The circulating level of C-reactive protein (CRP) is a strong predictor of cardiovascular disease and endothelial dysfunction. However, it is unknown whether CRP plays a causal role in these processes. We have discovered that CRP inhibits both endothelial NO synthase (eNOS) gene expression and processes regulating eNOS enzymatic activity, thereby decreasing the production of a key antiatheroslerotic molecule. The OBJECTIVE of the proposed research is to determine the mechanisms by which CRP downregulates eNOS expression and function, and the specific role of CRP in endothelial dysfunction and atherogenesis. In preliminary studies, CPR attenuated eNOS promoter activity by 90%.
Aim 1 is to determine the mechanism by which CRP downregulates eNOS gene transcription. CRP-responsive regulatory elements will be delineated by mutagenesis of the eNOS promoter, and relevant nuclear proteins will be identified in electrophoretic mobility shift assays. We also have found that brief (15 min) CRP exposure fully prevents agonist stimulation of eNOS.
Aim 2 is to determine the basis by which CRP attenuates the capacity to activate the enzyme. Studies will assess CRP effects on kinase cascades upstream of eNOS, eNOS phosphorylation, and intracellular [Ca2+].
Aim 3 is to determine the role of IgG Fcgamma receptors (FcgammaR),which bind CRP in other paradigms, and associated signaling molecules in CRP actions on eNOS. In initial studies, CRP effects on eNOS were mimicked by aggregated IgG; in addition, in isolated mouse carotid arteries eNOS expression fell by 89% with 24h CRP exposure, and 15min CRP treatment caused full blockade of acetylcholine-stimulated cGMP accumulation. As such, loss-of-function studies are feasible using arteries from FcgammaR-null mice. We have also shown that endothelial cells express both isoforms of SHIP (SH2 domain-containing inositol 5-phosphatase), which is recruited to inhibitory FcgammaRIIB in immune effector cells and which hydrolyzes PIP3, thereby blunting PI3 kinase action. The role of SHIP-l/2 will be determined in studies of SHIP activation by CRP and SHIP loss-of-function using dominant negative mutants.
Aim 4 is to determine the impact of CRP on eNOS expression, endothelial function an d atherogenesis in transgenic mice with diet-regulated CRP expression directed by the PEPCK promoter. Carotid blood flow responses and reendothelialization following electric injury will be tested at varying CRP levels. The impact of CRP on atherogenesis wall be determined an crosses of PEPCK-CRP and apoE mice. By meeting these aims, the proposed research will provide mechanistic links between CRP and endothelial dysfunction and vascular disease.
