C反应蛋白诱导内皮微颗粒损伤血管内皮功能的作用及机制研究

Inflammation and endothelial dysfucntion are the two most important pathophysiological mechanisms in atherosclerosis. Both of them can effect and promote the progress of each other. C-reactive protein(CRP) is the prototypic marker of inflammation and a valid marker of cardiovascular risk.Several studies have shown a significant relationship between CRP and endothelial dysfunction and demonstrated it can independently impair endothelial function. Endothelial microparticles, as a novel marker of endothelial dysfunction or injury, have been demonstrated as an important mediator leading endothelial dysfunction by the last studies.In our former research we found CRP injuried endothelial cells and then released endothelail microparticles via eNOS uncoupling. Other researches proved endothelial micropaticles can impair endothelial function. But whether endothelial microparticles induced by CRP can impair endothlial function and how it happens. We don't know. Studies demonstrated that CRP can impair endothelial cells through ERK1/2 signal pathway. In addition, CRP deduces eNOS acitivity, which in turn causes the reduction of NO production, and leads to endothelial dysfucntion. Hence,we put forward the hypothesis that CRP induces release of endothelial micropaticls and CRP-induced endothelial micropaticles impair endothelium-dependent vasodilation via ERK1/2-eNOS-NO signal pathway.To illuminate this hypothesis, we conduct this research. We would use cell biology, molecular biology, animal vascular function measurement and gene transfer technology and so on to study the mechanism in it. We would conduct in vitro cells research and in vivo animal model research. We hope to demonstrate that endothelial microparticles induced by CRP can impair endothelial function via ERK1/2-eNOS-NO signal pathway. Endothelial microparticles are one of the mediators between CRP and endothelial dysfunction. This helps us furtherly understand the relationship and mechanism between inflammation and endothelial dysfunction.

C反应蛋白是炎症因子也是独立的致内皮功能障碍损伤因素。内皮微颗粒是近年来提出的反映内皮损伤的敏感指标，新近研究证实它还是一种重要的致内皮损伤的介质。我们的前期研究发现C反应蛋白损伤内皮细胞生成内皮微颗粒。然C反应蛋白诱导生成的内皮微颗粒是否具有致内皮功能障碍的能力及其机制目前尚未明瞭。ERK1/2是C反应蛋白损伤内皮细胞的信号通路分子，eNOS活性降低致NO生成减少参与C反应蛋白致内皮功能障碍。我们提出C反应蛋白诱导内皮微颗粒损伤血管内皮舒张功能，ERK1/2-eNOS-NO信号通路是其分子机制之一。本项目拟从体外细胞培养以及在体动物模型两方面，结合细胞生物学、分子生物学、血管功能学、基因转染等技术，研究C反应蛋白诱导内皮微颗粒损伤内皮舒张功能及与ERK1/2－eNOS-NO信号通路的关系。提出内皮微颗粒是C反应蛋白致内皮功能障碍的中间介质，深化认识C反应蛋白损伤血管内皮功能的机制。

C反应蛋白（CRP）为代表的炎症因子在动脉粥样硬化性血管疾病的发生发展中起重要作用，炎症与血管内皮功能障碍既相互影响又相互作用。内皮微颗粒（EMPs）是循环中重要的远程信号传导介质，可致内皮功能障碍。我们前期的研究显示CRP可刺激内皮细胞生成释放EMPs。故EMPs能否是CRP和血管内皮功能障碍间的桥梁，介导了血管内皮舒张功能减退是本项目的研究重点且还将对相关机制进行探讨。研究拟通过体外实验观察CRP诱导EMPs损伤HUVECs及ERK1/2和eNOS在EMPs损伤HUVECs中的作用，并拟在动物实验模型验证ERK1/2－eNOS－NO通路在CRP诱导EMPs损伤血管内皮舒张功能中的作用。研究中我们发现即使在生理状态下，健康男性大学生循环中EMPs水平与基础血清超敏C反应蛋白呈正相关，提示CRP可能与循环EMPs生成释放增多有关。细胞实验中，我们观察到CRP-EMPs呈浓度依赖性影响ERK1/2和eNOS的蛋白表达及NO生成。300ug/ml以下浓度的CRP-EMPs剂量依赖性上调磷酸化ERK1/2和eNOS的蛋白表达，且NO生成增多。ERK1/2特异性抑制剂PD98059显著抑制了磷酸化ERK1/2和eNOS的蛋白表达，且抑制了NO生成，提示ERK1/2－eNOS途径参与了CRP-EMPs对人动脉内皮细胞损伤作用。进一步地我们在体外研究中还观察到EMPs刺激人主动脉内皮细胞后内皮细胞管状结构生成减少提示血管新生能力降低，伴随着Nortch1蛋白配体DLL4蛋白表达被上调及Nortch1蛋白和Cleaved Notch1蛋白表达上调。应用Norch蛋白抗体可减轻EMPs对内皮细胞管状结构生成的损伤，提示Norch通路参与了EMPs损伤内皮细胞血管新生能力的作用。临床研究中我们针对血压控制良好的高血压患者循环EMPs水平仍高的血管残余风险，给予单次急性中等强度运动，降低了循环EMPs水平，提示提高切应力可能减轻EMPs的损伤作用。汇总我们的研究结果，我们认为CRP-EMPs通过ERK1/2－eNOS途径损伤人主动脉动脉内皮细胞；且Norch通路参与了EMPs的致内皮细胞血管新生能力降低的作用。高血压患者微循环损伤，中等强度运动或许能通过降低循环EMPs水平并减弱EMPs的致内皮细胞血管新生能力降低作用改善高血压患者的内皮功能。