Abstract 826: PDZK1 is Required for HDL-Induced eNOS Activation and Endothelial Cell Migration
Circulating levels of high density lipoprotein (HDL) cholesterol are inversely related to the risk of cardiovascular disease. In addition to mediating reverse cholesterol transport, HDL and the HDL receptor scavenger receptor BI (SR-BI) initiate signaling in endothelium that promotes endothelial NO synthase (eNOS) activity and endothelial cell migration. We recently showed that signaling by SR-BI requires the C-terminal PDZ-interacting domain of the receptor, which interacts with the adaptor protein PDZK1 in liver. In the present study we determined if PDZK1 plays a role in HDL modulation of endothelial cell phenotype. Immunoblot analyses of bovine aortic endothelial cells (BAEC) revealed that PDZK1 is expressed in endothelium, and that it is found in both the plasma membrane and cytoplasmic fractions. Knockdown of PDZK1 by RNA interference (RNAi) abrogated eNOS activation by HDL, whereas enzyme stimulation by VEGF remained intact. Loss of PDZK1 in BAEC by RNAi did not alter SR-BI abundance. The expression of a dominant negative form of PDZK1 also prevented eNOS phosphorylation in BAEC in response to HDL. Furthermore, in studies of isolated endothelial cell plasma membranes, antibody to PDZK1 prevented eNOS activation by HDL. PDZK1 involvement in HDL modulation of endothelial cell migration, which is SR-BI-dependent and NO-independent, was also delineated in scratch assays. Following knockdown of PDZK1, migration with HDL was fully attenuated whereas migration stimulated by serum or VEGF was unaltered. To determine if PDZK1 regulates endothelial cell migration in vivo, carotid artery reendothelialization following perivascular electric injury was compared in PDZK1+/+ versus PDZK1−/− mice. Whereas the area of initial denudation evaluated by Evan’s blue dye incorporation was similar, PDZK1−/− had 4.5 times more remaining denudation 5d postinjury than PDZK1+/+, indicating markedly impaired reendothelialization. These cumulative findings reveal that PDZK1 is required for HDL-induced eNOS activation and for HDL-induced endothelial cell migration of relevance to the maintenance of intimal layer integrity. Thus, PDZK1 in endothelium plays an important role in HDL-related cardiovascular protection.