claims 1-16, the lipoprotein of any one of claims 30-46, the nanoparticle of any one of claims 42-46, or the nanodisk of any one of claims 43-46.

48. A method of treating a subject having a disease or disorder associated with vascular endothelial dysfunction, the method comprising administering the fusion protein of any one of claims 1-16, the lipoprotein of any one of claims 30-46, or the pharmaceutical composition of claim 47.

49. The method of claim 48, wherein the disease or disorder is thrombosis, or thrombotic inflammation.

50. The method of claim 48, wherein the disease or disorder is cardiovascular disease, metabolic disorder, autoimmune disease, inflammatory disease, infectious disease, ocular disorder, or cancer.

51. The method of claim 50, wherein the cardiovascular disease is cerebrovascular disease, hypercholesterolemia, atherosclerosis, stroke, heart failure, peripheral artery disease, acute liver failure, or acute kidney failure.

52. The method of claim 50, wherein the metabolic disorder is diabetes, diabetic nephropathy, or diabetic retinopathy.

53. The method of claim 50, wherein the autoimmune disease is rheumatoid arthritis, systemic lupus erythematosus, or an autoimmune syndrome.

54. The method of claim 50, wherein the inflammatory disease is an acute inflammatory disease, a chronic inflammatory disease, acute respiratory distress syndrome, or sepsis.

55. The method of claim 50, wherein the infectious disease is a respiratory virus infection.

56. The method of claim 50, wherein the ocular disorder is retinal vascular disease or age- related macular degeneration. 12124565.

157. A method of reducing inflammation in a subject, the method comprising administering the fusion protein of any one of claims 1-16, the lipoprotein of any one of claims 30-46, or the pharmaceutical composition of claim 47.

58. A method of reducing systemic inflammation in a subject, the method comprising administering the fusion protein of any one of claims 1-16, the lipoprotein of any one of claims 30-46, or the pharmaceutical composition of claim 47.

59. The method of claim 57 or 58, further comprising lipidating the fusion protein or the lipoprotein in vitro.

60. The method of claim 59, wherein the fusion protein or the lipoprotein is lipidated in vitro with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dimyristoyl-sn-glycero-3- phosphoglycerol (DMPG).

61. The method of claim 60, wherein the ratio of DMPC:DMPG is a 7:3 molar ratio.

62. The method of any one of claims 57-61, wherein lipidation of the fusion protein or the lipoprotein in vitro produces HDL-like nanoparticles.

63. The method of claim 62, wherein the HDL-like nanoparticles are between 8-12 nm in diameter.

64. The method of claim 57 or 58, wherein the method does not comprise lipidating the fusion protein or lipoprotein in vitro.

65. The method of any one of claims 48-64, the method comprising administering a therapeutically effective amount of the fusion protein, the lipoprotein, or the pharmaceutical composition.

66. The method of any one of claims 48-65, the method comprising administering the fusion protein, the lipoprotein, or the pharmaceutical composition intravenously. 12124565.

167. The method of claim 57 or claim 58, wherein the inflammation is associated with TNFalpha-induced NF-kappaB activation.

68. The method of claim 57 or claim 58, wherein the inflammation is associated with cardiovascular disease, metabolic disorder, autoimmune disease, inflammatory disease, infectious disease, ocular disorder, or cancer.

69. The method of claim 68, wherein the cardiovascular disease is cerebrovascular disease, hypercholesterolemia, atherosclerosis, stroke, heart failure, peripheral artery disease, acute liver failure, or acute kidney failure.

70. The method of claim 68, wherein the metabolic disorder is diabetes, diabetic nephropathy, or diabetic retinopathy.

71. The method of claim 68, wherein the autoimmune disease is rheumatoid arthritis, systemic lupus erythematosus, or an autoimmune syndrome.

72. The method of claim 68, wherein the inflammatory disease is an acute inflammatory disease, a chronic inflammatory disease, acute respiratory distress syndrome, or sepsis.

73. The method of claim 68, wherein the infectious disease is a respiratory virus infection.

74. The method of claim 68, wherein the ocular disorder is retinal vascular disease or age- related macular degeneration.

75. The method of claim 72, wherein the acute inflammatory disease is peritonitis.

76. The method of any one of claims 48-75, further comprising administrating an anti- thrombin agent.

77. The method of claim 76, wherein the anti-thrombin agent is Angiopoietin-1 (Ang-1) or Activated Protein C (APC). 12124565.

178. A method of purifying an ApoA1-ApoM fusion protein from a cell, the method comprising: (i) obtaining the cells of any one of claims 25-29; (ii) lysing the cells; (iii) obtaining inclusion bodies from the lysed cells; (iv) denaturing the inclusion bodies to release the ApoA1-ApoM fusion protein; and, (v) refolding the ApoA1-ApoM fusion protein.

79. The method of claim 78, wherein step (ii) is performed using polytron disruption.

80. The method of claim 78 or claim 79, wherein step (iv) is performed with a chaotropic reagent.

81. The method of any one of claims 78-80, wherein step (v) is performed by step-wise dialysis.

82. The method of any one of claims 78-81, wherein step (v) is performed without arginine. 12124565.1

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