Endothelial Barrier Function in Preeclampsia

描述（由申请人提供）：血管通透性增加是内皮功能障碍的重要组成部分，也是子痫前期（PE）的重要病理生理事件。在最初申请的研究方案“内皮屏障功能在子痫前期”中，我们对正常孕妇和PE患者的内皮细胞（ECs）进行了研究，发现无组织的EC连接蛋白vascular Endothelial (VE)-cadherin和紧密连接蛋白occludin是PE中内皮通透性增加的细胞基础。我们进一步证明，胎盘释放的因子具有破坏EC连接接触和增加内皮通透性的能力。为了确定胎盘释放的候选分子在PE期间诱导ECs炎症表型，我们发现胎盘来源的凝乳胰蛋白酶样蛋白酶（CLP/chymase）对血管内皮具有深远的影响。在我们的初步研究中，我们观察到CLP可以破坏内皮连接蛋白的分布并影响胎盘可溶性VEGF受体-1 （sFlt-1）的产生。在这个竞争性的续期拨款申请中，我们将进一步探索CLP调节PE中EC屏障功能的潜在细胞和分子机制。我们的中心假设是，胎盘来源的CLPs通过改变内皮连接组装和增加胎盘中sFlt-1的释放来介导PE中血管通透性的增加。我们将通过以下3个具体目标的实验来验证这一假设：1)确定胎盘源性CLP在调节内皮屏障功能中的作用；2)阐明胎盘源性蛋白酶诱导的内皮细胞粘附/紧密连接的破坏在多大程度上是由蛋白酶激活受体（PAR）介导的；3)探讨滋养细胞（TC） CLP活性的增强是否有助于PE患者胎盘中sFlt-1释放的增加及其机制。拟议的工作将利用来自正常妊娠和PE妊娠的tc和ec。PAR siRNA将用于转染EC，以研究胎盘源性clp诱导的EC完整性破坏与PE相关的机制。我们将进一步研究CLP对胎盘sFlt-1的影响。从所提出的工作中获得的结果应该加强目前对胎盘和EC功能障碍在PE发病机制中的作用的认识。

DESCRIPTION (provided by applicant): Increased vascular permeability is an important component of endothelial dysfunction and a significant pathophysiological event in preeclampsia (PE). In the original application of the research proposal "Endothelial Barrier Function in Preeclampsia", a study of endothelial cells (ECs) derived from normal pregnant women and from women with PE, we have found that disorganized EC junction protein vascular endothelial (VE)-cadherin and tight junction protein occludin are the cellular basis of increased endothelial permeability in PE. We further demonstrated that factors released from the placenta have the ability to disrupt EC junction contacts and increase endothelial permeability. In an effort to identify candidate molecules released from the placenta that induce an inflammatory phenotype in ECs during PE, we found that placenta-derived chymotrypsin-like protease (CLP/chymase) exerts profound effects on vascular endothelium. In our preliminary studies, we observed that CLP could disorganize endothelial junction protein distribution and affect placenta soluble VEGF receptor-1 (sFlt-1) production. In this competing renewal grant application, we will further explore the potential cellular and molecular mechanisms by which CLP regulates EC barrier function in PE. Our central hypothesis is that placenta-derived CLPs mediate the increased vascular permeability in PE by altering endothelial junction assembly and by increasing sFlt-1 release from the placenta. We will test this hypothesis by experiments outlined under 3 specific aims: 1) to determine the role of placental derived CLP in regulation of endothelial barrier function; 2) to elucidate to what extent the placenta-derived protease-induced disassembly of endothelial adhesion/tight junctions is mediated by proteinase-activated receptor (PAR) in PE; and 3) to explore whether enhanced trophoblast (TC) CLP activity contributes to increased sFlt-1 release from the placenta in PE and what mechanisms are involved. The proposed work will utilize TCs and ECs derived from normal and PE pregnancies. PAR siRNA will be used to transfect ECs to study mechanisms underlying the placenta-derived CLP-induced disruption of EC integrity that are relevant to PE. The influence of CLP on placental sFlt-1 will be studied. Results obtained from the proposed work should enhance current knowledge of the role of the placenta and EC dysfunction in the pathogenesis of PE.