辛伐斯坦通过claudin-5蛋白调节内皮细胞屏障功能

A significant and sustained increase in vascular permeability is a hallmark of acute inflammatory processes such as acute lung injury (ALI) and sepsis and is associated with significant mortality in these conditions. Recent data indicate that there are more than 100,000 cases of severe ALI per year in the United States with a high mortality rate of ~35-40%. Disruption of the pulmonary vascular endothelial barrier is a major pathophysiologic step in this process. Unfortunately, effective therapies for preserving or reconstituting the endothelial barrier are lacking.There are several different kinds of junctions to keep endothelial cells barrier function intact: tight junction, adherens junction, focal adhesion junction and other forms of junction such as PECAM-1 junction etc. Claudin(Cldn) is key component for the tight junction, which has 24 different subtypes in mice and human. Its molecular weight is 18-27 kD. The structure shows four times transmembrane, short cytoplasmic N-terminal,two extracellular loops and one C-terminal cytoplasmic domain. Different claudin subtpyes are specifically expressed in different tissue types,which indicates that claudin expression is under sophisticated regulation. For example, claudin 16 specifically expresses in the kidney and forms a non-selective cation channel. Mutation of claudin-16 expression results in wasting of magnesium and calcium. Claudin-2 (Cldn2), Cldn3, Cldn4, Cldn7, Cldn8, Cldn12 and Cldn15 are abundantly expressed in the duodenum, jejunum, ileum and/ or colon. Claudin-5 is the only one member of claudin- family expressing in endothelial cells. .Simvastatin, a cholesterol reducing drug has been demonstrated to have multiple mechanisms of action. One of them is the outstanding endothelial barrier enhancement effect accompanying attenuating inflammation of sepsis in animal models and human cases. Studies by our and several other groups have strongly indicated that statins changed endothelial barrier function via cytoskeleton rearrangement and are also involved in junction rearrangement. However, apparently these can not explain why simvastatin dramactically enhances endothelial cell junctions, including tight junction. preliminary data from Recent studies by us, and other groups, indicates that simvastatin and atorvastatin upregulate claudin-5 in endothelial cells.These exciting findings lead us to further characterize: (1) The mechanism of claudin-5 expression induced by simvastatin, (2) The impact of simvastatin on junctional assembly formation in cultured human pulmonary endothelial cells (EC) and in vivo models, and (3)Finally we investigate the potential novel regulatory mechanism which is more specifically targeted on claudin-5.

急性肺损伤是败血症中的标志性事件，肺血管内皮屏障的破坏是主要的病理生理因素。目前还没有有效的治疗手段保护或修复因急性肺损伤破坏的内皮屏障。我们发现一种降胆固醇药，辛伐斯坦(simvastatin)具有显著的抗炎症疗效，其抗炎症疗效部分通过改善肺血管内皮屏障功能，引起细胞骨架重建和增强内皮细胞连接。最近我们发现斯坦增加一个内皮细胞连接蛋白，claudin-5的表达。然而，它是否和辛伐斯坦改善肺血管内皮屏障功能有必然的联系尚不清楚。在这个项目中，我们用RNA静默，质粒转染技术和内皮细胞连接的结构和功能研究技术相结合，在细胞和动物模型上研究辛伐斯坦介导的claudin-5高表达对内皮连接功能的影响及对急性肺损伤的改善效果，以期能阐明辛伐斯坦诱导claudin-5表达的调节机制，为进一步开发更特异的抗急性肺损伤药物打下基础。

显著和持续增加血管通透性是急性炎症疾病，如急性肺损伤，败血症中的标志性事件。最近的资料显示美国每年有10万急性肺损伤的病人，其死亡率约35-40%。肺血管内皮屏障的破坏是主要的病理生理因素。然而，目前还没有有效的治疗手段保护或修复因急性肺损伤破坏的内皮屏障。在本项目中，我们首次阐明（1）在细胞和动物上用急性肺损伤模型，我们首次阐明claudin-5在claudin-5肺内皮屏障完整性中的重要作用。研究显示敲除claudin-5在培养的内皮细胞或动物模型上增加内皮屏障对小分子的染料的通透性；（2）我们探索并阐明了斯伐他汀的药物诱导claudin-5的高表达并保护内皮屏障的机制。我们确定了VE-cadherin-β-catenin 复合物在胞内和细胞核的转移及FOX-1在claudin-5表达中的重要作用；（3）通过化学抑制剂或siRNA 技术，我们在细胞模型上发现斯伐他汀诱导claudin-5 通过激活VEGF-AKT-GSK-β-catenin信号通路，抑制经典WNT-GSK-β-catenin通路等；（4）在此基础上，我们寻找更安全，更有效的药物诱导claudin-5在内皮细胞中的表达 ；（5）与此同时，我还拓展了内皮细胞一氧化氮合酶(Endothelial nitric oxide synthase, eNOS)在维护内皮屏障和功能的研究等新领域。