Abstract 12253: Disruption of Endothelial Adherens Junction by High Glucose Concentration is Mediated by HRas/PKC-β-Dependent VE-cadherin Tyrosine Phosphorylation
Hyperglycemia has been recognized as a primary factor in the development of diabetic Micro- and macrovascular diseases and endothelial barrier dysfunction, but the underlying biochemical mechanisms remain elusive. In the present study we determined the signal transductions that mediate the effects of high glucose (HG) on integrity of endothelial adherens junction (AJ). It is well established that tyrosine phosphorylation of VE-cadherin (VE-cad) leads to disruption of endothelial AJ and increased transendothelial migration (TEM) of leukocytes. Treatment of human umbilical vein endothelial cells (HUVECs) with high concentrations of glucose (25 mM) or hyperosmolar concentrations of mannitol or sodium chloride led to: 1-increased VE-cad tyrosine phosphorylation 2-dissociation of VE-cad-β-catenin complex 3-accumulation of β-catenin in nuclei of HUVECs and 4-increased TEM of monocytes. These processes were accompanied by increased phosphorylation of ERK and myosin light chain (MLC) and activation of endothelial Ras and protein kinase C (PKC). Over expression of constitutively active (CA) form of HRas or treatment of HUVECs with a PKC activator, PMA reproduced the effects of HG on VE-cad-β-catenin complex and TEM of monocytes. Inhibition of HRas/Raf-1MEKERK signaling cascade or PKC-β but not PKC-δ by overexpression of the dominant negative (DN) forms attenuated HG-induced tyrosine phosphorylation of VE-cad. The stimulating effect of CA-HRas on VE-cad tyrosine phosphorylation was attenuated when PKC-β was inhibited by DN-PKC-β. In contrast, PMA-induced tyrosine phosphorylation of VE-cad was inhibited by DN-PKC-β but not by DN-HRas, indicating a critical role for PKC-β in HRas-induced tyrosine phosphorylation of VE-cad. In addition, inhibition of MLC phoshorylation by the overexpression of a nonphosphorylatable DN form of MLC and inhibition of actin polymerization by cytochalasin D attenuated HG-induced tyrosine phosphorylation of VE-cad, suggesting a crucial role for MLC phosphorylation. Our study introduces a novel mechanism for breakdown of endothelial barrier by HG and indicates that HG-induced disruption of endothelial AJ is mediated by HRas/PKC-β signaling pathways and depends on phosphorylation of MLC.
- © 2012 by American Heart Association, Inc.