Method to induce neovascular formation and tissue regeneration
The present invention relates, e.g., to a method for inducing arteriogenesis, lymphangiogenesis, vasculogenesis, or cardiomyogenesis, and/or for inducing mitosis or proliferation of a smooth muscle cell, a skeletal muscle cell, or a cardiomyocyte, comprising administering to a cell or tissue in need thereof a dose of a polynucleotide that encodes a vascular endothelial growth factor (VEGF), or that encodes a polypeptide comprising an active site of the VEGF. The coding sequence is operably linked to an expression control sequence; and the dose is sufficient to induce arteriogenesis, lymphangiogenesis, vasculogenesis, or cardiomyogenesis, and/or to induce mitosis or proliferation of a smooth muscle cell, a skeletal muscle cell, or a cardiomyocyte. In preferred embodiments of the invention, the method is a method of tissue regeneration, particularly of cardiomyogenesis; and the polynucleotide (or a polypeptide encoded by such a polynucleotide) is administered into the myocardium.
1. A method for inducing cardiomyogenesis in a subject in need of such treatment, comprising administering directly to a cardiomyocyte or tissue comprising cardiomyocytes in the subject a dose of a polynucleotide that encodes the vascular endothelial growth factor, VEGF1-165, wherein the coding sequence is operably linked to a CMV promoter and is in a plasmid vector, the dose being in a sufficient amount, which is greater than about 0.04 mg/kg, to induce cardiomyogenesis.
2. The method of claim 1 , wherein the VEGF1-165 has the amino acid sequence:
Ala Pro Met Ala Glu Gly Gly Gly Gln
(SEQ ID NO: 1)
Asn His His Glu Val Val Lys Phe Met
Asp Val Tyr Gln Arg Ser Tyr Cys His
Pro Ile Glu Thr Leu Val Asp Ile Phe
Gln Glu Tyr Pro Asp Glu Ile Glu Tyr
Ile Phe Lys Pro Ser Cys Val Pro Leu
Met Arg Cys Gly Gly Cys Cys Asn Asp
Glu Gly Leu Glu Cys Val Pro Thr Glu
Glu Ser Asn Ile Thr Met Gln Ile Met
Arg Ile Lys Pro His Gln Gly Gln His
Ile Gly Glu Met Ser Phe Leu Gln His
Asn Lys Cys Glu Cys Arg Pro Lys Lys
Asp Arg Ala Arg Gln Glu Asn Pro Cys
Gly Pro Cys Ser Glu Arg Arg Lys His
Leu Phe Val Gln Asp Pro Gln Thr Cys
Lys Cys Ser Cys Lys Asn Thr Asp Ser
Arg Cys Lys Ala Arg Gln Leu Glu Leu
Asn Glu Arg Thr Cys Arg Cys Asp Lys
Pro Arg Arg.
3. The method of claim 1 , wherein the induced cardiomyogenesis is localized.
4. The method of claim 1 , wherein the cardiomyogenesis is induced in normoperfused tissue.
5. The method of claim 1 , wherein the cardiomyogenesis is induced in ischemic tissue.
6. The method of claim 1 , wherein the cardiomyogenesis is induced in myocardial tissue.
7. The method of claim 1 , wherein the cell or tissue is eukaryotic.
8. The method of claim 1 , wherein the cell or tissue is mammalian.
9. The method of claim 1 , wherein the cell or tissue is porcine or human.
10. The method of claim 1 , wherein the cell or tissue is human.
11. The method of claim 1 , wherein the polynucleotide is a genomic DNA, a cDNA, or a messenger RNA.
12. The method of claim 11 , wherein the polynucleotide encodes the polypeptide represented by SEQ ID NO: 1.
13. The method of claim 12 , wherein the polynucleotide is a cDNA.
14. The method of claim 1 , wherein the polynucleotide is administered to the cell or tissue in a liposome.
15. The method of claim 1 , wherein the subject exhibits signs or symptoms of, or suffers from, ischemic heart disease, myocardial infarction, myocardial ischemia, dilated cardiomyopathy, or heart failure.
16. The method of claim 1 , wherein the subject is a human patient.
17. The method of claim 1 , wherein the polynucleotide is in the form of a pharmaceutical composition.
18. The method of claim 1 , wherein the administration is intramuscular and is intramyocardial muscle administration.
19. The method of claim 18 , wherein the administration is transepicardial or transendocardial administration.
20. The method of claim 19 , wherein the administration is intramyocardial-transepicardial injection under direct visualization, or intramyocardial-transendocardial injection under fluoroscopic guidance.
21. The method of claim 18 , wherein the polynucleotide is administered by injection perpendicular to the plane of the area of injection.
22. The method of claim 18 , wherein the polynucleotide is administered by injection parallel to the plane of the area of injection.
23. The method of claim 18 , wherein the polynucleotide is administered by injection at an oblique angle in relation to the plane of the area of injection.
24. The method of claim 23 , wherein the angle in relation to the plane of the area of injection is between about 30° and about 90°.
25. The method of claim 18 , wherein the polynucleotide is administered by injections that are homogeneously or heterogeneously distributed in the area of injection.
26. The method of claim 1 , wherein the polynucleotide is administered in a single dose of between about 0.01 and about 0.36 nmoles /kg, wherein the nmoles are of the polynucleotide encoding the VEGF polypeptide.
27. The method of claim 26 , wherein the polynucleotide is administered in a single dose of about between about 0.01 and about 0.10 nmoles/kg.
28. The method of claim 26 , wherein the polynucleotide is administered in two or more doses, to achieve a total dose of between about 0.01 and about 0.36 nmoles /kg.
29. The method of claim 28 , wherein the polynucleotide is administered in two or more doses, to achieve a total dose of between about 0.01 and about 0.10 nmoles/kg.
30. The method of claim 1 , wherein the concentration of the plasmid vector is between about 0.5 and about 4 mg/mL.
31. A method for inducing mitosis or proliferation of a cardiomyocyte in a subject in need of such treatment, comprising administering directly to the cardiomyocyte cell a dose of a polynucleotide that encodes the vascular endothelial growth factor, VEGF 1-165, wherein the coding sequence is operably linked to a CMV promoter and is in a plasmid vector, the dose being in a sufficient amount, which is greater than about 0.04 mg/kg, to induce the mitosis or proliferation.
32. The method of claim 31 , wherein the VEGF1-165 has the amino acid sequence:
Ala Pro Met Ala Glu Gly Gly Gly Gln
(SEQ ID NO: 1)
Asn His His Glu Val Val Lys Phe Met
Asp Val Tyr Gln Arg Ser Tyr Cys His
Pro Ile Glu Thr Leu Val Asp Ile Phe
Gln Glu Tyr Pro Asp Glu Ile Glu Tyr
Ile Phe Lys Pro Ser Cys Val Pro Leu
Met Arg Cys Gly Gly Cys Cys Asn Asp
Glu Gly Leu Glu Cys Val Pro Thr Glu
Glu Ser Asn Ile Thr Met Gln Ile Met
Arg Ile Lys Pro His Gln Gly Gln His
Ile Gly Glu Met Ser Phe Leu Gln His
Asn Lys Cys Glu Cys Arg Pro Lys Lys
Asp Arg Ala Arg Gln Glu Asn Pro Cys
Gly Pro Cys Ser Glu Arg Arg Lys His
Leu Phe Val Gln Asp Pro Gln Thr Cys
Lys Cys Ser Cys Lys Asn Thr Asp Ser
Arg Cys Lys Ala Arg Gln Leu Glu Leu
Asn Glu Arg Thr Cys Arg Cys Asp Lys
Pro Arg Arg.
33. The method of claim 31 , wherein the cardiomyocyte is in a cardiac tissue.
34. The method of claim 33 , wherein the cell or tissue is eukaryotic.
35. The method of claim 33 , wherein the mitosis or proliferation is localized mitosis or proliferation.
36. The method of claim 33 , wherein the mitosis or proliferation induces tissue regeneration.
37. The method of claim 33 , wherein the tissue is normoperfused tissue.
38. The method of claim 33 , wherein the tissue is ischemic tissue.
39. The method of claim 33 , wherein the tissue is myocardial tissue.
40. The method of claim 33 , wherein the tissue is hypoperfused tissue.
41. The method of claim 26 , wherein the polynucleotide is administered in a single dose of greater than about 0.04 mg/kg.
42. The method of claim 41 , wherein the polynucleotide is administered in two or more doses, to achieve a total dose of greater than about 0.04 mg/kg.
43. The method of claim 1 , which is a method to reduce infarct size.
44. The method of claim 1 , wherein the cardiomyogenesis is induced in hypoperfused tissue.
45. The method of claim 1 , further wherein arteriogenesis is induced.