Inhibition of protein kinase Calpha prevents endothelial cell migration and vascular tube formation in vitro and myocardial neovascularization in vivo.
Although protein kinase C (PKC) activation is required for endothelial cell (EC) growth, migration, adhesion, and vessel formation, the role of individual PKC isoenzymes in these events is not defined. Because PKCalpha has been previously linked with enhanced EC migration and response to angiogenic growth factors, we characterized a specific phosphorothioate-modified 21-mer antisense PKCalpha (AS-PKCalpha). AS-PKCalpha (500 nmol/L) prevented the expression of PKCalpha protein by 90% in human ECs and did not reduce the expression of any other PKC isoenzyme. AS-PKCalpha reduced human EC migration by 64% compared with its control oligonucleotide in a "scratch" wounding assay, and AS-PKCalpha reduced human EC adhesion to the extracellular matrix protein vitronectin by 18%. Phosphorylation of mitogen-activated protein kinase (extracellular signal-regulated kinase 1/2) induced by vascular endothelial growth factor was inhibited by 30% in human ECs transfected with AS-PKCalpha. Compared with control, AS-PKCalpha also reduced the number of EC tubes formed in a 3D type I collagen gel assay by 37.5%. Finally, using an osmotic minipump, we infused AS-PKCalpha into mice in which myocardial infarction was induced by coronary ligation and found that the oligonucleotide was primarily taken up by intramyocardial blood vessels. Compared with the results with control oligonucleotide, AS-PKCalpha oligonucleotide inhibited the number of anti-PKCalpha-stained blood vessels by 48% and reduced the total vessel number by 72% as well. In conclusion, the expression of PKCalpha is required for full EC migration, adhesion to vitronectin, vascular endothelial growth factor-induced extracellular signal-regulated kinase activation, and tube formation and is likely to be of importance in myocardial angiogenesis in vivo after ischemia.
Wang
Aihong
A
Cardiovascular Division, Department of Medicine, Albert Einstein College of Medicine, Bronx, NY, USA.
Nomura
Motohiro
M
Patan
Sybill
S
Ware
J Anthony
JA
D000818:Animals; D002448:Cell Adhesion / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002465:Cell Movement / Q000187:drug effects; D002478:Cells, Cultured; D003331:Coronary Vessels / Q000187:drug effects / Q000378:metabolism; D016228:Endothelial Growth Factors / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D008026:Ligation; D008222:Lymphokines / Q000494:pharmacology; D048748:MAP Kinase Kinase Kinase 3; D020930:MAP Kinase Kinase Kinases / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009203:Myocardial Infarction / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology; D009204:Myocardial Revascularization; D018919:Neovascularization, Physiologic / Q000187:drug effects / Q000502:physiology*; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D009928:Organ Specificity; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D051571:Protein Kinase C-alpha; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D019096:Vitronectin / Q000378:metabolism
D016228:Endothelial Growth Factors; D007527:Isoenzymes; D008222:Lymphokines; D016376:Oligonucleotides, Antisense; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D019096:Vitronectin; C496628:PRKCA protein, human; C496630:Prkca protein, mouse; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D048748:MAP Kinase Kinase Kinase 3; D020930:MAP Kinase Kinase Kinases; C482625:MAP3K3 protein, human; C482626:Map3k3 protein, mouse