Downregulation of protein kinase Cδ activity enhances endothelial cell adaptation to hypoxia

Abstract

Background - Although protein kinase C (PKC) has been implicated in ischemic cell death, the role of individual PKC isoenzymes in the response of endothelial cells (ECs) to hypoxia is unknown. Methods and Results - To test the effect of hypoxia on the activity of individual PKC isoenzymes, human ECs were exposed to 95% N2 with 5% CO2 for 24 hours. This severe hypoxia reduced PKCδ specific activity in both human umbilical vein ECs (HUVECs) and a HUVEC-derived EC line (ECVs) significantly (80.5±5.7% and 55.5±8.6% of normoxia controls, respectively); the activities of PKCα and PKCε were unchanged. The protein levels of PKCα, PKCδ and PKCε were unchanged by hypoxia. To determine whether PKCδ downregulation by hypoxia was linked to EC function, ECVs in which PKCδ was stably overexpressed (PKCδ-ECs) were exposed to hypoxia. A significant increase in cell death was observed in PKCδ-ECs compared with controls (5.8±0.6% versus 2.3±0.4% at 24 hours, 13.2±1.2% versus 4.1±0.4% at 48 hours, P<0.05) during hypoxia. Neither the DNA laddering assay nor TUNEL staining revealed an increase in apoptosis of PKCδ-ECs exposed to hypoxia, suggesting a hypoxia-induced increase in nonapoptotic cell death of PKCδ-ECs. Inhibition of NO synthase with N(G)- monomethyl-L-arginine (L-NMMA) affected neither the decline in PKCδ activity nor the EC death induced by hypoxia. Conclusions - PKCδ activity is decreased by hypoxia by a mechanism that does not involve NO synthase; this downregulation appears to enhance EC survival during hypoxia by decreasing nonapoptotic cell death.