高血压患者血小板通过诱导内皮-间质转化促进心肌纤维化的分子机制研究

Hypertension-induced cardiac fibrosis is a leading cause of cardiac dysfunction and sudden death. Zeisberg et al., demonstrated that endothelial-to-mesenchymal transition (EndoMT) contributes to cardiac fibrosis in a mouse model of pressure-overload, suggesting that some of fibrobalsts from the fibroic area were originated from endothelial cells. The functions of platelets which are independent of coagulation and thrombosis have been gained a lot of attentions. Abnormalities in platelet function have been detected in hypertensive patients with unknown biological functions. In a mouse model of tumor, platelets were able to interact with tumor cells and then induce epithelial-to-mesenchymal transition. Whether platelets can induce endothelial cells transformation is unknown. We previously found platelet activity is positive correlated with PINP levels (serum fibrosis maker) in hypertensive patients. In addition, inhibition of platelet activation by clopidogrel prevents hypertension-induced cardiac fibrosis in the mouse pressure-overload model, especially in the peri-vascular areas. In this study, we will use cardiac MRI to further confirm whether platelet activity is correlated with cardiac fibrosis in hypertensive patients. Further, we are going to explore whether platelets contribute to cardiac fibrosis via EndoMT under the pressure-overload model. We will co-culture platelets and endothelial cells by using microfluidics to further explore the mechanism. Our study will provide promising evidence for anti-fibrosis therapy in hypertensive patients.

高血压所致心肌纤维化是心功能不全、心源性猝死的重要原因。研究发现，内皮-间质转化（EndoMT）来源的成纤维细胞在心肌纤维化过程中扮演重要角色。近年，血小板除凝血与血栓外的其它功能逐渐被人关注。高血压患者的血小板存在异常活化，但生物学效应不明。活化的血小板可诱导癌细胞向间质细胞转化，但能否诱导内皮细胞发生EndoMT尚缺乏报道。申请人前期研究发现，高血压患者血小板活化程度与血清I型前胶原肽（纤维化标志物）水平正相关；给予压力负荷小鼠抗血小板药物能够有效抑制心肌间及血管周围纤维化，且对血管周围纤维化的抑制效果更为显著。本项目拟通过临床研究探讨血小板活性与心肌纤维化程度的相关性；利用转基因小鼠压力负荷模型明确血小板能否诱导EndoMT，促进心肌纤维化；并基于微流控芯片技术在细胞水平探索血小板诱导EndoMT的机制，为临床预防和治疗心肌纤维化提供帮助。

高血压心肌纤维化是心衰的重要原因，内皮-间质转化（EndoMT）是心肌纤维化的可能机制。本项目研究发现：高血压患者血小板活化水平较普通人群升高，活化程度与血清心肌纤维化标志物水平正相关；将高血压患者的血小板与人脐静脉内皮细胞共培养，后者可发生形变。免疫印迹结果显示，与高血压患者血小板共培养的内皮细胞E-cadherin表达降低，vimentin和FSP-1表达升高。构建压力负荷小鼠模型后，给予模型小鼠饲喂氯吡格雷，发现氯吡格雷可抑制心肌纤维化进程、减少纤维化区域EndoMT来源成纤维细胞数量。我们利用微流控芯片建立了体外仿生血管模型，系列实验发现血小板活化水平与内皮细胞vimentin和α-SMA表达程度呈正相关趋势。以上结果支持活化的血小板可诱导EndoMT促进心肌纤维化。. 为探索血小板与内皮细胞结合的具体机制，课题组在微流控芯片模型体系内引入肿瘤细胞和细胞毒药物阿霉素作为协同干预变量，发现：肿瘤细胞可通过自身分泌功能提高血小板活化水平，此途径活化的血小板也可作用于内皮细胞，使之发生形变和功能变化；抗肿瘤药物阿霉素在影响肿瘤细胞活性的同时也损伤内皮细胞，激活血小板；在反应体系中，阿霉素使血小板黏附性增加，导致内皮细胞vimentin和α-SMA表达水平升高，进而加快EndoMT进程。. 为寻找心肌纤维化的干预手段，项目组利用已有的缺血性心肌病动物模型探讨粒细胞集落刺激因子（G-CSF）治疗心肌纤维化的可行性，发现适宜剂量的G-CSF治疗可增强缺血区域的心肌收缩能力、维持心肌细胞活性、减小缺血损伤面积、促进局部血管新生、减少细胞凋亡，使缺血区域内von Willebrand因子（vWF）、血管内皮生长因子（VEGF）和单核细胞趋化蛋白-1（MCP-1）表达上调，Akt1基因活性增强。以上结果说明适宜剂量的G-CSF有助于延缓心肌纤维化进程。