Treatment

Abstract

The invention discloses a group of anti-proliferative microRNAs (miRs). Wherein each member of the set (or a combination thereof) represents a target for modulating cell proliferation (and migration), affecting (or modulating) vascular remodeling, and treating various vascular complications, vascular injuries, vascular diseases, and, for example, disorders, diseases, syndromes, and / or conditions affecting the blood vessels and / or vascular systems of the human or animal body.

Description

Technical Field

[0001] The present disclosure provides compositions, medicaments, and methods for treating or preventing post-operative vascular complications and / or injury or damage. Background Art

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

[0003] There is no treatment for late vein graft failure, except for percutaneous coronary intervention or repeat bypass surgery, which is considered challenging and high-risk. Therefore, most efforts have focused on prevention (statins and antiplatelet drugs such as aspirin), but these do not address the root cause of the problem, which is early pathological remodeling of the graft after implantation that creates the necessary environment to promote superimposed atherosclerosis; therefore, prevention of early pathological graft remodeling will result in improved vein graft patency in the long term. Some surgical options (such as total arterial revascularization) have also been shown to reduce vein graft failure rates, but most are difficult to perform outside of a few specialized centers and have associated risks to the patient. If successful, the proposed approach has the potential to be widely implemented. Summary of the invention

[0004] The present disclosure is based on the discovery of a group of anti-proliferative microRNAs (miRs), wherein each member of the group (or a combination thereof) represents a target for regulating cell proliferation (and migration), affecting (or regulating) vascular remodeling, and treating various vascular complications, vascular injuries, vascular diseases, and (e.g.) disorders, diseases, syndromes and / or conditions affecting the blood vessels and / or vascular system of the human or animal body.

[0005] In the context of the present disclosure, any of the disclosed miRs can be targeted, for example, to regulate their expression levels in cells. For example, the expression level of any of the disclosed miRs can be increased or decreased as needed. Without wishing to be limited by theory, it has been shown that the expression level of any of the disclosed miRs is associated with potential beneficial therapeutic effects. For example, and again without wishing to be limited by theory, it has been found that it is possible to modify or regulate the proliferation of vascular smooth muscle cells by targeting one or more of the disclosed miRs. This may help to modify the architecture / structure of the vascular wall after injury. Therefore, the present disclosure provides a group of miRs, each of which can be targeted as a means of regulating (e.g., inhibiting) vascular smooth muscle cell proliferation, and / or treating or preventing (vascular) complications, (vascular) injuries and / or (vascular) diseases characterized by vascular smooth muscle cell proliferation.

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

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

[0008] Vascular smooth muscle cells (VSMC) and VSMC-derived cells are the main source of plaque cells and extracellular matrix in all stages of atherosclerosis. Therefore, the various compounds / compositions, drugs, uses and methods described herein can also be applied to, for example, 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 VSMC may include human saphenous vein SMC (HSVSMC). Furthermore, throughout this specification, the terms "comprise" and / or "comprising" may be used to indicate that an embodiment "comprises" the noted features, and therefore may also include other features. However, in the context of the present disclosure, the terms "comprise" and "comprising" encompass embodiments that "consist essentially of" or "consist of" the relevant features.

[0010] In a first aspect, the present disclosure provides miR modulators for use in:

[0011] Regulate VSMC proliferation;

[0012] Treating or preventing a disease or condition characterized by VSMC proliferation;

[0013] Treat or prevent vein graft failure / late vein graft failure;

[0014] Treat or prevent post-surgical vein graft failure / late vein graft failure;

[0015] Treating or preventing vein graft failure / late vein graft failure following coronary artery bypass surgery that includes, for example, the use of the saphenous vein;

[0016] Treat or prevent atherosclerosis;

[0017] Treat, prevent or modulate vascular remodeling;

[0018] Treat or prevent vascular damage; and / or

[0019] Treat or prevent vascular damage in which VSMC proliferation is an important phenotype.

[0020] In a second aspect, the present disclosure provides the following method:

[0021] Regulate VSMC proliferation;

[0022] Treating or preventing a disease or condition characterized by VSMC proliferation;

[0023] Treat or prevent vein graft failure / late vein graft failure;

[0024] Treat or prevent post-surgical vein graft failure / late vein graft failure;

[0025] Treating or preventing vein graft failure / late vein graft failure following coronary artery bypass surgery that includes, for example, the use of the saphenous vein;

[0026] Treat or prevent atherosclerosis;

[0027] Treat, prevent or modulate vascular remodeling;

[0028] Treat or prevent vascular damage; and / or

[0029] Treat or prevent vascular damage with VSMC proliferation as an important phenotype; the method comprises administering a miR regulator to a subject in need. The regulator can be administered in a therapeutically effective or regulating amount. The subject in need can be a human or animal subject.

[0030] In a third aspect, the present disclosure provides the use of a miR modulator for the preparation of a medicament for use in:

[0031] Regulate VSMC proliferation;

[0032] Treating or preventing a disease or condition characterized by VSMC proliferation;

[0033] Treat or prevent vein graft failure / late vein graft failure;

[0034] Treat or prevent post-surgical vein graft failure / late vein graft failure;

[0035] Treating or preventing vein graft failure / late vein graft failure following coronary artery bypass surgery that includes, for example, the use of the saphenous vein;

[0036] Treat or prevent atherosclerosis;

[0037] Treat, prevent or modulate vascular remodeling;

[0038] Treat or prevent vascular damage; and / or

[0039] Treat or prevent vascular damage in which VSMC proliferation is an important phenotype.

[0040] It should be noted that the phrase "diseases or conditions characterized by VSMC proliferation" may include, for example, in-stent restenosis, arteriovenous fistula, intimal thickening (hyperplasia), vascular occlusion, and complications caused by restricted / restricted blood flow. Therefore, the various methods, uses, or drugs disclosed herein may be used for:

[0041] Treat or prevent in-stent restenosis;

[0042] Treat or prevent arteriovenous fistulas;

[0043] Treat or prevent intimal thickening (hyperplasia);

[0044] Treating or preventing vascular occlusion; and / or

[0045] Treat or prevent complications caused by restricted / restricted blood flow.

[0046] Vascular remodeling is often abnormal, and thus the uses, methods, and medicaments described herein can be used as a means of inhibiting or preventing certain vascular remodeling events / processes. Abnormal vascular remodeling may lead to the development of vascular pathologies such as atherosclerosis, pulmonary hypertension, aortic aneurysm, and intimal hyperplasia caused by saphenous vein graft failure. Therefore, the various methods, uses, or medicaments disclosed herein can be used for:

[0047] Treat or prevent pulmonary hypertension;

[0048] Treating or preventing aortic aneurysms; and / or

[0049] Treating, preventing or modulating (eg, preventing or inhibiting) intimal hyperplasia resulting from saphenous vein graft failure.

[0050] Various methods, uses or medicaments disclosed herein can be applied or administered to human or animal subjects, including, for example:

[0051] A subject suffering from a disease or condition characterized by VSMC proliferation;

[0052] Subjects with vein graft failure / late vein graft failure;

[0053] A subject susceptible to or predisposed to a disease or condition characterized by VSMC proliferation;

[0054] Subjects susceptible or predisposed to vein graft failure / late vein graft failure;

[0055] Subjects undergoing or recovering from coronary artery bypass surgery, including subjects undergoing or recovering from coronary artery bypass surgery using the saphenous vein;

[0056] Subjects with atherosclerosis;

[0057] Subjects who are susceptible or prone to atherosclerosis;

[0058] Subjects with vascular damage; and / or

[0059] Subjects who are susceptible to or are prone to vascular injury, including vascular injury in which VSMC proliferation is an important phenotype.

[0060] The term "modulation" as applied to "VSMC proliferation" may encompass any increase or decrease in the rate or occurrence / incidence of VSMC proliferation events.

[0061] Therefore, the miR modulators of the present disclosure can be used as a means to inhibit (prevent or suppress) or stimulate (encourage or increase) VSMC proliferation events. In one teaching, the miR modulators of the present disclosure can be used to inhibit (prevent or suppress) VSMC proliferation events.

[0062] The extent of regulation effected by the miR modulators of the present disclosure can be assessed relative to "normal" or "control" levels of VSMC proliferation, which levels may occur in healthy / normal tissue that does not exhibit pathologies associated with abnormal VSMC proliferation.

[0063] Those skilled in the art will be familiar with the term "microRNA" (or "miR"). MicroRNAs are small non-coding RNA molecules that affect the regulation of gene expression. They are produced by gene sequences or intron / exon sequences; many are encoded by intergenic sequences. Specific examples of suitable miR modulators are described below, but the miR modulators of the present disclosure can be any molecule or compound that is capable of increasing or inhibiting (reducing) the expression of a particular miR (e.g., one or more miRs described herein).

[0064] In the context of the present disclosure, the term microRNA or "miR" includes any one or more miRs selected from the group consisting of:

[0065] (i) miR-1827

[0066] (ii) miR-332a-3p

[0067] (iii) miR-449b-5p

[0068] (iv) miR-491-3p

[0069] (v) miR-4774-3p

[0070] (vi) miR-5681b and

[0071] (vii) miR-892b.

[0072] Thus, a "miR modulator" is any compound or molecule. It has been noted that modulating the expression (e.g., overexpression) of any one or more miRs disclosed herein (including any one or more of the miRs (i)-(vii) listed above) does not (at the same time) induce deleterious effects, such as apoptosis or senescence in VSMCs.

[0073] Furthermore, the inventors have noted that modulating the expression of miR-892b produces the disclosed effects simultaneously in HSVSMCs, HSVECs, and PSVSMCs.

[0074] Therefore, the present disclosure provides modulators (e.g., modulators that increase expression) of one or more miRs listed in (i)-(vii) above, for use in any one or more therapeutic applications described herein, including:

[0075] Regulate VSMC proliferation;

[0076] Treating or preventing a disease or condition characterized by VSMC proliferation;

[0077] Treat or prevent vein graft failure / late vein graft failure;

[0078] Treat or prevent post-surgical vein graft failure / late vein graft failure;

[0079] Treating or preventing vein graft failure / late vein graft failure following coronary artery bypass surgery that includes, for example, the use of the saphenous vein;

[0080] Treat or prevent atherosclerosis;

[0081] Treat or prevent vascular damage; and / or

[0082] Treat or prevent vascular damage in which VSMC proliferation is an important phenotype.

[0083] Of the miRs listed above (also referred to 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 wishing to be bound by theory, targeting these miRs may limit unnecessary damage to or within the host endothelium and / or off-target effects; one skilled in the art will appreciate that this is extremely important for the various therapeutic uses described herein, including, for example, prevention of early vein graft failure.

[0084] Accordingly, the present disclosure provides modulators (e.g., modulators that increase expression) of one or more of the following:

[0085] miR-1827,

[0086] miR-332a-3p,

[0087] miR-5681b, and / or

[0088] miR-892b,

[0089] The regulator is used for the following purposes:

[0090] Regulate VSMC proliferation;

[0091] Treating or preventing a disease or condition characterized by VSMC proliferation;

[0092] Treat or prevent vein graft failure / late vein graft failure;

[0093] Treat or prevent post-surgical vein graft failure / late vein graft failure;

[0094] Treating or preventing vein graft failure / late vein graft failure following coronary artery bypass surgery that includes, for example, the use of the saphenous vein;

[0095] Treat or prevent atherosclerosis;

[0096] Treat or prevent vascular damage; and / or

[0097] Treat or prevent vascular damage in which VSMC proliferation is an important phenotype.

[0098] The present disclosure also provides the use of a modulator (e.g., a modulator that increases expression) of one or more of miR-1827, miR-332a-3p, miR-5681b, and / or miR-892b for the preparation of a medicament for:

[0099] Regulate VSMC proliferation;

[0100] Treating or preventing a disease or condition characterized by VSMC proliferation;

[0101] Treat or prevent vein graft failure / late vein graft failure;

[0102] Treat or prevent post-surgical vein graft failure / late vein graft failure;

[0103] Treating or preventing vein graft failure / late vein graft failure following coronary artery bypass surgery that includes, for example, the use of the saphenous vein;

[0104] Treat or prevent atherosclerosis;

[0105] Treat or prevent vascular damage; and / or

[0106] Treat or prevent vascular damage in which VSMC proliferation is an important phenotype.

[0107] More specifically, modulators (e.g., modulators that increase expression of miR-1827, miR-332a-3p, miR-5681b, and / or miR-892b) can be used in the following methods:

[0108] Regulate VSMC proliferation;

[0109] Treating or preventing a disease or condition characterized by VSMC proliferation;

[0110] Treat or prevent vein graft failure / late vein graft failure;

[0111] Treat or prevent post-surgical vein graft failure / late vein graft failure;

[0112] Treating or preventing vein graft failure / late vein graft failure following coronary artery bypass surgery that includes, for example, the use of the saphenous vein;

[0113] Treat or prevent atherosclerosis;

[0114] Treat or prevent vascular damage; and / or

[0115] Treat or prevent vascular damage in which VSMC proliferation is an important phenotype;

[0116] The method comprises administering the modulator to a subject in need thereof. The modulator can be administered in a therapeutically effective amount and / or in an amount that increases expression of the relevant miR.

[0117] The present disclosure also provides a modulator of miR-892b (e.g., a modulator that increases expression), for use in:

[0118] Regulate VSMC proliferation;

[0119] Treating or preventing a disease or condition characterized by VSMC proliferation;

[0120] Treat or prevent vein graft failure / late vein graft failure;

[0121] Treat or prevent post-surgical vein graft failure / late vein graft failure;

[0122] Treating or preventing vein graft failure / late vein graft failure following coronary artery bypass surgery that includes, for example, the use of the saphenous vein;

[0123] Treat or prevent atherosclerosis;

[0124] Treat or prevent vascular damage; and / or

[0125] Treat or prevent vascular damage in which VSMC proliferation is an important phenotype.

[0126] Also disclosed is the use of a modulator of miR-892b (e.g., a modulator that increases expression) for preparing a medicament for:

[0127] Regulate VSMC proliferation;

[0128] Treating or preventing a disease or condition characterized by VSMC proliferation;

[0129] Treat or prevent vein graft failure / late vein graft failure;

[0130] Treat or prevent post-surgical vein graft failure / late vein graft failure;

[0131] Treating or preventing vein graft failure / late vein graft failure following coronary artery bypass surgery that includes, for example, the use of the saphenous vein;

[0132] Treat or prevent atherosclerosis;

[0133] Treat or prevent vascular damage; and / or

[0134] Treat or prevent vascular damage in which VSMC proliferation is an important phenotype.

[0135] Modulators (e.g., modulators that increase miR-892b expression) can be used in the following methods:

[0136] Regulate VSMC proliferation;

[0137] Treating or preventing a disease or condition characterized by VSMC proliferation;

[0138] Treat or prevent vein graft failure / late vein graft failure;

[0139] Treat or prevent post-surgical vein graft failure / late vein graft failure;

[0140] Treating or preventing vein graft failure / late vein graft failure following coronary artery bypass surgery that includes, for example, the use of the saphenous vein;

[0141] Treat or prevent atherosclerosis;

[0142] Treat or prevent vascular damage; and / or

[0143] Treat or prevent vascular damage in which VSMC proliferation is an important phenotype;

[0144] The method comprises administering the modulator to a subject in need thereof. The modulator can be administered in a therapeutically effective amount and / or in an amount that increases miR-892b expression.

[0145] The miR modulators for any use or method described herein can take the form of inhibitors of one or more miRs described herein. The term "miR inhibitor" can include compounds or molecules that inhibit or reduce the expression, function and / or activity of miRs (including, for example, one or more miRs described herein).

[0146] Modulators of the present disclosure may include miR promoters, i.e., molecules that increase the expression of relevant miRs (in cells). The term "miR promoter" may include compounds or molecules that increase the expression, function and / or activity of miRs (including, for example, one or more miRs described herein).

[0147] miR promoters can include, for example, miR mimics—i.e., nucleic acids encoding the relevant miR for expression in cells. One skilled in the art will appreciate that, when introduced into a cell, the nucleic acid encoding a particular miR will provide additional copies of that miR—additional copies to supplement any natural copy (or copies) expressed by the cell, such that the end result is overexpression of that miR in the cell.

[0148] Suitable miR mimics can include double-stranded RNA molecules that mimic the mature miR duplex.

[0149] Nucleic acids encoding related miRs may include stem-loop miRNAs.

[0150] The nucleic acid may encode any miR described herein (including any of the miRs listed in (i)-(vii) herein).

[0151] The nucleic acid encoding the miR for expression may also comprise (or be operably linked to) a promoter element and a polyA element. The promoter element and the polyA element may 'flank' the miR encoding nucleic acid sequence.

[0152] Nucleic acids can be provided in the form of vectors for delivery to cells.

[0153] The vector may include a viral vector.

[0154] The vector may include an adenoviral vector, such as HAdV5 or an adeno-associated virus (such as AAV1, AAV2, AAV3, AAV4 or AAV5), or a lentivirus.

[0155] The miR promoter (eg, a nucleic acid encoding a miR) can be packaged or contained in a viral, adenoviral, or AAV5 vector.

[0156] 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-based nucleic acids, including PNA), carbohydrates, lipids, antibodies (including antigen-binding fragments thereof), and the like.

[0157] Any miR modulators disclosed herein can be directly applied to the vessel wall to be treated (e.g., a vessel wall that has been surgically repaired and / or a vessel wall that shows signs of disease and / or injury or damage). The miR modulators disclosed herein can be directly applied to the saphenous vein wall. The miR modulators disclosed herein can be directly applied to the vessel wall within a 30-minute clinical window, such as during CABG, when SVG is available between resection and implantation. The miR modulators disclosed herein can be packaged in a vector (e.g., a viral (adenoviral) vector) for administration.

[0158] The present disclosure provides adenoviral vectors comprising sequences for expressing one or more of the following miRs in a cell:

[0159] (i) miR-1827

[0160] (ii) miR-332a-3p

[0161] (iii) miR-449b-5p

[0162] (iv) miR-491-3p

[0163] (v) miR-4774-3p

[0164] (vi) miR-5681b and

[0165] (vii) miR-892b.

[0166] The present disclosure provides compositions comprising a miR modulator of the present disclosure and one or more excipients.

[0167] The present disclosure also provides pharmaceutical compositions comprising the miR modulators of the present disclosure and one or more pharmaceutically acceptable excipients.

[0168] The compositions or pharmaceutical compositions of the present disclosure may comprise a miR modulator that is a mimetic of one or more of the following miRs:

[0169] (i) miR-1827

[0170] (ii) miR-332a-3p

[0171] (iii) miR-449b-5p

[0172] (iv) miR-491-3p

[0173] (v) miR-4774-3p

[0174] (vi) miR-5681b and

[0175] (vii) miR-892b.

[0176] The composition or pharmaceutical composition can be used for (i) the following purposes, or (ii) the following methods:

[0177] Regulate VSMC proliferation;

[0178] Treating or preventing a disease or condition characterized by VSMC proliferation;

[0179] Treat or prevent vein graft failure / late vein graft failure;

[0180] Treat or prevent post-surgical vein graft failure / late vein graft failure;

[0181] Treating or preventing vein graft failure / late vein graft failure following coronary artery bypass surgery that includes, for example, the use of the saphenous vein;

[0182] Treating or preventing atherosclerosis; and / or

[0183] Treat or prevent blood vessel damage.

[0184] When the composition or pharmaceutical composition is used in a method of treatment, the composition or pharmaceutical composition may be administered to a subject in need thereof (as defined herein).

[0185] Furthermore, the composition or pharmaceutical composition can be used for the preparation of a medicament for:

[0186] Regulate VSMC proliferation;

[0187] Treating or preventing a disease or condition characterized by VSMC proliferation;

[0188] Treat or prevent vein graft failure / late vein graft failure;

[0189] Treat or prevent post-surgical vein graft failure / late vein graft failure;

[0190] Treating or preventing vein graft failure / late vein graft failure following coronary artery bypass surgery that includes, for example, the use of the saphenous vein;

[0191] Treating or preventing atherosclerosis; and / or

[0192] Treat or prevent blood vessel damage. DETAILED DESCRIPTION

[0193] Now, the present disclosure will be described in further detail with reference to the following drawings.

[0194] Figure 1: High-throughput miRNA screening identifies novel miRNAs that regulate VSMC proliferation. A, Schematic diagram of the high-throughput miRNA screening design. B, Density plot showing the distribution of miRNAs tested in the screen based on the effect of the tested miRNAs on VSMC proliferation, expressed as fold change normalized to miR-CTRL transfected VSMCs. C, Microscope images of human VSMCs stained with DAPI (blue) and EdU (green) after treatment with each of the 7 candidate miRNAs and miR-CTRL. D, Scatter plot of fold change in PASMC proliferation (miR mimics relative to miR-CTRL). Each dot represents a miRNA. Anti = antiproliferation, Pro = proproliferation.

[0195] Figure 2: In vitro evaluation of the effect of overexpression of 7 novel candidate miRNAs in HSVSMC proliferation. A, schematic diagram of the experimental design of overexpressing miRNAs in HSVSMCs using miR mimics. B, representative fluorescence-activated cell sorting (FACS) diagrams showing the uptake of 5-ethynyl-2'-deoxyuridine (EdU) in HSVSMCs transfected with 7 miRNA mimics or miR-CTRL. The gate (vertical line) represents EdU-positive cells. C, flow cytometry quantification of EdU incorporation in HSVSMCs after transfection with each of the 7 novel candidate miRNAs and subsequent stimulation with platelet-derived growth factor ββ (PDGF-ββ) / interleukin 1-α (IL-1α) (n=3-4). Multiple corrections were performed using mixed effects analysis and Dunnett's test to obtain statistical analysis and p values, in which the mean values ​​of all columns were always compared with the mean values ​​of the miR-CTRL columns. N numbers correspond to different biological replicates. The p value is included in the figure.

[0196] Figure 3: In vitro evaluation of the effects of overexpression of seven novel candidate miRNAs in HSVSMCs. A, Representative images of the scratch assay showing IL-1a / PDGF-bb stimulated HSVSMCs at 0 and 24 hours after scratching after transfection with miRNA mimics, miR-CTRL, and mimic control (n=3). B, Quantification of relative migration distance of IL-1a / PDGF-bb stimulated HSVSMCs obtained by 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 induction of apoptosis. D, Quantification of senescence-associated (SA) β-galactosidase (β-gal) activity after transfection with miR-mimics (measured at 405 nm, n=3). Bleomycin (1µg / mL) was used as a positive control for induction of senescence. In all cases, transformation of non-normal data was performed using Iman-Conover rank-order analysis followed by multiple correction using repeated measures (RM)-ANOVA and Dunnett's test to obtain statistical analysis and p-values, where 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 included in the figures. "Ns" means "not significant".

[0197] Figure 4: RNA sequencing and bioinformatics analysis revealed candidate targets of 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 shows the GO terms of all differentially expressed genes due to overexpression of each miRNA, ranked based on enrichment. C, Most important gene ontology (GO) terms of 125 commonly down-regulated genes in 4A, ranked by enrichment fold. D. Heat map of all 99 genes included in the GO terms annotated as “cell cycle” among the 125 commonly down-regulated genes in 4A (expressed as Z-scores of log2[FPKM+1]). E, Schematic diagram of the KEGG cell cycle pathway, with genes significantly down-regulated by overexpression of the candidate miRNA highlighted in green.

[0198] Figure 5: miRNAs act through different candidate target groups including cell cycle-related genes. A, Schematic diagram of the bioinformatics pipeline used to identify possible functional miRNA-mRNA interactions. B. Table showing the number of: i. downregulated genes, ii. predicted targets of miRNAs collected from the multimiR tool, and iii. the intersection of i. and ii., which represent candidate targets. C. Figure 5APipeline described in , ranked based on the levels of putatively targeted miRNAs, Heatmap of all candidate miRNA targets listed in 5B (as z-scores of log2[FPKM+1]). D, STRING schematic of the association between commonly down-regulated genes and candidate targets of each miRNA annotated under the “cell cycle” GO term (168 genes).

[0199] Figure 6: In vitro evaluation of the effects of 7 novel candidate miRNAs in HSVECs. A, Schematic diagram of the experimental design for overexpression of miRNAs in HSVECs using miR-mimics. B, Flow cytometric quantification of EdU incorporation in HSVECs after transfection with each of the 7 novel candidate miRNAs and subsequent stimulation with 10% FBS (n=3). Non-normal data were transformed using the Iman-Conover rank order, followed by multiple correction using RM-ANOVA and Dunnett's test to obtain statistical analysis and p-values, where the means of all columns were always compared to the mean of the miR-CTRL column. N numbers correspond to different biological replicates. p-values ​​are included in the figure. "Ns" means "not significant". C, from Figure 4A Heatmap of cell cycle process enriched groups of 125 commonly downregulated genes (as z-scores of log2[FPKM+1]).

[0200] Figure 7: In vitro evaluation of the effects of 7 novel candidate miRNAs in PSVSMCs. A, Schematic diagram of the experimental design for overexpressing miRNAs in PSVSMCs using miR-mimetics. B, Flow cytometry quantification of EdU incorporation in PSVSMCs after transfection with each of the 7 novel candidate miRNAs and stimulation with 10% FBS (n=4). Non-normal data were transformed using the Iman-Conover rank, followed by multiple corrections using RM-ANOVA and Dunnett's test to obtain statistical analysis and p-values, in which the mean values ​​of all columns were always compared to the mean values ​​of the miR-CTRL columns. N numbers correspond to different biological replicates. P values ​​are included in the figure. "Ns" means "not significant".

[0201] Figure 8: In vitro and ex vivo adenovirus-mediated overexpression of miRNA candidates. A, schematic representation of the miRNA overexpression strategy for ex vivo saphenous vein organ culture. B, vector diagram of adenovirus serotype 5 (Ad5) expressing the stem-loop of a candidate miRNA (taking human miR-892b in the schematic as an example) under a cytomegalovirus promoter (CMV), wherein the candidate miRNA includes a polyadenylic acid (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 (HSVSMC) or human venous tissue. Viruses expressing the microRNA of interest will be incubated with cells or tissues at a specific multiplicity of infection (MOI) to induce overexpression of mature microRNAs after processing the stem-loop. In addition, an adenovirus vector expressing LacZ will be used as a negative control.

[0202] Figure 9: Expression of candidate miRNAs in basal HSVSMCs, saphenous vein tissue, and after mimic overexpression in HSVSMCs. A, Basal expression of each selected miRNA in quiescent primary HSVSMCs was quantified by reverse transcription quantitative PCR (RT-qPCR) in HSVSMCs and shown as ΔCt after normalization using the endogenous expression control gene RNU48. B, Basal expression of each selected miRNA in RNA from human saphenous vein tissue lysates and shown as ΔCt after normalization using the endogenous expression control gene RNU48. C, Relative quantification of each selected miRNA candidate in primary HSVSMCs after transfection with 50 nM miRNA mimics. 0.2% = 48 h under basal conditions after quiescence, PDGF / IL1a = 48 h stimulation with IL-1α / PDGF-ββ, Mock = Mock-transfected HSVSMCs.

[0203] Figure 10: Principal component analysis of RNA sequencing datasets in HSVSMCs. A, Plot showing the principal component analysis (PCA) of RNA sequencing datasets of miRNA overexpressing HSVSMCs and controls performed on all patients. Different colors represent paired samples: green shades correspond to controls, red shades correspond to miRNA overexpression conditions, and quiescent HSVSMC conditions are represented in purple. Different shapes represent different patient origins of HSVSMCs used. B, Individual PCA plots for each patient. Color coding of samples is consistent with Fig. 10A similar.

[0204] Fig.11: Top GO term plot of miR-323a-3p, miR-449b-5p, miR-491-3p and miR-892b candidate targets (>20 genes) showing the 10 most significantly enriched GO terms for targets of miR-323a-3p, miR-449b-5p, miR-491-3p and miR-892b.

[0205] Fig.12 : Validation of screening 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 7 miRNA mimics or miR-CTRL and "simulated" transfection control.Multiple corrections were performed using repeated measures (RM) ANOVA and Dunnett's test for statistical analysis.The p-values ​​for comparison between miRNA mimics processing and miR-CTRL processing are included in the figure.N numbers correspond to different biological replicates.

[0206] Example

[0207] Functional screening identified novel miRNAs that selectively inhibit vascular smooth muscle cell proliferation.

[0208] summary

[0209] Aberrant vascular smooth muscle cell (VSMC) proliferation in response to vascular injury is a key driver of pathological remodeling of the vessel wall and a major underlying factor in vascular disease. Therefore, targeting VSMC proliferation represents a promising therapeutic opportunity.

[0210] Purpose

[0211] To systematically identify novel miRNAs that selectively exert antiproliferative effects in VSMCs and investigate their therapeutic value in pathological vascular remodeling.

[0212] Methods and Results

[0213] In a high-throughput in vitro functional screen, a library of 2000 human miRNA mimics was evaluated for their role in regulating VSMC proliferation. The following were selected for further evaluation: miR-1827, miR-4774-3p, miR-5681b, miR-449b-5p, miR-491-3p, miR-323a-3p, and miR-892b. Functional validation of these seven candidates in primary human saphenous vein smooth muscle cells (HSVSMCs) showed that their overexpression was able to significantly reduce proliferation without inducing apoptosis and senescence, with six of them also significantly reducing migration. RNA sequencing of HSVSMCs after transfection with the corresponding miRNA mimics revealed that overexpression of the seven candidate miRNAs resulted in transcriptome changes significantly associated with cell cycle regulation. By interrogating the candidate targets of each miRNA, we observed that these miRNAs act through mechanisms distinct from each other that converge in the regulation of cell cycle processes.

[0214] introduction

[0215] Vascular remodeling is an essential process of adaptive structural changes in the vessel wall. It involves changes in vessel wall thickness that confer increased vascular resistance in response to pathological, hemodynamic, or iatrogenic injury triggers. However, this process is often abnormal, leading to the development of vascular pathologies such as atherosclerosis, pulmonary hypertension, aortic aneurysms, and intimal hyperplasia caused by saphenous vein graft failure. An important component of the etiology of the vascular remodeling process is the transition of intrinsic VSMCs from a differentiated and quiescent state to a dedifferentiated, pro-proliferative, and pro-migratory phenotype. 1-3 At the molecular level, this is induced by the release of growth factors and inflammatory cytokines, such as platelet-derived growth factor BB (PDGF-BB) and interleukin-1a (IL-1A), following damage to the endothelial cell layer in the vascular lumen and the subsequent inflammatory response. 4 This makes targeting VSMC proliferation an attractive therapeutic strategy to prevent adverse vascular remodeling in response to injury. Therapeutic approaches based on reducing VSMC proliferation have been successful preclinically and show promising clinical results, as demonstrated in animal studies and clinical trials testing the use of antiproliferative agents in drug-eluting stents used during coronary angioplasty. 5 However, these approaches suffer from the disadvantage of interfering with the reendothelialization process, an essential process to counteract the subsequent pathological vascular remodeling events triggered by endothelial cell injury and denudation. In saphenous vein grafts, in vivo induction of E2F family transcription factors, which act as activators or inducers of cell cycle-related genes, successfully prevented intimal hyperplasia preclinically. Nevertheless, clinical trials failed due to the failure to observe a reduction in vein graft failure. 6These results highlight the need to develop novel therapeutic approaches for vascular diseases associated with vascular remodeling.

[0216] MicroRNAs (miRNAs) are small noncoding 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), inducing their translational repression or degradation. miRNAs have been shown to play key roles in various biological contexts. 7 In cardiovascular physiology, dysregulation of several miRNAs has been implicated in the development of disease-associated VSMC phenotypic switches by regulating various homeostatic processes in vascular cells. 8 , where single miRNAs often regulate VSMC function across multiple vascular disease contexts. A notable example of such regulation is the SMC-enriched miR-143 / 145 cluster, whose roles in neointimal lesion formation, pulmonary hypertension, and atherosclerosis are well described. 9,10 A single miRNA can regulate the expression of multiple target mRNA transcripts, which are often involved in the same signaling pathway or biological process. The ability to modulate their abundance to induce downstream transcriptome changes using miRNA mimics, inhibitors, or viral vector-mediated overexpression of miRNA loci has led to the development of novel therapeutic approaches in a variety of disease settings, including cardiovascular disease. 11,12 In the past, there have been many attempts to attenuate the effects of vascular injury by modulating the endogenous levels of previously studied VSMC miRNA regulators. 13 However, none of these approaches have entered clinical translation.

[0217] result

[0218] Functional screening identified miRNAs that effectively blocked proliferation of early passage VSMCs.

[0219] In vitro high-throughput miRNA screening was performed using a library containing 2000 different human miRNA mimics annotated in miRbase v21 to identify miRNAs that exert antiproliferative effects in VSMCs (Figure 1A). Primary human VSMCs of early passage were transfected with a library of miRNA mimics for 72 hours under basal conditions. Next, high-content fluorescent image analysis was performed to measure proliferation by quantitative incorporation of the thymidine analog 5-ethynyl-2'-deoxyuridine (EdU), as well as cell viability by assessing cell number.

[0220] Of all the miRNAs tested, 1141 (55.8%) reduced proliferation, while 903 (44.2%) miRNAs promoted proliferation, with the largest increase reaching 5.84-fold compared to transfection with a miRNA mimic control (miR-CTRL) (Figure 1B). 14 miRNAs reduced proliferation to 0%, of which 7 were considered the most interesting candidates based on the following:

[0221] i) their ability to not significantly reduce cell viability as identified based on a deviation from the mean exceeding -1.65 standard deviations (z-score for cell number > -1.65, p=0.1), and

[0222] ii) they are novel in any cardiovascular context literature.

[0223] These miRNAs are: miR-1827, miR-4774-3p, miR-5681b, miR-449b-5p, miR-491-3p, miR-323a-3p, and miR-892b ( Figure 1C ).

[0224] Overexpression of the selected miRNA candidates reduced the proliferation and migration of stimulated HSVSMCs without inducing apoptosis and senescence.

[0225] Saphenous vein grafts are suitable for ex vivo therapeutic intervention during coronary artery bypass surgery. This is because the therapeutic window created between resection and implantation can allow ex vivo manipulation to prevent remodeling, as achieved in gene therapy studies in our laboratory. 14 Therefore, this clinical setting was used to evaluate the effects of seven miRNA candidates identified through the initial screen. The endogenous expression profiles of the seven candidate miRNAs were characterized in primary human saphenous vein SVMCs (HSVSMCs) using reverse transcription quantitative polymerase chain reaction (RT-qPCR) under basal conditions and in whole human saphenous vein tissue. Figure 9A-9B As shown, no or very low expression of each of the seven novel miRNAs was observed in HSVSMCs and human saphenous vein, respectively, as indicated by the delta cycle threshold (Ct) values ​​normalized to the housekeeping gene RNU48 (higher ΔCt values ​​indicate lower expression). In addition, RT-qPCR in HSVSMCs transfected with miR-CTRL confirmed significant overexpression of the miRNAs ( Fig. 9B ).

[0226] Next, we tested whether overexpression of candidate miRNAs in stimulated HSVSMCs resulted in a reduction in proliferation. After 48 h of serum starvation to induce quiescence, HSVSMCs were transiently transfected with mimics of seven miRNAs and then stimulated with IL-1α / PDGF-ββ for 48 h (Figure 2A). Flow cytometry quantification of EdU incorporation (Figure 2B) showed that, compared with miR-CTRL, transfection of seven miRNA mimics significantly reduced IL-1α / PDGF-ββ-induced proliferation by 83.7% to 98.2% (Figure 2B). Figure 2C The decreased proliferation in HSVSMCs was consistent with and further confirmed the antiproliferative effects exerted by the candidate miRNAs on human SMCs in the high-throughput screen.

[0227] We also sought to evaluate whether overexpression of novel candidate miRNAs affects the migration of HSVSMCs. Figure 3A-3B As shown, six miRNAs significantly reduced the migration rate of IL-1a / PDGF-bb stimulated HSVSMCs assessed by scratch wound assay.

[0228] To test whether candidate miRNAs could induce deleterious phenotypic effects that could explain the reduced proliferation and migration observed in HSVSMCs, the effects of miRNA overexpression on apoptosis and senescence, as measured by Caspase-3 activity in HSVSMCs, were measured compared with treatment with the proteasome inhibitor MG-115. 15 No miRNA was found to induce apoptosis ( Figure 3C This is consistent with the non-significant effect on cell number observed in the miRNA screen, which could indicate a putative toxic effect induced by the miRNA. 16 Compared with HSVSMC, there was no trend of increased senescence-associated (SA) β-galactosidase activity ( Figure 3D ). These results indicate that overexpression of the candidate miRNAs reduced IL-1α / PDGF-ββ-induced HSVSMC proliferation and migration without causing HSVSMC apoptosis and senescence.

[0229] RNA sequencing in HSVSMCs revealed regulation of a common network of cell cycle genes upon overexpression of all seven miRNAs.

[0230] In order to understand the role of the 7 miRNA candidates in HSVSMCs at the transcriptome level, RNA sequencing was performed on IL-1α / PDGF-ββ stimulated, proliferating HSVSMCs with overexpression of candidate miRNAs compared to miR-CTRL, and quiescent HSVSMC samples. Principal component analysis (PCA) showed a clear aggregation of IL-1A / PDGF-BB stimulated HSVSMCs compared to quiescent HSVSMCs (Figure 10). PCA analysis also showed that there was no overlap between miRNA mimics treated HSVSMCs and their relative IL-1A / PDGF-BB controls (indicated by green hues in Figure 10), where miRNA mimics overexpressed HSVSMC samples (red hues) remained different from quiescent HSVSMCs. This analysis reveals how candidate miRNA overexpression leads to different transcriptome changes in HSVSMCs as described below. Fig. 10B This highlights a similar pattern observed between patients, where the effects of IL-1A / PDGF-BB stimulation (with and without miRNA overexpression) in HSVSMCs clearly separated these samples from quiescent HSVSMCs, with additional separation between samples undergoing both IL-1A / PDGF-BB stimulation and miRNA overexpression compared to IL-1A / PDGF-BB stimulated controls.

[0231] As mentioned above 17 Differential expression analysis was performed to identify genes that were at least two-fold significantly differentially expressed between miRNA mimics overexpression and miR-CTRL samples. Due to the separation of samples based on patient origin in the PCA plot (Figure 10), we corrected for patient differences in differential gene expression analysis and detected a total of 389 to 1033 differentially expressed genes after miRNA overexpression ( Figure 4A For each list of differentially expressed genes corresponding to each candidate miRNA overexpression, gene ontology analysis was performed, and it was found that the top 10 enriched gene ontology (GO) terms for each miRNA overexpression were exclusively related to cell cycle-related processes ( Figure 4B ) 18,19,20 This is consistent with the strong effects of candidate miRNA overexpression on HSVSMC proliferation and reveals that the seven miRNAs appear to regulate similar antiproliferative pathways downstream.

[0232] Since the regulation of similar pathways by the seven miRNAs indicated the regulation of common genes, we evaluated the overlap of genes differentially regulated by each miRNA. We identified three common up-regulated genes and 125 common down-regulated genes by all seven candidate miRNAs. Typically, miRNAs downregulate mRNA expression, so we focused on the 125 common down-regulated genes to investigate the transcriptome changes shared by the seven candidate miRNAs. GO term enrichment analysis found that the most enriched GO terms for 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 ), and 17 of them were associated with the KEGG “cell cycle pathway”, which includes major components required for cell cycle progression ( Figure 4E ) 21,22,23 This analysis revealed that overexpression of candidate miRNAs resulted in the downregulation of a common set of 125 genes that were primarily enriched in cell cycle-related processes.

[0233] Novel miRNAs have different downstream candidate targets that are involved in the cell cycle.

[0234] To characterize the mechanism of action of each candidate miRNA, we aimed to identify their direct targets in IL-1A / PDGF-BB-stimulated proliferating HSVSMCs by developing a bioinformatics pipeline for predicting functional miRNA-mRNA interactions for each candidate miRNA. We used multimiR 24 , a prediction toolkit that compiled several target prediction algorithms to obtain a comprehensive list of predicted targets for each candidate novel miRNA. After these predicted target lists were created, we filtered them for genes that were significantly downregulated after overexpression of each miRNA alone compared to the miR-CTRL condition ( Figure 5A ).

[0235] This resulted in the identification of candidate targets ranging from 3 to 173 per individual candidate miRNA ( Figure 5B ), if the total targets of all 7 miRNAs are added together, there are a total of 493 candidate targets. These candidate targets have the potential to both interact with existing miRNA-3'UTRs and to cause downregulation of gene expression. 93.5% of the 493 candidate targets identified were unique to a specific miRNA, while only 32 genes (6.5%) were candidate targets of 2 or more miRNAs ( Figure 5C). The genes most frequently targeted by candidate miRNAs were IGF2BP3 and TIMP3 (each targeted by 4 miRNAs). The large number of individual targets and the lack of complete overlap of candidate targets suggest that each miRNA has a unique mechanism of action.

[0236] For miRNAs with more than 20 candidate targets (i.e., miR323-3p, miR491-3p, miR-449b-5p, and miR892b), we evaluated the gene ontology terms associated with these genes ( Fig.11 ) to understand whether a specific miRNA plays a key role in a specific biological process by targeting several genes from this process. It was shown that the targets of miR-323a-3p were only associated with significantly enriched GO terms related to "cell cycle", indicating that miR-323-3p is a putative regulator of multiple cell cycle-related genes downstream. For miR-892b, we identified enriched GO terms related to "spindle microtubules to centromeres", a process related to cell division, indicating that the anti-proliferative phenotype induced by miR-892b in HSVSMCs may be related to downstream effects on the spindle. Other significantly enriched GO terms found for miR-323-3p, miR-892b, and the other two candidate miRNAs with more than 20 candidate targets included "response to glucose", "movement", "response to calcium", and "lipid transport".

[0237] Next, we evaluated whether each candidate miRNA could target any of the following genes: i. The set of 125 commonly down-regulated genes identified by differential expression analysis above ( Fig. 6A ), or ii. any gene annotated under the "cell cycle" GO term. Figure 5D As shown in the boxes of different shapes in Figure 2, miR-323-3p, miR-449b-5p, and miR-892b are able to directly target candidate genes from the 125 commonly down-regulated genes. Next, we attempted to use the GO term annotation “cell cycle” to reveal whether any of the 7 miRNA candidate targets may be involved in cell cycle process, progression, and / or regulation. It was found that each miRNA had at least one candidate target related to “cell cycle” ( Figure 5D ).

[0238] Thus far, the data reveal potential pathways and biological processes affected by mRNA repression following overexpression of our seven novel miRNAs in HSVSMCs. All seven miRNAs induced transcriptome changes by targeting minimally overlapping sets of candidate genes, suggesting that downstream mechanisms of action differ between miRNAs. The overall transcriptome changes produced upon overexpression of each novel miRNA revealed convergence in affecting a core network of cell cycle-related genes.

[0239] Overexpression of individual miRNA candidates differentially regulated HSVSMCs and HSVECs.

[0240] It is critical to protect endothelial function and test whether interventions may interfere with HSVEC healing; therefore, we were interested in whether our miRNA candidates could induce phenotypic modulation in HSVECs. To test the effects of miRNA candidates on HSVEC proliferation, we induced quiescence for 12 h, transfected with miRNA mimics, and subsequently stimulated with FBS for 48 h ( Fig. 6A Flow cytometry quantification of EdU incorporation showed non-significant changes in proliferation after transfection with miR-1827, miR-323a-3p, and miR-5681b compared with miR-CTRL ( Figure 6B ). The cells were stimulated with FBS and transfected with miR-CTRL or candidate miRNA mimics (according to Fig. 6A Schematic diagram of the RNA sequencing dataset in HSVEC (see Figure 2) Figure 4A We observed that while these genes were significantly differentially regulated between miR-CTRL conditions and miRNA mimics treatment in HSVSMCs, there were no unique patterns and high variability at the transcriptome level in HSVECs ( Figure 6C ).

[0241] Overexpression of miR-892b, miR-491-3p, miR-449b-5p, and miR-4774-3p could also significantly reduce the proliferation of PSVSMCs in vitro.

[0242] Testing the effects of novel miRNA candidates on the proliferation of porcine saphenous vein smooth muscle cells (PSVSMC) can provide necessary information on potential therapeutic efficacy using large mammalian models of vascular injury, such as pigs. PSVSMC were quiescent in basal medium for 48 hours, transfected and stimulated with FBS ( Fig. 7A Candidates miR-892b, miR-491-3p, miR-449b-5p, and miR-4774-3p significantly reduced FBS-induced PSVSMC proliferation ( Figure 7B Among our seven miRNA candidates, only miR-323a-3p was annotated in the porcine genome. 26, sequences highly similar to human pro- and mature miR-491-3p and miR-449b-5p were identified in the porcine genome, although no miRbase annotation indicated that these two mature miRNAs could be expressed and function in PSVSMCs. Therefore, these two miRNAs may be conserved. However, conservation is not required for significant phenotypic effects, as shown by the significant effect of miR-892b on PSVSMC proliferation.

[0243] These results suggest that all seven novel candidate miRNAs are also able to reduce adverse disease-inducing phenotypes in other vascular cell types relevant to the vein graft environment, giving them further therapeutic and translational significance.

[0244] miRNA overexpression strategy for isolated saphenous vein transplantation therapy

[0245] To evaluate the effects of candidate miRNA overexpression, a reproducible model of saphenous vein injury and culture associated with increased VSMC proliferation and migration was used. 27,28 Venous segments were exposed to miRNA mimics for 30 min, then stretched and cultured for 7 days. Following treatment (as well as our untreated controls), tissues were collected and either: i) lysed for RNA isolation and qPCR quantification of candidate miRNA expression, or ii) fixed for subsequent staining for the VSMC marker myosin heavy chain 11 (Myh11), the proliferation marker PCNA, and mature miRNA candidates using miRNA in situ hybridization ( Fig. 8A ). To provide proof of principle that candidate miRNA overexpression reduces VSMC proliferation in this system, we focused on miR-892b because it has the desired effect in both HSVSMC, HSVEC, and PSVSMC. However, other miRNA candidates remain interesting therapeutic agents and their efficacy will be further evaluated. In addition, an adenoviral vector (Ad5) of the candidate miR-892b will also be tested ( Figure 8B -C) to validate the overexpression of miRNAs after viral transduction of HSVSMCs, as well as to validate the antiproliferative phenotype of miRNAs in these cells after adenoviral vector-mediated overexpression. These two approaches will allow us to select the most effective strategy for overexpression of candidate miRNAs in isolated saphenous vein segments.

[0246] discuss

[0247] Abnormally proliferating VSMCs play a central role 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 handling as well as hemodynamic stress after implantation leads to the activation of intrinsically quiescent VSMCs, characterized by increased proliferation and mobility. Here, we aimed to identify novel miRNA therapeutics based on their ability to reduce VSMC proliferation. By filtering the most antiproliferative miRNA candidates, which were also novel in the literature, we generated a list of seven miRNA candidates and evaluated their functional roles as inhibitors of cell proliferation and migration, without simultaneously inducing apoptosis and 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 reduced the pathological VSMC phenotype associated with vascular remodeling, but also had no significant effect on the proliferation of HSVECs or significantly reduced the proliferation of PSVSMCs. This makes them attractive potential therapeutics that deserve further investigation in the context of saphenous vein injury and ex vivo organ culture and subsequent injury-induced neointimal hyperplasia in vivo.

[0248] For the first time, a systematic and unbiased approach was used to identify therapeutic miRNA candidates, specifically in the context of vascular remodeling, through a high-throughput, functional miRNA mimic screen. The miRNA library used contained mimics corresponding to the entire set of human mature miRNA sequences annotated in miRbase version 21. Therefore, our candidate selection was not limited by the existing miRNA literature and enabled the identification of novel potential miRNA therapeutics. The overexpressed miRNA sequences we screened included individually annotated -3p and -5p miRNA strands from all known miRNA loci, based on evidence that miRNA strands originating from the same miRNA locus may have different or even opposing effects. We hypothesized that since -3p and -5p sequences carry different seed sequences, their downstream mRNA targets and subsequent effects on VSMC phenotype may also be different. 29 .

[0249] Since our goal was to specifically identify miRNAs that are able to reduce VSMC proliferation associated with states of pathological vascular injury, early passage VSMCs derived from patients with pulmonary hypertension and therefore characterized by a high basal proliferation rate were used for miRNA screening. After the initial miRNA screening step, we performed a round of validation using a panel of different cell types to increase confidence in the miRNA screening results. For further downstream evaluation of miRNA candidates, we used an in vitro model of saphenous vein graft injury and currently use an ex vivo model of saphenous vein graft injury.

[0250] By using RNA sequencing, we were able to detect changes induced by overexpression of a single miRNA at the transcriptome level.

[0251] Since we observed a strong antiproliferative phenotype in HSVSMCs overexpressing miRNAs in vitro, we were interested in whether candidate miRNAs led to downstream regulation of cell cycle-related processes at the transcriptome level. We identified strong convergence in cell cycle regulation by inducing downregulation of a core network of 125 genes. However, this common overall effect on the transcriptome appeared to be induced in distinct ways by each miRNA, as evidenced by their distinct candidate target genes.

[0252] After identifying these candidate miRNAs, analyzing their induced transcriptome changes by RNA sequencing, and evaluating their functions in different types of SMCs and vascular cells, we performed further work to evaluate their effects in vitro using HSVECs and PSVSMCs, and in silico using RNA sequencing of HSVECs as well as human vascular tissues. All miRNAs induced a significant reduction in PSVSMC proliferation, and / or had no significant effect on HSVEC proliferation. These results interestingly demonstrate the feasibility of using the porcine vein graft failure model to test miRNA candidates, and that some miRNAs produce specific antiproliferative effects on HSVSMCs, which may interfere with the healing of HSVECs and impair the preservation of the endothelial layer, respectively. Candidate miR-892b was selected only to provide proof of concept for ex vivo miRNA delivery and in situ reduction of VSMC proliferation in human primary tissues, based on its lack of significant inhibitory effect on HSVEC proliferation while significantly reducing PSVSMC proliferation. However, all miRNAs remain interesting candidates from the perspective of translational interest and deserve further evaluation.

[0253] Although VSMC proliferation can contribute to the important process of adaptive vascular remodeling after vascular injury, it can also lead to intimal thickening, subsequent vascular occlusion, and restricted blood flow. 30 The specific molecular mechanisms underlying the potential differences between beneficial and abnormal VSMC proliferation induced by injury have not been explored. Therefore, for the purpose of this study, we evaluated the impact of miRNAs on the overall amount of VSMC proliferation observed in our ex vivo model of HSV. In this context, further studies are needed to provide a novel framework to specifically target abnormally proliferating VSMCs present in the injured vessel wall. References

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Claims

1. A miR modulator for use in: (i) Regulate 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-surgical vein graft failure / late vein graft failure; (v) treating or preventing vein graft failure / late vein graft failure following coronary artery bypass surgery involving, for example, the use of the saphenous vein; (vi) treating or preventing atherosclerosis; (vii) treating, preventing or modulating vascular remodeling; (viii) treating or preventing vascular damage; and / or (ix) Treat or prevent vascular damage in which VSMC proliferation is an important phenotype.

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

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

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

5. The miR modulator according to any preceding claim, wherein the miR modulator increases the expression of one or more of the following miRs: (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.

6. The miR modulator according to any preceding claim, wherein the miR modulator increases the expression of one or more of the following miRs: (i) miR-892b; (ii) miR-1827; (iii) miR-332a-3p; and / or (iv) miR-5681b.

7. The miR modulator of any preceding claim, wherein the miR modulator increases expression of miR-892b.

8. A miR modulator for use as a medicament, wherein the miR modulator increases the expression of one or more of the following miRs: (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.

9. A modulator for use according to claim 8, wherein the miR modulator increases the expression of any one or more of the following miRs: (i) miR-892b; (ii) miR-1827; (iii) miR-332a-3p; and / or (iv) miR-5681b.

10. The modulator for use according to claim 8, wherein the miR modulator increases the expression of any one or more of the following miRs: (i) miR-332a-3p; (ii) miR-449b-5p; (iii) miR-4774-3p; and / or (iv) miR-5681b.

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

12. A miR modulator for use according to any preceding claim, wherein the miR modulator comprises, consists essentially of or consists of a miR mimic.

13. A miR modulator for use according to any preceding claim, wherein the miR modulator is provided in the form of a vector for delivery to a cell.

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

15. The miR modulator of any preceding claim, wherein the miR modulator reduces abnormal proliferation and / or migration of VSMCs.

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