Compositions and methods for treating peripheral arterial disease
The present application discloses roles for miR-93 in treating hypoxia and ischemia. Endothelial cells (HUVEC) and myocytes (C2C12) expressed miR-93 and up-regulated miR-93 in response to hypoxia and serum starvation. Over-expression of miR-93 in HUVECs promoted cell proliferation, prevented hypoxia-induced apoptosis, and enhanced endothelial cell tube formation. miR-93 knockdown in HUVECs resulted in increased hypoxia-induced apoptosis and decreased tube formation. Over-expression or knockdown of miR-93 in myocytes resulted in reduced or increased hypoxia-induced apoptosis, respectively. Down-regulation of miR-93 in C57BL/6 mice with antagomiR resulted in attenuated perfusion recovery (% non-ischemic leg at day-21: Scramble 85.22.9 vs. AntagomiR-93 67.96). Over-expression of miR-93 in BALB/C mice improved perfusion recovery (% non-ischemic leg at day 21: PremiR-93 757.5 vs. Scramble 59.62.5). The present invention encompasses the use of miR-93 and regulation of miR-93 to treat and prevent hypoxia, ischemia, and other injuries, diseases, disorders, and conditions associated with ischemia.
1. A method of treating or preventing a disease, disorder, injury, or condition associated with skeletal muscle ischemia, wherein said disease, disorder, injury, or condition is peripheral arterial disease, said method comprising administering to a subject a pharmaceutical composition comprising an effective amount of an agonist of miRNA expression, levels, or activity, a pharmaceutically-acceptable carrier, wherein said miRNA is miR-93, and optionally an additional therapeutic agent, thereby treating said disease, disorder, or condition associated with ischemia.
2. The method of claim 1 , wherein said agonist of miR-93 is selected from the group of isolated nucleic acids consisting of a nucleic acid comprising a nucleic acid sequence encoding a precursor miR-93, a nucleic acid comprising a nucleic acid sequence encoding a mature miR-93, a nucleic acid comprising a precursor miR-93, and a nucleic acid comprising a mature miR-93, and biologically active fragments or homologs thereof.
3. The method of claim 2 , wherein said isolated nucleic acid is a precursor miR-93, and biologically active fragments or homologs thereof.
4. The method of claim 2 , wherein said isolated nucleic acid is a mature miR-93, and biologically active fragments or homologs thereof.
5. The method of claim 2 , wherein said isolated nucleic acid is a deoxyribonucleic acid.
6. The method of claim 2 , wherein said isolated nucleic acid is a ribonucleic acid.
7. The method of claim 3 , wherein said sequences encoding miR-93 or comprising mir-93 microRNA are selected from the group consisting of SEQ NOs:1, 2, 5, 6, 7, 8, 9, and 10, and biologically active fragments and homologs thereof.
8. The method of claim 1 , wherein said agonist increases miR-93 expression, levels, or activity.
9. The method of claim 8 , wherein said agonist is an miR-93 mimic.
10. The method of claim 7 , wherein said isolated nucleic acid is encoded by a vector.
11. The method of claim 10 , wherein said vector is an expression vector selected from an miRNA expression vector or AAV expression vector.
12. The method of claim 11 , wherein said expression vector is an miRNA expression vector.
13. The method of claim 10 , wherein said isolated nucleic acid is operably-linked to a cell-specific promoter.
14. The method of claim 10 , wherein a lipid vehicle comprises said isolated nucleic acid.
15. The method of claim 1 , wherein said additional therapeutic agent comprises an anti-ischemia agent.
16. The method of claim 1 , wherein said method decreases expression of at least one cell cycle pathway gene.
17. The method of claim 16 , wherein said cell cycle pathway genes are selected from the group consisting of p21, E2F-1, and p53.
18. The method of claim 16 , wherein expression of at least two of p21, EF-1, and p53 decreases.
19. The method of claim 1 , wherein expression or levels of at least one of VEGF-A, PTEN, MCM-7, TGF-β1, Epiregulin, BMP-2, ATP8b, Dusp-4, and integrin β8 do not change.
20. The method of claim 16 , wherein said expression is in skeletal muscle cells.
21. The method of claim 1 , wherein said agonist is incorporated into a skeletal muscle cell or an endothelial cell.
22. The method of claim 1 , wherein said agonist incorporates into at least one skeletal muscle cell and at least one endothelial cell.
23. The method of claim 1 , wherein said method enhances perfusion recovery.
24. The method of claim 1 , wherein said method enhances the angiogenic response to ischemia.
25. The method of claim 1 , wherein said method stimulates cell proliferation.
26. The method of claim 25 , wherein said cell is an endothelial cell or a skeletal muscle cell.
27. The method of claim 1 , wherein said method increases capillary density.
28. The method of claim 1 , wherein said method reduces apoptosis.
29. The method of claim 28 , wherein said apoptosis is hypoxia-induced apoptosis.
30. The method of claim 1 , wherein said administration is by a route selected from the group consisting of oral, buccal, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, vaginal, ophthalmic, pulmonary, rectal, intrasternal injection, kidney dialytic infusion, and parenteral.
31. The method of claim 30 , wherein said administration is intramuscular.
32. The method of claim 1 , wherein said agonist is administered at a frequency selected from the group consisting of at least once a day, twice a day, three times a day, four times a day, once a week, twice a week, once a month, and twice a month.
33. The method of claim 1 , wherein said subject is a human.
34. The method of claim 1 , wherein at least two agonists are administered.
35. The method of claim 1 , wherein said treatment is prophylactic.
36. The method of claim 1 , wherein said ischemia is selected from the group consisting of vascular ischemia, peripheral arterial disease, and brain ischemia.