CRP, eNOS and Endothelial Dysfunction

CRP、eNOS 和内皮功能障碍

基本信息

批准号：
7149130
负责人：
PHILIP W SHAUL
金额：
$ 33.28万
依托单位：
UT SOUTHWESTERN MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2003
资助国家：
美国
起止时间：
2003-12-23 至 2008-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7149130
关键词：
1-Phosphatidylinositol 3-Kinase 5&apos Flanking Region Acceleration Acetylcholine Acute Acute-Phase Proteins Address Agonist Animal Model Antithymoglobulin Aorta Apolipoprotein E Arginine Arterial Fatty Streak Arteries Attenuated Base Sequence Binding Biological Assay Blood flow C-reactive protein COS-7 Cell Calcium Calmodulin Carbohydrates Cardiovascular Diseases Carotid Arteries Cell model Citrulline Cultured Cells Cyclic GMP Dactinomycin Development Diet Dominant-Negative Mutation Dose Down-Regulation Effector Cell Electrophoretic Mobility Shift Assay Elevation Endothelial Cells Endothelium Enzymes Functional disorder Gene Expression Gene Expression Process Genes Genetic Transcription Genetically Engineered Mouse Hydrolysis Immune Immune system Immunoblotting Immunoglobulin G Impairment In Vitro Knockout Mice Knowledge Link Luciferases MEKs Mediating Membrane Mitogen-Activated Protein Kinase Kinases Mitogen-Activated Protein Kinases Molecular Mus Mutagenesis Nitric Oxide Synthase Nuclear Nuclear Protein Nuclear Proteins Phosphatidylinositols Phosphoenolpyruvate Carboxylase Phosphoinositide-3-Kinase, Catalytic, Gamma Polypeptide Phosphoric Monoester Hydrolases Phosphorylation Phosphotransferases Play Polymerase Chain Reaction Process Production Protein Binding Protein C Protein Isoforms Protein Tyrosine Kinase Proteins Recruitment Activity Regulation Regulatory Element Reporter Research Role Shock from electric current Signal Transduction Signaling Molecule Standards of Weights and Measures Testing Time Transcriptional Regulation Transgenic Mice Vascular Diseases Vascular Endothelial Growth Factors atherogenesis attenuation base cysteine rich protein falls human NOS3 protein inositol-1,4,5-trisphosphate 5-phosphatase loss of function mutant prevent programs promoter protein expression receptor reconstitution research study response src Homology Region 2 Domain

项目摘要

DESCRIPTION (provided by applicant): 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.

描述（由申请人提供）：C反应蛋白（CRP）的循环水平是心血管疾病和内皮功能障碍的有力预测指标。但是，尚不清楚CRP在这些过程中是否起因果作用。我们发现CRP抑制了调节eNOS酶活性的调节eNOS酶促活性的内皮NO合酶（ENOS）基因表达和过程，从而减少了关键的抗抗氧化治疗分子的产生。拟议研究的目的是确定CRP下调eNOS表达和功能的机制，以及CRP在内皮功能障碍和动脉粥样硬化中的特定作用。在初步研究中，心肺复苏术使eNOS启动子活性减少了90％。目的1是确定CRP下调eNOS基因转录的机制。通过eNOS启动子的诱变，将描绘出CRP响应性调节元件，并且将在电泳迁移率转移测定中鉴定相关的核蛋白。我们还发现，短暂的（15分钟）CRP暴露充分阻止了eNOS的激动剂刺激。目标2是确定CRP减弱激活酶的能力的基础。研究将评估CRP对eNOS，eNOS磷酸化和细胞内[Ca2+]上游激酶级联反应的影响。 AIM 3是确定IgG Fcgamma受体（FCGAMMAR）的作用，该受体在其他范式中结合CRP，以及相关的信号分子在CRP对eNOS上的作用。在最初的研究中，CRP对eNOS的影响通过聚集的IgG模仿。此外，在孤立的小鼠颈动脉中，ENOS表达在24H CRP暴露时下降了89％，而15分钟的CRP处理导致乙酰胆碱刺激的CGMP积累的完全阻断。因此，使用FCGAMMAR-NULL小鼠的动脉可行的功能丧失研究是可行的。我们还表明，内皮细胞表达船舶的同工型（含SH2域的含肌醇5-磷酸酶），该酶在免疫效应细胞中被募集以抑制性fcgammariib，并水解PIP3，从而使PI3激酶的作用钝化。船-L/2的作用将在CRP和使用显性负突变体功能丧失的船舶激活研究中确定。 AIM 4是确定CRP对ENOS表达，内皮功能和Dytherogenogy在转基因小鼠中的影响，其饮食调节的CRP表达由PEPCK启动子指导。颈动脉血流反应和电损伤后的再粘膜化将在不同的CRP水平下进行测试。 CRP对动脉粥样硬化壁的影响确定了PEPCK-CRP和APOE小鼠的交叉。通过达到这些目标，拟议的研究将提供CRP和内皮功能障碍与血管疾病之间的机械联系。