Abstract 626: Shear Stress Regulates Angiotensin Type One Receptor Expression in Endothelial Cells

Abstract

Several studies have shown that angiotensin II tends to favour atherosclerotic lesion formation and progression, acting mainly through angiotensin type one receptors (AT1R). Conversely, shear stress (SS) confers atheroprotective effects in the vasculature. However, whether SS regulates endothelial AT1R expression remains unknown. Using en face staining of C57BL/6 mouse aortas, we found a pronounced expression of AT1R in the inner regions of the aortic arch and at collateral artery branch points, known to have low flow and to be atheroprone, compared with neighboring high SS regions. Furthermore, staining of vessels from hypercholesterolemic LDLR−/−mice demonstrated a clear expression of AT1R only in endothelium overlying plaques. Using HUVECs exposed to a laminar SS of 15dynes/cm2, we observed a biphasic decrease in AT1R protein expression characterized by a first reduction at 1 hr (31+4% of static control, p<0.001), partial recovery at 3 hr (65+9%), and a second more prolonged decrease at 6, 12 and 24 hrs (48+9%, 36+9%, 33+5% of static control, respectively)(p<0.001). AT2R levels remained unchanged in these conditions. Shear-induced downregulation of AT1R was abolished by treatment with L-NAME (10–7M), suggesting that NO regulates AT1R in sheared endothelial cells. In accordance, stimulating static HUVECs with a NO donor for 1 hour decreased AT1R protein expression. The acute loss of AT1R at 1 hour SS suggested a rapid endocytosis and degradation of the protein. Indeed, immunocytochemical staining demonstrated a clear colocalisation of AT1R with caveolin-1 in HUVECs submitted to 5 mins SS, similar to that observed in angII-stimulated cells. Cholesterol depletion with methyl-beta-cyclodextrin prevented AT1R-caveolin colocalisation. No colocalisation of AT1R with clathrin was observed. In comparison, AT2R did not undergo endocytosis under any conditions tested. Finally, RT-PCR revealed a significant (p<0.001) decrease of AT1R mRNA in HUVECs exposed to SS during 6 (4+1% of static control), 12 (4+1%) and 24 hours (15+4%), suggesting a transcriptional downregulation of AT1R at length. Hence, our results demonstrate that SS may convey some of its atheroprotective effects through degradation and downregulation of AT1R in endothelial cells.