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Abstract

A cohort of anti-proliferative microRNAs (miRs) is disclosed, each member of the cohort (or combinations thereof) being a target for regulating cell proliferation (and migration) and affecting (or modulating) vascular remodeling, as well as the treatment of various vascular complications, vascular injuries, vascular diseases, and disorders, diseases, syndromes, and / or conditions affecting the blood vessels and / or vasculature of (for example) the human or animal body.

Description

[Technical Field]

[0001] The present application provides compositions, medicaments, and methods for use in the treatment or prevention of post-surgical vascular complications and / or injury or damage. [Background technology]

[0002] Abnormal proliferation of vascular smooth muscle cells is known to be involved in acute responses to vascular injury (eg, coronary artery bypass surgery using the saphenous vein) and chronic responses (eg, the development of atherosclerosis).

[0003] The only treatments for late vein graft failure are percutaneous coronary intervention or repeat bypass surgery, which are considered difficult and risky. Consequently, many efforts have focused on prevention (statins and antiplatelet drugs, e.g., aspirin), but these efforts do not address the elusive principle that early pathological graft remodeling after transplantation creates the necessary environment for promoting subsequent atherosclerosis. Therefore, preventing early pathological graft remodeling would improve long-term vein graft patency. Some surgical protocols, such as total arterial revascularization, have also been shown to reduce the incidence of vein graft failure, but they are often difficult to perform outside of specialized centers and carry associated risks for patients. If successful, the approach proposed here has the potential for widespread implementation. Summary of the Invention

[0004] The present disclosure is based on the discovery of a cohort of anti-proliferative microRNAs (miRs), each member of which (or combinations thereof) is a target for regulating cell proliferation (and migration) and impacts (or modulates) vascular remodeling and the treatment of various vascular complications, injuries, diseases, and disorders, diseases, syndromes, and / or conditions affecting the blood vessels and / or vasculature of (for example) the human or animal body.

[0005] Within the context of the present disclosure, any of the disclosed miRs may be targeted, for example, by modulating their intracellular expression levels. By way of example, the expression level of any of the disclosed miRs may be increased or decreased as needed. Without intending to be bound by theory, it has been shown that the expression level of any of the disclosed miRs is associated with potentially beneficial therapeutic effects. For example, and again without intending to be bound by theory, it has been found that targeting one or more of the disclosed miRs can alter or regulate vascular smooth muscle cell proliferation. This can be useful for modifying the composition / structure of the vascular wall after injury. Thus, the present disclosure provides a cohort of miRs, each of which can be targeted as a means for regulating (e.g., inhibiting) vascular smooth muscle cell proliferation and / or for the treatment or prevention of (vascular) complications, (vascular) injuries, and / or (vascular) diseases characterized by vascular smooth muscle cell proliferation.

[0006] The present disclosure provides compounds and compositions for a variety of therapeutic applications, uses of the compounds and compositions for the manufacture of therapeutically effective medicaments, and methods of treating various diseases, disorders, and conditions.

[0007] One particular application of the compounds / compositions, medicaments, uses and methods described herein may be in the treatment or prevention of vein graft failure / late vein graft failure.

[0008] Vascular smooth muscle cells (VSMCs) and VSMC-derived cells are the major source of plaque cells and extracellular matrix in all stages of atherosclerosis. As such, the compounds / compositions, medicaments, uses, and methods described herein may further be applied, for example, to the treatment or prevention of diseases or complications associated with vascular plaque formation and / or atherosclerosis.

[0009] It should be noted that as used herein, the term VSMCs can encompass human saphenous vein SMCs (HSVSMCs). Furthermore, throughout this specification, the terms "comprise" and / or "comprising" are used to indicate that an embodiment "comprises" the described features and, as such, may also include other features. However, in the context of this disclosure, the terms "comprise" and / or "comprising" encompass embodiments that "consist essentially of" or "consist of" the relevant features.

[0010] In a first aspect, the present disclosure provides a method for producing a pharmaceutical composition comprising: regulation of VSMC proliferation; Treating or preventing a disease or condition characterized by VSMC proliferation; Treatment or prevention of vein graft failure / late vein graft failure; Treatment or prevention of postoperative vein graft failure / late vein graft failure; Treatment or prevention of vein graft failure / late vein graft failure after coronary artery bypass surgery, including, for example, the use of the saphenous vein; Treatment or prevention of atherosclerosis; Treating, preventing, or modulating vascular remodeling; Treatment or prevention of vascular injury; and / or Treatment or prevention of atherosclerosis, a vascular injury in which VSMC proliferation is a key phenotype miR-modulating agents for use in

[0011] In a second aspect, the present disclosure provides a method for manufacturing a pharmaceutical composition comprising: regulation of VSMC proliferation; Treating or preventing a disease or condition characterized by VSMC proliferation; Treatment or prevention of vein graft failure / late vein graft failure; Treatment or prevention of postoperative vein graft failure / late vein graft failure; Treatment or prevention of vein graft failure / late vein graft failure after coronary artery bypass surgery, including, for example, the use of the saphenous vein; Treatment or prevention of atherosclerosis; Treating, preventing, or modulating vascular remodeling; Treatment or prevention of vascular injury; and / or Treatment or prevention of atherosclerosis, a vascular injury in which VSMC proliferation is a key phenotype The method of Methods are provided that include administering an miR-modulating agent to a subject in need thereof. The modulating agent can be administered in a therapeutically effective amount or a therapeutically modulating amount. The subject in need thereof can be a human or animal subject.

[0012] In a third aspect, the present disclosure provides a method for producing a pharmaceutical composition comprising: regulation of VSMC proliferation; Treating or preventing a disease or condition characterized by VSMC proliferation; Treatment or prevention of vein graft failure / late vein graft failure; Treatment or prevention of postoperative vein graft failure / late vein graft failure; Treatment or prevention of vein graft failure / late vein graft failure after coronary artery bypass surgery, including, for example, the use of the saphenous vein; Treatment or prevention of atherosclerosis; Treating, preventing, or modulating vascular remodeling; Treatment or prevention of vascular injury; and / or Treatment or prevention of atherosclerosis, a vascular injury in which VSMC proliferation is a key phenotype The present invention provides the use of a miR-modulating agent for the manufacture of a medicament for use in a method for treating a cancer.

[0013] It should be noted that the phrase "diseases or conditions characterized by VSMC proliferation" can encompass, for example, in-stent restenosis, arteriovenous fistula, intimal hyperplasia, vascular occlusion, and complications resulting from inhibited / restricted blood flow. As such, various methods, uses, or medicaments of the present disclosure can be used to: Treatment or prevention of in-stent restenosis; Treatment or prevention of arteriovenous fistulas; Treatment or prevention of intimal hyperplasia (hyperplasia); Treatment or prevention of vascular obstruction; and / or Treating or preventing complications resulting from restricted / restricted blood flow It can be used for.

[0014] Vascular remodeling is often abnormal, and therefore the uses, methods, and medicaments described herein can be utilized as a means to inhibit or prevent certain vascular remodeling events / processes. Abnormal vascular remodeling can lead to the development of vascular pathologies such as atherosclerosis, pulmonary hypertension, aortic aneurysms, and intimal thickening, which is the basis for saphenous vein graft failure. Therefore, the various methods, uses, or medicaments of the present disclosure can be used to: Treatment or prevention of pulmonary hypertension; Treatment or prevention of aortic aneurysms; and / or Treating, preventing, or modulating (e.g., preventing or inhibiting) intimal hyperplasia underlying saphenous vein graft failure It can be used for.

[0015] The various methods, uses, or medicaments of the present disclosure may be, for example: a subject suffering from a disease or condition characterized by VSMC proliferation; subjects with vein graft failure / late vein graft failure; a subject susceptible to or prone to a disease or condition characterized by VSMC proliferation; Subjects who are susceptible or prone to vein graft failure / late vein graft failure; subjects undergoing or recovering from coronary artery bypass surgery, including subjects undergoing or recovering from coronary artery bypass surgery using the saphenous vein; subjects with atherosclerosis; Subjects prone / predisposed to atherosclerosis; subjects suffering from vascular injury; and / or Subjects prone to or susceptible to vascular injury, including vascular injury in which VSMC proliferation is an important phenotype The composition may be applied or administered to a human or animal subject, including

[0016] "Modulation" when used in reference to "VSMC proliferation" can encompass any increase or decrease in the rate or occurrence / occurrence of VSMC proliferative events.

[0017] Thus, the miR-modulating agents of the present disclosure can be utilized as a means to inhibit (prevent or suppress) or stimulate (promote or increase) VSMC proliferation events. As one teaching, the miR-modulating agents of the present disclosure can be used to inhibit (prevent or suppress) VSMC proliferation events.

[0018] The degree of modulation due to the influence of the miR modulating agents of the present disclosure may be assessed relative to a "normal" or "control" level of VSMC proliferation, such as would occur in healthy / normal tissue not exhibiting a pathology associated with abnormal VSMC proliferation.

[0019] Those skilled in the art will be familiar with the term "microRNA" (or "miR"). MicroRNAs are small, non-coding RNA molecules that influence the regulation of gene expression. MicroRNAs are generated from either gene sequences or intron / exon sequences, and many are encoded by intergenic sequences. Specific examples of suitable miR modulating agents are described below, but a miR modulating agent of the present disclosure can be any molecule or compound that can increase or inhibit (decrease) the expression of a particular miR (e.g., one or more miRs described herein).

[0020] Within the context of the present disclosure, the term microRNA or "miR" refers to: (i) miR-1827, (ii) miR-332a-3p, (iii) miR-449b-5p, (iv) miR-491-3p, (v) miR-4774-3p, (vi) miR-5681b, and (vii) miR-892b The present invention encompasses any one or more miRs selected from the group consisting of:

[0021] As such, an "miR-modulating agent" is any compound or molecule for which modulation of the expression (e.g., overexpression) of any one or more of the miRs described herein (including any one or more of miRs (i)-(vii) above) has been determined not to (concomitantly) induce deleterious effects, such as apoptosis or senescence in VSMCs.

[0022] Furthermore, the present inventors have confirmed that regulating the expression of miR-892b simultaneously induces the effects of the present disclosure in HSVSMCs, HSVECs, and PSVSMCs.

[0023] Thus, the present disclosure: regulation of VSMC proliferation; Treating or preventing a disease or condition characterized by VSMC proliferation; Treatment or prevention of vein graft failure / late vein graft failure; Treatment or prevention of postoperative vein graft failure / late vein graft failure; Treatment or prevention of vein graft failure / late vein graft failure after coronary artery bypass surgery, including, for example, the use of the saphenous vein; Treatment or prevention of atherosclerosis; Treatment or prevention of vascular injury; and / or Treatment or prevention of atherosclerosis, a vascular injury in which VSMC proliferation is a key phenotype The present invention provides a modulator (e.g., a modulator that increases expression) of one or more miRs listed as (i)-(vii) above for any one or more of the therapeutic uses described herein, including:

[0024] Of the miRs listed above (known as "miR targets"), miR-1827, miR-332a-3p, miR-5681b, and / or miR-892b have been shown to have no significant effect on HSVEC proliferation, making these particular miRNAs of particular therapeutic interest. Without intending to be bound by theory, targeting these miRs may limit unwanted damage and / or off-target effects to or in the recipient's endothelium. Those skilled in the art will appreciate that this is crucial for the various therapeutic applications described herein, including, for example, the prevention of premature vein graft failure.

[0025] Thus, the present disclosure: regulation of VSMC proliferation; Treating or preventing a disease or condition characterized by VSMC proliferation; Treatment or prevention of vein graft failure / late vein graft failure; Treatment or prevention of postoperative vein graft failure / late vein graft failure; Treatment or prevention of vein graft failure / late vein graft failure after coronary artery bypass surgery, including, for example, the use of the saphenous vein; Treatment or prevention of atherosclerosis; Treatment or prevention of vascular injury; and / or Treatment or prevention of atherosclerosis, a vascular injury in which VSMC proliferation is a key phenotype For use in miR-1827, miR-332a-3p, miR-5681b, and / or miR-892b One or more modulators of (e.g., modulators that increase expression of) are provided.

[0026] The present disclosure provides: regulation of VSMC proliferation; Treating or preventing a disease or condition characterized by VSMC proliferation; Treatment or prevention of vein graft failure / late vein graft failure; Treatment or prevention of postoperative vein graft failure / late vein graft failure; Treatment or prevention of vein graft failure / late vein graft failure after coronary artery bypass surgery, including, for example, the use of the saphenous vein; Treatment or prevention of atherosclerosis; Treatment or prevention of vascular injury; and / or Treatment or prevention of atherosclerosis, a vascular injury in which VSMC proliferation is a key phenotype For the manufacture of medicines for miR-1827, miR-332a-3p, miR-5681b, and / or miR-892b Further provided are uses of one or more modulators of (eg, modulators that increase expression of).

[0027] More specifically, a modulator, e.g., a modulator that increases expression of miR-1827, miR-332a-3p, miR-5681b, and / or miR-892b, regulation of VSMC proliferation; Treating or preventing a disease or condition characterized by VSMC proliferation; Treatment or prevention of vein graft failure / late vein graft failure; Treatment or prevention of postoperative vein graft failure / late vein graft failure; Treatment or prevention of vein graft failure / late vein graft failure after coronary artery bypass surgery, including, for example, the use of the saphenous vein; Treatment or prevention of atherosclerosis; Treatment or prevention of vascular injury; and / or Treatment or prevention of atherosclerosis, a vascular injury in which VSMC proliferation is a key phenotype The method may be for use in a method of treating a disease comprising administering a modulating agent to a subject in need thereof. The modulating agent may be administered in a therapeutically effective amount and / or in an amount that increases expression of the corresponding miR.

[0028] The present disclosure provides: regulation of VSMC proliferation; Treating or preventing a disease or condition characterized by VSMC proliferation; Treatment or prevention of vein graft failure / late vein graft failure; Treatment or prevention of postoperative vein graft failure / late vein graft failure; Treatment or prevention of vein graft failure / late vein graft failure after coronary artery bypass surgery, including, for example, the use of the saphenous vein; Treatment or prevention of atherosclerosis; Treatment or prevention of vascular injury; and / or Treatment or prevention of atherosclerosis, a vascular injury in which VSMC proliferation is a key phenotype Further provided are modulators of miR-892b (eg, modulators that increase expression) for use in.

[0029] moreover, regulation of VSMC proliferation; Treating or preventing a disease or condition characterized by VSMC proliferation; Treatment or prevention of vein graft failure / late vein graft failure; Treatment or prevention of postoperative vein graft failure / late vein graft failure; Treatment or prevention of vein graft failure / late vein graft failure after coronary artery bypass surgery, including, for example, the use of the saphenous vein; Treatment or prevention of atherosclerosis; Treatment or prevention of vascular injury; and / or Treatment or prevention of atherosclerosis, a vascular injury in which VSMC proliferation is a key phenotype Also disclosed is the use of a modulator of miR-892b (eg, a modulator that increases expression) for the manufacture of a medicament for treating a disease.

[0030] A modulator, e.g., a modulator that increases expression of miR-892b, regulation of VSMC proliferation; Treating or preventing a disease or condition characterized by VSMC proliferation; Treatment or prevention of vein graft failure / late vein graft failure; Treatment or prevention of postoperative vein graft failure / late vein graft failure; Treatment or prevention of vein graft failure / late vein graft failure after coronary artery bypass surgery, including, for example, the use of the saphenous vein; Treatment or prevention of atherosclerosis; Treatment or prevention of vascular injury; and / or Treatment or prevention of atherosclerosis, a vascular injury in which VSMC proliferation is a key phenotype The method may be for use in a method comprising administering a modulating agent to a subject in need thereof. The modulating agent may be administered in a therapeutically effective amount and / or in an amount that increases expression of miR-892b.

[0031] An miR-modulating agent for any use or method described herein may take the form of an inhibitor of one or more miRs described herein. The term "MiR inhibitor" can encompass compositions or molecules that inhibit or reduce the expression, function, and / or activity of a miR, including, for example, one or more miRs described herein.

[0032] A modulating agent of the present disclosure may comprise an miR promoter, which is a molecule that increases expression of a corresponding miR (in a cell). The term "miR promoter" can encompass compositions or molecules that increase the expression, function, and / or activity of a miR, including, for example, one or more miRs described herein.

[0033] An miR promoter can, for example, contain a miR mimic—a nucleic acid that encodes a corresponding miR for expression in a cell. Those skilled in the art will understand that a nucleic acid encoding a particular miR, when introduced into a cell, will give rise to additional copies of that miR (additional copies that supplement any native copy(s) expressed by the cell) ultimately resulting in overexpression of that miR in the cell.

[0034] A suitable miR mimic can comprise a double-stranded RNA molecule that mimics the mature miR duplex.

[0035] The nucleic acid encoding the corresponding miR can comprise a stem-loop miRNA.

[0036] The nucleic acid can encode any miR described herein (including any of the miRs listed herein as (i) through (vii)).

[0037] A nucleic acid encoding an miR for expression may further comprise (or be operably linked to) a promoter element and a polyA element. The promoter element and polyA element may be "adjacent" to the miR encoding nucleic acid sequence.

[0038] The nucleic acid may be provided in the form of a vector for delivery to a cell.

[0039] The vector may comprise a viral vector.

[0040] The vector may comprise an adenoviral vector, such as HAdV5 or an adeno-associated virus (e.g., AAV1, AAV2, AAV3, AAV4, or AAV5), or a lentivirus.

[0041] The miR promoter, eg, a nucleic acid encoding a miR, can be packaged or included in a viral, adenoviral, or AAV5 vector.

[0042] MiR inhibitors suitable for use in the present disclosure may include, for example, small organic / inorganic molecules, proteins, peptides, amino acids, nucleic acids (including RNA, DNA, and / or synthetic or peptide nucleic acids (PNAs)), carbohydrates, lipids, antibodies (including antigen-binding fragments thereof), and the like.

[0043] Any miR modulating agent of the present disclosure may be administered directly to the vessel wall undergoing treatment (e.g., a vessel wall that has been surgically repaired and / or shows signs of disease and / or injury or damage). The miR modulating agent of the present disclosure may be administered directly to the saphenous vein wall. The miR modulating agent of the present disclosure may be administered directly to the vessel wall within the 30-minute therapeutic window during CABG, for example, when SVG is available between harvest and transplant. The miR modulating agent of the present disclosure may be packaged and administered within a vector, for example, a viral (adenoviral) vector.

[0044] The present disclosure provides the following miRs in cells: (i) miR-1827, (ii) miR-332a-3p, (iii) miR-449b-5p, (iv) miR-491-3p, (v) miR-4774-3p, (vi) miR-5681b, and (vii) miR-892b The present invention provides an adenovirus vector comprising a sequence for expression of one or more of the following:

[0045] The present disclosure provides compositions comprising a miR modulating agent of the present disclosure and one or more excipients.

[0046] The present disclosure further provides pharmaceutical compositions comprising a miR modulating agent of the present disclosure and one or more therapeutically acceptable excipients.

[0047] The composition or pharmaceutical composition of the present disclosure may comprise the following miRs: (i) miR-1827, (ii) miR-332a-3p, (iii) miR-449b-5p, (iv) miR-491-3p, (v) miR-4774-3p, (vi) miR-5681b, and (vii) miR-892b The miR modulator may comprise a miR modulating agent that is a mimic of one or more of:

[0048] The composition or pharmaceutical composition comprises regulation of VSMC proliferation; Treating or preventing a disease or condition characterized by VSMC proliferation; Treatment or prevention of vein graft failure / late vein graft failure; Treatment or prevention of postoperative vein graft failure / late vein graft failure; Treatment or prevention of vein graft failure / late vein graft failure after coronary artery bypass surgery, including, for example, the use of the saphenous vein; Treatment or prevention of atherosclerosis; and / or Treatment or prevention of vascular injury It may be for use in (i) or for use in the method of (ii).

[0049] Where the composition or pharmaceutical composition is for use in a method of treatment, the composition or pharmaceutical composition may be administered to a subject (as defined herein) in need thereof.

[0050] Furthermore, the composition or pharmaceutical composition comprises: regulation of VSMC proliferation; Treating or preventing a disease or condition characterized by VSMC proliferation; Treatment or prevention of vein graft failure / late vein graft failure; Treatment or prevention of postoperative vein graft failure / late vein graft failure; Treatment or prevention of vein graft failure / late vein graft failure after coronary artery bypass surgery, including, for example, the use of the saphenous vein; Treatment or prevention of atherosclerosis; and / or Treatment or prevention of vascular injury The compound may be used in the manufacture of a medicament for

[0051] Detailed Description The present disclosure will be described in further detail with reference to the following drawings, in which: [Brief explanation of the drawings]

[0052] [Figure 1]Figure 1 shows a high-throughput miRNA screen to identify novel miRNAs that regulate VSMC proliferation. A is a schematic diagram of the high-throughput miRNA screen design. B shows the distribution of miRNAs tested in the screen based on their effect on VSMC proliferation, expressed as fold change after normalization to VSMCs transfected with miR-CTRL (control). C is a micrograph of human VSMCs stained with DAPI (blue) and EdU (green) after treatment with each of the seven candidate miRNAs and miR-CTRL. D is a scatter plot of the fold change in PASMC proliferation (miR mimic vs. miR-CTRL). Each point represents one miRNA. Anti = growth inhibitory, Pro = growth promoting.

[0053] [Figure 2] Figure 2 shows an in vitro evaluation of the effect of overexpression of seven novel candidate miRNAs on HSVSMC proliferation. (A) Schematic diagram of the experimental design for miRNA overexpression using miR mimics in HSVSMCs. (B) Representative fluorescence-activated cell sorting (FACS) plots showing 5-ethynyl-2'-deoxyuridine (EdU) incorporation in HSVSMCs transfected with seven miRNA mimics or miR-CTRL. The gate (vertical line) indicates EdU-positive cells. (C) Flow cytometry quantification of EdU incorporation in HSVSMCs after transfection with each of the seven novel candidate miRNAs and subsequent stimulation with platelet-derived growth factor ββ (PDGF-ββ) / interleukin 1α (IL-1α) (n = 3–4). Statistical analysis and p values were obtained using mixed-effects analysis and Dunnett's test to correct for multiplicity. The mean of all columns was always compared to the mean of the miR-CTRL column. The n numbers correspond to different biological replicates. The p values are indicated on the graphs.

[0054] [Figure 3]Figure 3 shows the in vitro evaluation of the effects of overexpression of seven novel candidate miRNAs in HSVSMCs. (A) Representative images of scratch assays (n = 3) showing HSVSMCs stimulated with IL-1a / PDGF-BB at 0 and 24 h after scratching after transfection with miRNA mimics, miR-CTRL, and mock control. (B) Quantification of relative migration distance of HSVSMCs stimulated with IL-1a / PDGF-BB obtained using the ImageJ MRI wound healing tool (n = 3). (C) Quantification of caspase 3 activity after miRNA overexpression (measured at 405 nm, n = 3). The proteasome inhibitor MG-115 was used as a positive control for apoptosis induction. (D) Quantification of senescence-associated (SA) β-galactosidase (β-gal) activity after miR mimic transfection (measured at 405 nm, n = 3). Bleomycin (1 μg / mL) was used as a positive control for senescence induction. In all cases, Iman-Conover rank transformation was used to transform non-normal data, followed by repeated measures (RM)-ANOVA and Dunnett's test to correct for multiplicity. The mean of all columns was always compared to the mean of the miR-CTRL column. n numbers correspond to different biological replicates. p values are indicated on the graphs. "ns" means "non-significant."

[0055] [Figure 4]Figure 4: RNA sequencing and bioinformatics analysis revealed candidate targets for seven candidate miRNAs related to cell cycle, migration, and glycosylation. A. Number of differentially expressed genes up- and down-regulated after overexpression of each miRNA. B. Gene Ontology (GO) term analysis showing GO terms for all differentially expressed genes by overexpression of each miRNA, ranked based on enrichment. C. The most significant GO terms for the 125 commonly downregulated genes in Figure 4A, ranked by fold enrichment. D. Heatmap (as z-scores of log2[FPKM+1]) of all 99 genes included in the GO term annotated as "cell cycle" among the 125 commonly downregulated genes in Figure 4A. E is a schematic diagram of the KEGG cell cycle pathway, with genes significantly downregulated by candidate miRNA overexpression highlighted in green.

[0056] [Figure 5]Figure 5: miRNAs function through distinct sets of candidate targets, including cell cycle-related genes. A is a schematic diagram of the bioinformatics pipeline used to identify potential functional miRNA-mRNA interactions. B is a table showing i. downregulated genes, ii. a summary of predicted targets of the miRNAs by the multimiR tool, and iii. the number of intersections between i. and ii. (which represent candidate targets). C is a heatmap (as z-scores of log2[FPKM+1]) of all candidate targets of the miRNAs listed in 5B, sorted horizontally by their predicted targets based on the pipeline described in 5A. D is a STRING diagram of the relationship between commonly downregulated genes and candidate targets (168 genes) of each miRNA annotated with the "cell cycle" GO term.

[0057] [Figure 6] Figure 6 shows in vitro evaluation of the effects of seven novel candidate miRNAs in HSVECs. (A) Schematic of the experimental design for miRNA overexpression using miR mimics in HSVECs. (B) Flow cytometry quantification of EdU incorporation in HSVECs after transfection with each of the seven novel candidate miRNAs and subsequent stimulation with 10% FBS (n = 3). Statistical analysis results and p-values were obtained using Iman-Conover rank transformation for non-normal data, followed by RM-ANOVA and Dunnett's test for multiplicity correction. The mean of all columns was always compared to the mean of the miR-CTRL column. n numbers correspond to different biological replicates. p-values are indicated on the graph. "ns" means "non-significant." (C) Heatmap of the cell cycle process-enriched set of 125 commonly downregulated genes in Figure 4A (as z-scores of log2[FPKM+1]).

[0058] [Figure 7] Figure 7 shows the in vitro evaluation of the effects of seven novel candidate miRNAs in PSVSMCs. (A) Schematic diagram of the experimental design for miRNA overexpression using miR mimics in PSVSMCs. (B) Flow cytometry quantification of EdU incorporation in PSVSMCs after transfection with each of the seven novel candidate miRNAs and stimulation with 10% FBS (n = 4). Statistical analysis results and p-values were obtained using Iman-Conover rank transformation for non-normal data, followed by RM-ANOVA and Dunnett's test for multiplicity correction. The mean of all columns was always compared to the mean of the miR-CTRL column. n corresponds to different biological replicates. p-values are indicated on the graph. "ns" means "non-significant."

[0059] [Figure 8] Figure 8 shows adenovirus-mediated overexpression of candidate miRNAs in vitro and ex vivo. (A) Schematic diagram of the miRNA overexpression strategy for ex vivo organ culture of saphenous veins. (B) Vector map of adenovirus serotype 5 (Ad5) expressing stem-loop sequences of candidate miRNAs (human miR-892b is used as an example) driven by the cytomegalovirus (CMV) promoter, including a polyadenosine (polyA) tail at the end of the miRNA stem-loop sequence. (C) Schematic diagram of the infection strategy in primary human saphenous vein smooth muscle cells (HSVSMCs) or human venous tissue. Viruses expressing the target miRNA are incubated with cells or tissues at a specific multiplicity of infection (MOI), inducing overexpression of the mature miRNA following stem-loop processing. Additionally, an adenovirus vector expressing LacZ is used as a negative control.

[0060] [Figure 9] Figure 9 shows the expression of candidate miRNAs in basal HSVSMCs, i.e., saphenous vein tissue, followed by overexpression of the mimics in HSVSMCs. (A) Basal expression of each selected miRNA in quiescent primary HSVSMCs was quantified by quantitative reverse transcription PCR (RT-qPCR) in HSVSMCs after normalization with the engogenous expression control gene RNU48 and visualized as delta Ct. (B) Basal expression of each selected miRNA in RNA derived from human saphenous vein tissue lysate was normalized with the engogenous expression control gene RNU48 and visualized as delta Ct. (C) Relative quantification of each selected miRNA candidate in primary HSVSMCs after transfection with 50 nM of the miRNA mimic. 0.2% = quiescent HSVSMCs in basal conditions for 48 hours; PDGF / IL1a = IL-1α / PDGF-ββ stimulation for 48 hours; Mock = mock-transfected HSVSMCs.

[0061] [Figure 10] Figure 10 shows principal component analysis of RNA sequencing datasets in HSVSMCs. (A) Principal component analysis (PCA) of RNA sequencing datasets in miRNA-overexpressing HSVSMCs and controls for all patients. Different colors indicate paired samples: green for control, red for miRNA-overexpressing, and purple for quiesced HSVSMCs. Different shapes indicate different patients from which the HSVSMCs were derived. (B) PCA plots for each patient, respectively. Samples are color-coded as in Figure 10A.

[0062] [Figure 11]Figure 11 shows the top GO terms of miR-323a-3p, miR-449b-5p, miR-491-3p, and miR-892b candidate targets (>20 genes). The graph shows the 10 most significantly enriched GO terms for targets of miR-323a-3p, miR-449b-5p, miR-491-3p, and miR-892b.

[0063] [Figure 12] Figure 12 shows the validation of the screen in PASMC, CASMC, and HUVSMC. High-throughput microscopic imaging quantification of EdU incorporation in PASMC (n = 4), CASMC (n = 3), and HUVSMC (n = 3) transfected with seven miRNA mimics or miR-CTRL, as well as in a "mock" transfected control. Statistical analysis was performed using repeated measures (RM)-ANOVA and Dunnett's test to correct for multiplicity. p values for comparisons between miRNA mimic and miR-CTRL treatments are indicated on the graph. n corresponds to different biological replicates. DETAILED DESCRIPTION OF THE INVENTION

[0064] Functional screening identifies novel miRNAs that selectively inhibit vascular smooth muscle cell proliferation.

[0065] overview Aberrant vascular smooth muscle cell (VSMC) proliferation in response to vascular injury is a key driver of pathological remodeling in the vessel wall, a major underlying cause of vascular disease. Therefore, targeting VSMC proliferation represents a promising therapeutic opportunity.

[0066] the purpose To systematically identify novel miRNAs that selectively exhibit antiproliferative effects in VSMCs and explore their therapeutic value in pathological vascular remodeling.

[0067] Methods and Results A library of 2,000 human miRNA mimics was evaluated for their regulatory effects on VSMC proliferation in a high-throughput in vitro functional screen. miR-1827, miR-4774-3p, miR-5681b, miR-449b-5p, miR-491-3p, miR-323a-3p, and miR-892b were selected for further evaluation. Functional validation of these seven candidates in primary human saphenous vein smooth muscle cells (HSVSMCs) demonstrated that their overexpression significantly suppressed proliferation without inducing apoptosis or senescence, and six of them also significantly suppressed migration. RNA sequencing of HSVSMCs after transfection with each miRNA mimic revealed that expression of these seven candidate miRNAs resulted in transcriptome changes significantly associated with cell cycle regulation. Examination of the candidate targets of each miRNA revealed that, individually, these miRNAs function through distinct mechanisms that converge in regulating cell cycle processes.

[0068] Introduction Vascular remodeling is a critical process of adaptive structural changes in the vascular wall. It involves changes in vessel wall thickness, resulting in increased vascular resistance in response to pathological, hemodynamic, or iatrogenic insults. However, this process is often abnormal, leading to the development of vascular pathologies such as atherosclerosis, pulmonary hypertension, aortic aneurysms, and intimal hyperplasia, which underlie saphenous vein graft failure. Essential to the pathogenesis of the vascular remodeling process is the switch of resident VSMCs from a differentiated, quiescent phenotype to a dedifferentiated, hyperproliferative, and hypermigratory phenotype. 1-3 At the molecular level, it is triggered by injury to the endothelial cell layer in the vascular lumen and the subsequent inflammatory response, which releases growth factors and proinflammatory cytokines, such as platelet-derived growth factor BB (PDGF-BB) and interleukin-1α (IL-1A). 4This makes targeting VSMC proliferation an attractive therapeutic strategy for preventing adverse vascular remodeling in response to injury. Therapeutic approaches based on inhibiting VSMC proliferation have been successful preclinically and have shown promising clinical results, as demonstrated by animal and clinical studies testing the use of antiproliferative agents in drug-eluting stents used in coronary angioplasty. 5 However, a pitfall of these approaches is the inhibition of reendothelialization, a process essential for preventing subsequent pathological vascular remodeling events that lead to subsequent endothelial cell injury and denudation. In saphenous vein grafts, decoys of E2F family transcription factors (which are activators or cell cycle-related genes) have been successful in preventing intimal hyperplasia in vivo preclinically. Nevertheless, related clinical trials have failed because no suppression of vein graft failure events was observed. 6 These results highlight the need for the development of novel therapeutic approaches for vascular remodeling-related vascular diseases.

[0069] MicroRNAs (miRNAs) are small non-coding RNA molecules (20–24 nucleotides in length) that regulate gene expression by imperfectly base-pairing with regions in the 3'UTR of target messenger RNAs (mRNAs) and inducing their translational repression or degradation. miRNAs have been shown to play important roles in a range of biological contexts. 7 In cardiovascular physiology, dysregulation of several miRNAs has been linked to the occurrence of disease-related VSMC phenotypic switching by controlling various homeostatic processes in vascular cells. 8 Often, a single miRNA regulates VSMC function across diverse vascular disease contexts. A notable example of such regulation is the SMC-enriched miR-143 / 145 cluster, whose roles in neointimal lesion formation, pulmonary arterial hypertension, and atherosclerosis are well described. 9,10) Individual miRNAs can regulate the expression of multiple target mRNA transcripts, often associated with the same signaling pathway or biological process. The ability to modulate mRNA transcript abundance and induce downstream transcriptomic changes by using miRNA mimics, inhibitors, or viral vector-mediated overexpression of miRNA loci has led to the development of novel therapeutic approaches in the context of diverse diseases, including cardiovascular disease. 11,12 To date, numerous attempts have been made to modulate the endogenous levels of previously studied VSMC miRNA regulators in an effort to attenuate the effects of vascular injury. 13 However, none of them have progressed to clinical trials. [Example]

[0070] result Functional screening identifies miRNAs that effectively block early passage VSMC proliferation. To identify miRNAs that exhibit antiproliferative effects in VSMCs, we performed an in vitro high-throughput miRNA screen using a library containing mimics of 2,000 different human miRNAs annotated in miRbase v21 (Figure 1A). Early-passage primary human VSMCs were transfected with the miRNA mimic library for 72 hours under basal conditions. High-content fluorescence imaging analysis was then performed to measure proliferation by quantifying the incorporation of the thymidine analog 5-ethynyl-2'-deoxyuridine (EdU) and cell viability by counting cell number.

[0071] Of all the miRNAs tested, 1141 (55.8%) inhibited proliferation, while 903 (44.2%) promoted proliferation, with the degree of promotion reaching a maximum of 5.84-fold compared to transfection with the miRNA mimic control (miR-CTRL) (Figure 1B). 14 miRNAs inhibited proliferation to 0%. i) the ability to not significantly reduce cell viability as determined by a deviation from the mean of more than -1.65 standard deviations (cell count z-score > -1.65, p = 0.1); and ii) Novelty in the literature in any cardiovascular context Based on these criteria, seven of the most interesting candidates were selected for further consideration.

[0072] The selected miRNAs are miR-1827, miR-4774-3p, miR-5681b, miR-449b-5p, miR-491-3p, miR-323a-3p, and miR-892b (Figure 1C).

[0073] Overexpression of selected miRNA candidates suppresses the proliferation and migration of stimulated HSVSMCs without inducing apoptosis or senescence. Saphenous vein grafts are suitable for ex vivo therapeutic intervention during coronary artery bypass surgery because of the therapeutic window provided between harvest and transplantation during which ex vivo manipulation can prevent remodeling, as demonstrated in gene therapy studies in our laboratory. 14Therefore, we utilized this clinical setting to evaluate the effects of seven miRNA candidates identified in our initial screening. The endogenous expression profiles of the seven candidate miRNAs were analyzed in primary human saphenous vein VSMCs (HSVSMCs) and whole human saphenous vein tissue under basal conditions using quantitative reverse transcription-polymerase chain reaction (RT-qPCR). As shown in Figure 9A-B, the expression observed for each of the seven novel miRNAs in HSVSMCs and human saphenous vein was either absent or very low, as indicated by the delta cycle threshold (ΔCt) values after normalization by the housekeeping gene RNU48 (high ΔCt values indicate low expression). Furthermore, RT-qPCR in HSVSMCs after transfection with mimics confirmed significant overexpression of the miRNAs compared to miR-CTRL (Figure 9B).

[0074] Next, we tested whether overexpression of candidate miRNAs in stimulated HSVSMCs suppresses proliferation. After 48 hours of serum starvation to induce quiescence, HSVSMCs were transiently transfected with seven miRNA mimics and then stimulated with IL-1α / PDGF-ββ for 48 hours (Figure 2A). Flow cytometry quantification of EdU incorporation (Figure 2B) demonstrated that IL-1α / PDGF-ββ-induced proliferation was significantly suppressed by transfection with the seven miRNA mimics, ranging from 83.7 to 98.2%, compared with miR-CTRL (Figure 2C). This suppression of proliferation in HSVSMCs is consistent with and further supports the antiproliferative effects of candidate miRNAs on human SMCs in high-throughput screening.

[0075] We further investigated whether overexpression of novel candidate miRNAs affects HSVSMC migration. As shown in Figure 3A-B, six miRNAs significantly reduced the migration rate of HSVSMCs stimulated with IL-1α / PDGF-BB in a scratch wound assay.

[0076] To determine whether candidate miRNAs could induce adverse phenotypic effects that could explain the observed suppression of proliferation and migration in HSVSMCs, we measured the effects of miRNA overexpression on apoptosis and senescence, as measured by caspase 3 activity in HSVSMCs treated with the proteasome inhibitor MG-115. 15 None of the miRNAs induced apoptosis compared with bleomycin-treated cells (Figure 3C). This is consistent with the fact that no significant effect on cell number was observed in the miRNA screen, which would suggest a putative toxic effect induced by the miRNA. Furthermore, senescence-associated (SA) β-galactosidase activity was significantly reduced in bleomycin-treated cells. 16 No trend toward an increase was observed compared with HSVSMCs (Figure 3D). These results demonstrate that overexpression of candidate miRNAs suppresses IL-1α / PDGF-ββ-induced HSVSMC proliferation and migration without inducing apoptosis or senescence of HSVSMCs.

[0077] RNA sequencing in HSVSMCs reveals the regulation of a common network of cell cycle genes after overexpression of all seven miRNAs. To understand the effects of the seven miRNA candidates at the transcriptome level in HSVSMCs, we investigated the effects of the seven miRNA candidates on proliferating HSVSMCs overexpressing the candidate miRNAs stimulated with IL-1α / PDGF-ββ. 4RNA sequencing was performed on miR-CTRL and miR-CTRL, as well as quiescent HSVSMC samples. Principal component analysis (PCA) demonstrated distinct clusters of IL-1a / PDGF-BB-stimulated HSVSMCs compared with quiescent HSVSMCs (Figure 10). PCA analysis further demonstrated no overlap between miRNA mimic-treated HSVSMCs and their corresponding IL-1A / PDGF-BB controls (shown in green in Figure 10), and miRNA mimic-overexpressing HSVSMC samples (shown in red) also differed from quiescent HSVSMCs. This analysis reveals how overexpression of candidate miRNAs leads to distinct transcriptome changes in HSVSMCs, as described below. In Figure 10B, the similar patterns observed between patients are highlighted, with the effects of IL-1A / PDGF-BB stimulation (both with and without miRNA overexpression) in HSVSMCs clearly separating from those in quiescent HSVSMCs, and also separating between samples with simultaneous IL-1A / PDGF-BB stimulation and miRNA overexpression and IL-1A / PDGF-BB-stimulated controls.

[0078] As previously reported 17 To identify genes whose expression significantly differed by at least two-fold between miRNA mimic overexpression and miR-CTRL samples, we performed differential expression analysis. Because the samples were separated based on patient of origin in the PCA plot (Figure 10), we corrected for patient variation in differential gene expression analysis and detected a total of 389 to 1,033 differentially expressed genes after miRNA overexpression (Figure 4A). We performed gene ontology analysis for each list of differentially expressed genes corresponding to the overexpression of each candidate miRNA. We found that the top 10 enriched gene ontology (GO) terms for each miRNA overexpression were exclusively associated with cell cycle-related processes (Figure 4B). 18,19,20This is consistent with the strong effect of candidate miRNA overexpression on HSVSMC proliferation, indicating that the seven miRNAs appear to regulate similar downstream antiproliferative pathways.

[0079] Since the regulation of similar pathways by the seven miRNAs suggests common gene regulation, we evaluated the overlap of differentially regulated genes for each miRNA. We identified three commonly up-regulated genes and 125 commonly down-regulated genes by all seven candidate miRNAs. Essentially, miRNAs down-regulate mRNA expression. 7 Therefore, we focused on the 125 commonly down-regulated genes to examine the transcriptome changes common to the seven candidate miRNAs. GO term enrichment analysis revealed that the most enriched GO terms of these 125 common genes were related to cell cycle and mitosis (Figure 4C). We found that 99 of the 125 down-regulated genes were associated with the "cell cycle" GO term annotation (Figure 4D), while 17 of them were associated with the KEGG "cell cycle pathway," which includes key components required for cell cycle progression (Figure 4E). 21,22,23 This analysis suggests that overexpression of candidate miRNAs downregulates a common set of 125 genes that are most enriched for cell cycle-related processes.

[0080] Each of the novel miRNAs has distinct candidate downstream targets related to the cell cycle. To clarify the mechanism of action of each candidate miRNA, we aimed to identify its direct targets in IL-1A / PDGF-BB-stimulated proliferative HSVSMCs by a bioinformatics pipeline developed to predict functional miRNA-mRNA interactions for each candidate miRNA. We used multimiR, a prediction tool package that compiles several target prediction algorithms and obtains a comprehensive list of predicted targets for each novel candidate miRNA. 24 After generating a list of such predicted targets, we filtered for genes that were individually significantly downregulated after overexpression of each miRNA compared to the miR-CTRL condition (Figure 5A).

[0081] This resulted in the identification of a range of 3 to 173 candidate targets per individual candidate miRNA (Figure 5B), totaling 493 candidate targets when all targets of all seven miRNAs were combined. These candidate targets were characterized by both the potential presence of miRNA-3'UTR interactions and the resulting downregulation of gene expression. Of the 493 candidate targets identified, 93.5% were unique to a single miRNA, whereas only 32 genes (6.5%) were candidate targets for two or more miRNAs (Figure 5C). The genes targeted by the most candidate miRNAs were IGF2BP3 and TIMP3 (each targeted by four miRNAs). The high number of unique targets and the lack of complete overlap in candidate targets suggest that each miRNA has a distinct mechanism of action.

[0082] To understand whether a particular miRNA plays an important role in a specific biological process by targeting several genes from that process, we evaluated the Gene Ontology terms associated with miRNAs with more than 20 candidate targets (i.e., miR323-3p, miR491-3p, miR-449b-5p, and miR892b) (Figure ​(Figure11). 11). The miR-323a-3p target was only associated with significantly enriched GO terms related to "cell cycle," revealing that miR-323-3p is a putative regulator of various downstream cell cycle-related genes. For miR-892b, we identified an enriched GO term related to "spindle microtubules to kinetochore" (a process related to cell division), suggesting that the antiproliferative phenotype induced by miR-892b in HSVSMCs may be linked to downstream effects on the mitotic spindle. Other significantly enriched GO terms found for miR-323-3p, miR-892b, and two other candidate miRNAs with more than 20 candidate targets were “response to glucose,” “exercise,” “response to calcium,” and “lipid transport.”

[0083] We next evaluated whether each candidate miRNA could target either the set of 125 commonly downregulated genes identified by the differential expression analysis (Figure 6A) described above, or any genes annotated with the GO term "cell cycle." miR-323-3p, miR-449b-5p, and miR-892b can directly target candidates from the 125 commonly downregulated genes, as indicated by the differently-appearing boxes in Figure 5D. Next, we attempted to determine whether any of the seven miRNA candidate targets could be involved in cell cycle process, progression, and / or regulation using the GO term annotation "cell cycle." It was shown that each miRNA had at least one candidate target related to "cell cycle" (Figure 5D).

[0084] Taken together, this data reveals potential pathways and biological processes affected by overexpression of our seven novel miRNAs followed by mRNA inhibition in HSVSMCs. All seven miRNAs induced transcriptome changes by targeting minimally overlapping sets of candidate genes, suggesting that downstream mechanisms of action differ among miRNAs. The overall transcriptome changes resulting from overexpression of each novel miRNA demonstrate convergence toward effects on core cell cycle-related gene networks.

[0085] Overexpression of individual miRNA candidates differentially regulates HSVSMCs and HSVECs. It is important to maintain endothelial function and examine whether intervention may hinder HSVEC healing. Therefore, we focused on whether the miRNA candidates of the present invention can regulate the phenotype in HSVEC. To test the effect of the miRNA candidates on HSVEC proliferation, we induced quiescence for 12 hours, transfected with miRNA mimics, and then stimulated with FBS for 48 hours (Fig. 6A). Flow cytometry quantification of EdU incorporation showed that there was no significant change in proliferation after transfection with miR-1827, miR-323a-3p, and miR-5681b compared to miR-CTRL (Fig. 6B). By examining the regulation of the cell cycle process-enriched set of 125 commonly downregulated genes shown in Figure 4A in RNA-sequencing datasets of HSVECs stimulated with FBS and transfected with either miR-CTRL or mimics for candidate miRNAs (according to the schematic diagram in Figure 6A), we confirmed that in HSVSMCs, the regulation of these genes was clearly different between miR-CTRL conditions and miRNA mimic treatment, whereas in HSVECs, neither a distinctive pattern nor high variability was observed at the transcriptome level (Figure 6C).

[0086] Overexpression of miR-892b, miR-491-3p, miR-449b-5p, and miR-4774-3p could also significantly suppress PSVSMC proliferation in vitro. By testing the effects of novel miRNA candidates on porcine saphenous vein smooth muscle cell (PSVSMC) proliferation, we can obtain fundamental information regarding the potential efficacy of using large mammalian models of vascular injury (e.g., pigs). PSVSMCs were quiescent in basal medium for 48 hours, transfected, and stimulated with FBS (Figure 7A). Candidates miR-892b, miR-491-3p, miR-449b-5p, and miR-4774-3p significantly suppressed FBS-induced proliferation of PSVSMCs (Figure 7B). Of our seven miRNA candidates, only miR-323a-3p was annotated in the pig genome. UCSC Genome Browser 26 Using the genomic DNA sequencing technique, we identified sequences in the pig genome that share high homology with human pre- and mature miR-491-3p and miR-449b-5p (although there is no miRbase annotation suggesting that these two mature miRNAs may be expressed and functional in PSVSMCs). Therefore, these two miRNAs appear to be conserved. However, conservation is not required for significant phenotypic effects, as demonstrated by the significant effect of miR-892b on PSVSMC proliferation.

[0087] These results indicate that all seven novel candidate miRNAs are also capable of suppressing unwanted disease-inducing phenotypes in additional vascular cell types relevant to the vein graft setting, making them of further therapeutic and clinical interest.

[0088] miRNA overexpression strategy for ex vivo engineering of saphenous vein grafts Use a reproducible model of saphenous vein injury and culture to evaluate the effects of candidate miRNA overexpression, which is associated with increased VSMC proliferation and migration. 27,28Vein segments were exposed to miRNA mimics for 30 minutes, then stretched and cultured for 7 days. After treatment (as well as for the untreated control), tissues were harvested and either i) lysed for RNA isolation and quantification of candidate miRNA expression by qPCR or ii) fixed for subsequent staining of the VSMC marker myosin heavy chain 11 (Myh11), the proliferation marker PCNA, and mature miRNA candidates using miRNA in situ hybridization (Figure 8A). To demonstrate proof of principle for suppressing VSMC proliferation in this system after overexpression of candidate miRNAs, we focused on miR-892b, as it simultaneously demonstrated desirable effects in HSVSMCs, HSVECs, and PSVSMCs. However, other miRNA candidates remain interesting as therapeutic agents, and their effects will be pursued for further evaluation. Additionally, adenoviral vectors (Ad5) will be tested for candidate miR-892b to validate miRNA overexpression after viral transduction in HSVSMCs, as well as to validate the antiproliferative phenotype of the miRNA after adenoviral vector-mediated overexpression in these cells (Figure 8B-C). Both of these approaches will enable the selection of the most efficient overexpression strategy for candidate miRNAs in ex vivo saphenous vein segments.

[0089] Consideration Abnormally proliferative VSMCs are central players in the development of pathological vascular remodeling following stimuli that disrupt the integrity of the vessel wall. In saphenous vein graft failure, vascular trauma caused by surgical manipulation and post-transplant vascular mechanical stress leads to the activation of resident, quiescent VSMCs, characterized by increased proliferation and migration rates. Here, we aimed to identify novel miRNA therapeutics based on their ability to suppress VSMC proliferation. By screening for the most antiproliferative miRNA candidates (which were also characterized by their novelty in the literature), we created a list of seven miRNA candidates and evaluated their functional roles as inhibitors of cell proliferation and migration without simultaneously inducing apoptosis or senescence. To further analyze and evaluate their potential therapeutic efficacy, we used an ex vivo model of saphenous vein graft injury. This study successfully identified potential therapeutic candidates that not only suppress the pathological VSMC phenotype associated with vascular remodeling but also do not significantly affect HSVEC proliferation or significantly suppress PSVSMC proliferation. This makes them attractive potential therapeutic agents worthy of further investigation in the context of saphenous vein injury and ex vivo tissue culture, as well as subsequent injury-induced neointimal hyperplasia in vivo.

[0090] First, we used a systematic and unbiased approach to identify therapeutic miRNA candidates, particularly in the context of vascular remodeling, by high-throughput functional miRNA mimic screening. The miRNA library used contained mimics corresponding to the entire range of human mature miRNA sequences annotated in miRbase version 21. Therefore, our candidate selection was not limited by the existing miRNA literature and led to the identification of novel potential miRNA therapeutics. The overrepresented miRNA sequences in our screen included individually annotated -3p and -5p miRNA strands from all known miRNA sites, based on evidence that miRNA strands from the same miRNA site can have different, even opposing, effects. We hypothesized that because -3p and -5p sequences have different seed sequences, their downstream mRNA targets and subsequent effects on VSMC phenotype may also potentially differ. 29 .

[0091] Because our goal was to specifically identify miRNAs capable of suppressing VSMC proliferation associated with pathological vascular injury, we used early-passage VSMCs derived from patients with pulmonary arterial hypertension, which are characterized by a high basal proliferation rate, for miRNA screening to increase confidence in the results. Following the initial miRNA screening step, we performed a series of validation studies using a panel of different cell types. For further downstream evaluation of miRNA candidates, we have used an in vitro model of saphenous vein graft injury and are currently using an ex vivo model.

[0092] RNA sequencing allowed us to analyze the changes induced by overexpression of individual miRNAs at the transcriptome level.

[0093] Because we observed a strong antiproliferative phenotype in HSVSMCs overexpressing miRNAs in vitro, we were interested in whether candidate miRNAs could downstream regulate cell cycle-related processes at the transcriptome level. We identified a strong convergence toward cell cycle control by inducing downregulation of a central network of 125 genes. However, this common global effect on the transcriptome appears to be induced by different means for each miRNA, as indicated by its individual candidate target genes.

[0094] Following the identification of these candidate miRNAs, profiling their induced transcriptome changes by RNA sequencing, and evaluating their function across different types of SMCs and vascular cells, we conducted further studies to evaluate their effects in vitro using HSVECs and PSVSMCs, in silico using RNA sequencing of HSVECs, and in human vascular tissues. All miRNAs induced significant inhibition of PSVSMC proliferation and / or had no significant effect on HSVEC proliferation. These results intriguingly demonstrate the feasibility of using a porcine model of vein graft failure to test miRNA candidates, as well as the HSVSMC-specific antiproliferative effects exerted by some miRNAs, which may inhibit HSVEC healing and prevent the preservation of the endothelial layer, respectively. Candidate miR-892b was selected solely to demonstrate proof of concept for ex vivo miRNA delivery and in situ inhibition of VSMC proliferation in human primary tissues, based on its lack of significant inhibitory effects on HSVEC proliferation while simultaneously significantly suppressing PSVSMC proliferation. However, from the perspective of clinical translational interest, all miRNAs remain interesting candidates and merit further evaluation.

[0095] Although VSMC proliferation may be involved in the important process of adaptive vascular remodeling after vascular injury, it can also lead to intimal hyperplasia, subsequent vascular occlusion, and reduced blood flow. 30 The specific molecular mechanisms underlying the potential differences between injury-induced beneficial and aberrant VSMC proliferation remain to be explored. Consequently, for the purposes of this study, we evaluated the effects of miRNAs on the overall amount of VSMC proliferation observed in our ex vivo HSV model. Further research in this context is needed to provide a novel framework for the specific targeting of aberrantly proliferating VSMCs present in the injured vascular wall.

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Efficacy and Safety of Edifoligide, an E2F Transcription Factor Decoy, for Prevention of Vein Graft Failure Following Coronary Artery Bypass Graft Surgery. JAMA, 294(19), p.2446. 7. Bartel Dp. Micrornas: Genomics, Biogenesis, Mechanism, And Function. Cell. 2004 Jan 23;116(2):281-97. Doi: 10.1016 / S0092-8674(04)00045-5. Pmid: 14744438 8. Lu Y, Thavarajah T, Gu W, Cai J, Xu Q. Impact Of Mirna In Atherosclerosis. Arterioscler Thromb Vasc Biol. 2018 Sep;38(9):E159-E170. Doi: 10.1161 / Atvbaha.118.310227. Pmid: 30354259; Pmcid: Pmc6795547 9. Vacante F, Denby L, Sluimer JC, Baker AH. The function of miR-143, miR-145 and the MiR-143 host gene in cardiovascular development and disease. Vascul Pharmacol. 2019 Jan;112:24-30. doi: 10.1016 / j.vph.2018.11.006. Epub 2018 Nov 29. PMID: 30502421; PMCID: PMC6395947. 10. Vacante, F., Rodor, J., Lalwani, M., Mahmoud, A., Bennett, M., De Pace, A., Miller, E., Van Kuijk, K., de Bruijn, J., Gijbels, M., Williams, T., Clark, M., Scanlon, J., Doran, A., Montgomery, R., Newby, D., Giacca, M., O’Carroll, D., Hadoke, P., Denby, L., Sluimer, J. and Baker, A., 2021. CARMN Loss Regulates Smooth Muscle Cells and Accelerates Atherosclerosis in Mice. Circulation Research, 128(9), pp.1258-1275. 11. Rupaimoole, R., Slack, F. Microrna Therapeutics: Towards A New Era For The Management Of Cancer And Other Diseases. Nat Rev Drug Discov 16, 203-222 (2017). Https: / / Doi.Org / 10.1038 / Nrd.2016.246 12. Gabisonia, K., Prosdocimo, G., Aquaro, G.D. Et Al. Microrna Therapy Stimulates Uncontrolled Cardiac Repair After Myocardial Infarction In Pigs. Nature 569, 418-422 (2019). Https: / / Doi.Org / 10.1038 / S41586-019-1191-6 13. Qin, S. and Zhang, C., 2011. MicroRNAs in Vascular Disease. Journal of Cardiovascular Pharmacology, 57(1), pp.8-12. 14. Wan, S., George, S., Berry, C. 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Alexa A, Rahnenfuhrer J (2022). topGO: Enrichment Analysis for Gene Ontology. R package version 2.48.0. 21. Kanehisa, M. and Goto, S.; KEGG: Kyoto Encyclopedia of Genes and Genomes. Nucleic Acids Res. 28, 27-30 (2000). 22. Kanehisa, M; Toward understanding the origin and evolution of cellular organisms. Protein Sci. 28, 1947-1951 (2019) 23. Kanehisa, M., Furumichi, M., Sato, Y., Ishiguro-Watanabe, M., and Tanabe, M.; KEGG: integrating viruses and cellular organisms. Nucleic Acids Res. 49, D545-D551 (2021). 24. Ru Y, Kechris Kj, Tabakoff B, Et Al. The MultimiR R Package And Database: Integration Of Microrna-Target Interactions Along With Their Disease And Drug Associations. Nucleic Acids Res. 2014;42(17):E133. Doi:10.1093 / Nar / Gku631 25. Jensen LJ, Kuhn M, Stark M, Chaffron S, Creevey C, Muller J, Doerks T, Julien P, Roth A, Simonovic M, Bork P, von Mering C. STRING 8--a global view on proteins and their functional interactions in 630 organisms. 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Claims

1. (i) Regulation of VSMC proliferation; (ii) treating or preventing a disease or condition characterized by VSMC proliferation; (iii) treatment or prevention of vein graft failure / late vein graft failure; (iv) treatment or prevention of post-operative vein graft failure / late vein graft failure; (v) Treatment or prevention of vein graft failure / late vein graft failure after coronary artery bypass surgery, including, for example, the use of the saphenous vein; (vi) treating or preventing atherosclerosis; (vii) treating, preventing, or modulating vascular remodeling; (viii) treatment or prevention of vascular injury; and / or (ix) treating or preventing vascular injury in which VSMC proliferation is an important phenotype A miR modulator for use in

2. The disease or condition characterized by VSMC proliferation is (i) in-stent restenosis; (ii) arteriovenous fistula; (iii) intimal thickening (hyperplasia); (iv) vascular occlusion; and / or (v) Complications resulting from restricted / restricted blood flow The miR-modulating agent for use according to claim 1, comprising one or more selected from:

3. The miR modulator for use according to claim 1 , wherein the vascular remodeling is abnormal vascular remodeling.

4. The regulator is (i) atherosclerosis; (ii) pulmonary hypertension; (iii) aortic aneurysm; and / or (iv) Intimal hyperplasia underlying saphenous vein graft failure The miR modulator for use according to claim 1, which is used to treat or prevent

5. The miR modulating agent is an miR: (i) miR-892b; (ii) miR-1827; (iii) miR-332a-3p; (iv) miR-449b-5p; (v) miR-491-3p; (vi) miR-4774-3p; and / or (vii) miR-5681b The miR modulating agent of any one of claims 1 to 4, which increases the expression of one or more of:

6. The miR modulating agent is an miR: (i) miR-892b; (ii) miR-1827; (iii) miR-332a-3p; and / or (iv) miR-5681b The miR modulating agent of any one of claims 1 to 5, which increases the expression of one or more of:

7. The miR modulator of any one of claims 1 to 6, wherein the miR modulator increases expression of miR-892b.

8. 1. A miR modulator for use as a pharmaceutical, the miR modulator comprising: (i) miR-892b; (ii) miR-1827; (iii) miR-332a-3p; (iv) miR-449b-5p; (v) miR-491-3p; (vi) miR-4774-3p; and / or (vii) miR-5681b a miR modulator that increases the expression of one or more of the following:

9. The miR modulating agent is an miR: (i) miR-892b; (ii) miR-1827; (iii) miR-332a-3p; and / or (iv) miR-5681b The method of claim 8, wherein the modulator increases the expression of any one or more of the following:

10. The miR modulating agent is an miR: (i) miR-332a-3p; (ii) miR-449b-5p; (iii) miR-4774-3p; and / or (iv) miR-5681b The method of claim 8, wherein the modulator increases the expression of any one or more of the following:

11. The use of claim 8 , wherein the miR modulator increases expression of miR-892b.

12. The miR-modulating agent for use according to any one of claims 1 to 11, wherein the miR-modulating agent comprises, consists essentially of, or consists of a miR mimic.

13. The miR modulating agent for use according to any one of claims 1 to 12, wherein the miR modulating agent is provided in the form of a vector for delivery to a cell.

14. The miR modulating agent for use according to claim 13 , wherein the vector is a viral vector.

15. The miR-modulating agent of any one of claims 1 to 14, wherein the miR-modulating agent inhibits the abnormal proliferation and / or migration of VSMCs.

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