Abstract 4143744: Cross-talk between oxidized LDL, oxidative stress and renin-angiotensin-aldosterone system: Impact on endothelial function and atherosclerosis

Introduction/Background: Hypertension and hypercholesterolemia are important risk factors of endothelial dysfunction and atherosclerosis. Previous studies suggested a crosstalk between an activated renin-angiotensin-aldosterone system (RAAS), reactive oxygen species (ROS) and oxidized low-density lipoproteins (oxLDL) in atherosclerosis, but the underlying molecular mechanisms are not well understood. Research Question/Hypothesis: Can we identify novel signaling pathways controlling the molecular crosstalk of the RAAS with ROS and oxLDL in endothelial dysfunction and atherosclerosis? Methods/Approach: The impact of AT 1 R blockade on oxLDL-induced superoxide anion formation and endothelial dysfunction was studied in human umbilical artery endothelial cells and aortic rings of wild-type mice by chemiluminescence and Mulvany myograph. We cloned 5’-terminal deletions of the AT 1 R promoter and assessed the luciferase activity in human endothelial cells. Oct-1 binding to the human AT 1 R promoter region was studied by EMSA. Further assays included real-time PCR, confocal microscopy, Western blotting, G protein reporter assays, phospho-ERK1/2 antibodies and specific siRNAs. The data were validated in heart of obese C57BL/6 mice and cardiac and aortic tissue of AT 1a /AT 1b double knockout mice in vivo . Results/Data: AT 1 R promoter activation studies upon Ang II- and oxLDL-stimulation in endothelial cells revealed that Ang II and oxLDL activate AT 1 R signaling through G protein Gα 12/13 , followed by activation of ERK1/2 MAP kinases, and transcription and translation of Oct-1, resulting in up-regulation of AT 1 R, LOX-1 and NOX2 expression, which could be antagonized by specific inhibitors at each step of the identified signaling cascade. AT 1 R blockade improved oxLDL-induced endothelial dysfunction in aortic rings of wild-type mice. Male C57BL/6 mice fed a high-fat diet exhibited upregulation of Oct-1 levels in cardiac tissues, compared to normal controls, while AT 1a /AT 1b double knockout mice demonstrated downregulation of Oct-1, AT 1 R, LOX-1, and NOX2 on mRNA and protein level in cardiac and aorta tissue, thus confirming the identified signaling cascade in vivo . Conclusions: Oct-1 is an essential transcription factor for Ang II- and oxLDL-induced upregulation of AT 1 R and LOX-1 expression in endothelium, thus identifying a novel molecular cross-talk of oxLDL with ROS signaling and the RAAS contributing to development of endothelial dysfunction and atherosclerosis.