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.
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