Fibulin-5/β1-integrin 信号通路在醛固酮诱导血管平滑肌细胞凋亡中的作用

Primary aldosteronism is the most common reason of secondary hypertension. Adrenal aldosterone-producing adenoma (APA), the most important type of primary aldosteronism, is treated mainly by surgical resection. However, even though APA is removed, a part of patients still suffer from resistant hypertension possibly due to irreversible vascular remodeling, the exact mechanism of which is still unclear. Aldosterone-induced cardiovascular damage is currently a hot research at home and abroad. Our previous study found that aldosterone induced both apoptosis of vascular smooth muscle cells(VSMCs) and extracelluar matrix protein Fibulin-5 deposition in the aorta in vivo. Similar to fibronectin, Fibulin-5 can affect cell survival by combining with β1-integrin via its RGD motif, and may play an important role in aldosterone-induced VSMC apoptosis. In the present project, the effect of aldosterone on VSMC apoptosis in vitro will be first observed; then, Fibulin-5 overexpression or suppression in VSMCs is achieved by transfecting Fibulin-5 eukaryotic expression vector or fibulin-5 siRNA plasmid to observe the effect of Fibulin-5 on aldosterone-induced VSMC apoptosis; next, adding fibronectin to the experimental environment to verify the competitive relationship in function between Fibulin-5 and fibronectin; finally, the effect of Fibulin-5 on β1-integrin signal pathway is examined by using β1-integrin inhibitory or stimulatory antibodies, in order to systematically reveal the effect of Fibulin-5 on aldosterone-induced VSMC apoptosis and the mechanism wherein.

醛固酮对心血管系统的损害为国内外研究的热点。我们前期研究发现，醛固酮（Aldo）在体内诱导主动脉血管平滑肌细胞(VSMC)凋亡和细胞外基质蛋白Fibulin-5沉积。与fibronectin类似，Fibulin-5也可以与β1-integrin结合影响细胞生存，其可能是Aldo诱导VSMC凋亡的重要介质。本项目拟首先观察Aldo在体外对VSMC凋亡的影响；然后转染fibulin-5表达载体或siRNA质粒，使VSMC过表达或沉默Fibulin-5，观察Fibulin-5在Aldo诱导VSMC凋亡中的作用；随后,加入fibronectin,验证Fibulin-5与fibronectin功能上的竞争关系；最后应用β1-integrin激活或抑制抗体，观察Fibulin-5对β1-integrin信号通路的影响，从而系统揭示Fibulin-5在Aldo诱导VSMC凋亡中的作用及其机制。

肾上腺醛固酮腺瘤是原发性醛固酮增多症最重要的类型，占70％～80％，手术治疗效果不加，成为长期困扰临床的难题，血管重构可能是其中原因之一。研究发现醛固酮（aldosterone, Aldo）是导致血管重构的重要原因，而Fibulin-5可能在Aldo诱导血管重构中扮演重要的角色，我们前期研究发现结扎Fibulin-5基因敲除小鼠的颈动脉，伴随严重的新内膜形成及增厚的外膜。而在Aldo的作用下，大鼠主动脉Fibulin-5沉积显著增加，提示Fibulin-5在Aldo诱导血管重构中可能具有重要作用。主要研究内容，通过流式细胞术筛选出醛固酮诱导主动脉平滑肌细胞VSMCs凋亡的最佳浓度100μM，1000μM，用于后续实验，构建Fibulin-5干扰及过表达慢病毒，检测干扰及过表达Fibulin-5后研究Aldo对VSMC细胞的影响，加入外源性Fibulin-5后研究VSMC细胞的凋亡情况，从而得出Fibulin-5对醛固酮诱导的细胞凋亡有抑制作用，而当醛固酮浓度较高时，Fibulin-5的抑制活性可能达到饱和，从而对其抑制活性减弱。我们进一步研究发现Fibulin-5过表达细胞与醛固酮和Fibronectin共同孵育时，凋亡水平与对照相比，显著增加；与之相对应的Fibulin-5干扰细胞与醛固酮和Fibronectin共同孵育时，凋亡水平下降。在高浓度醛固酮诱导作用下，其干扰效果由于作用饱和而不显著。由此推测，Fibronectin在醛固酮诱导的细胞凋亡过程中能够拮抗Fibulin-5的作用，促进醛固酮诱导的细胞凋亡。β1-integrin通路抑制剂Ofloxacin能够诱导VSMC细胞凋亡，且有剂量依赖效果，Fibulin-5过表达或干扰细胞中Ofloxacin的加入均导致细胞凋亡率增加，故Ofloxacin能够加强醛固酮诱导的细胞凋亡，而该作用可能是Fibulin-5非依赖性的。Fibulin-5过表达细胞与醛固酮和β1-integrin通路抑制剂Ofloxacin共同孵育时，抑凋亡基因Bcl-2的表达与对照细胞相比，显著升高，Bax基本表达降低；而Fibulin-5敲低细胞与醛固酮和β1-integrin通路抑制剂Ofloxacin共同孵育时，Bax及 Bcl-2的表达与对照细胞相比没有显著变化。