RHO-ASSOCIATED KINASE (ROCK) REGULATES ENDOTHELIAL NITRIC OXIDE SYNTHASE IN BLOOD OUTGROWTH ENDOTHELIAL CELLS

Abstract

Human blood outgrowth endothelial progenitor cells (BOECs), derived from peripheral blood mononuclear cells express endothelial protein profiles (i.e. CD31, 144, 146, KDR, and vWF). Endothelial nitric oxide synthase (eNOS) is an important regulator of vascular tone and loss of eNOS activity is a hallmark of endothelial dysfunction. Previous studies have demonstrated that the extracellular matrix (ECM) proteins negatively affect eNOS expression and activity, and a recent study has shown that BOECs deposit extensive amounts of ECM proteins. Rho-associated kinase (ROCK) plays a critical role in the regulation of cytoskeleton and ECM production. In this study, we have investigated the role of ROCK on eNOS expression and activity in BOECs. Human BOECs were cultured with ROCK inhibitors (Y27632 or Fasudil) and ECM proteins (type I collagen (Col. I) and fibronectin (FN)) deposition were measured. After 24 hours of culture, BOECs deposit an organized mesh-like structure of Col. I. This collagen structure was not seen in cells treated with Y27632 or fasudil. To explore the regulation of ROCK on eNOS, we have measured eNOS expression (Western Blot), nitric oxide (4-Amino-5-Methylamino-2',7'-Difluorofluorescein), nitrotyrosine (Western Blot) and superoxide (Dihydroethidium) levels after Y27632 treatment. We found a significant increase in eNOS protein expression (1.37 ± 0.11 fold change, p < 0.01) in BOECs after incubating with Y27632. Interestingly, the nitrotyrosine and superoxide levels were both markedly reduced (0.60 ± 0.10 fold change in Nitrotyrosine, p < 0.01) to a similar level as superoxide dismutase treated group. Taken together, this result suggests that ROCK inhibition may negatively regulate ECM production and potentially enhance eNOS expression while at the same time limiting peroxynitrite mediated nitrosylation, suggesting an enhanced eNOS bioactivity in BOECs. Human blood outgrowth endothelial progenitor cells (BOECs), derived from peripheral blood mononuclear cells express endothelial protein profiles (i.e. CD31, 144, 146, KDR, and vWF). Endothelial nitric oxide synthase (eNOS) is an important regulator of vascular tone and loss of eNOS activity is a hallmark of endothelial dysfunction. Previous studies have demonstrated that the extracellular matrix (ECM) proteins negatively affect eNOS expression and activity, and a recent study has shown that BOECs deposit extensive amounts of ECM proteins. Rho-associated kinase (ROCK) plays a critical role in the regulation of cytoskeleton and ECM production. In this study, we have investigated the role of ROCK on eNOS expression and activity in BOECs. Human BOECs were cultured with ROCK inhibitors (Y27632 or Fasudil) and ECM proteins (type I collagen (Col. I) and fibronectin (FN)) deposition were measured. After 24 hours of culture, BOECs deposit an organized mesh-like structure of Col. I. This collagen structure was not seen in cells treated with Y27632 or fasudil. To explore the regulation of ROCK on eNOS, we have measured eNOS expression (Western Blot), nitric oxide (4-Amino-5-Methylamino-2',7'-Difluorofluorescein), nitrotyrosine (Western Blot) and superoxide (Dihydroethidium) levels after Y27632 treatment. We found a significant increase in eNOS protein expression (1.37 ± 0.11 fold change, p < 0.01) in BOECs after incubating with Y27632. Interestingly, the nitrotyrosine and superoxide levels were both markedly reduced (0.60 ± 0.10 fold change in Nitrotyrosine, p < 0.01) to a similar level as superoxide dismutase treated group. Taken together, this result suggests that ROCK inhibition may negatively regulate ECM production and potentially enhance eNOS expression while at the same time limiting peroxynitrite mediated nitrosylation, suggesting an enhanced eNOS bioactivity in BOECs.