Normal pregnancy is accompanied by extensive changes in the blood coagulation and fibrinolytic system. Preeclampsia is a moor obstetrical problem characterized by hypertension, proteinuria, increased activation of blood coagulation, and decreased activity of the fibrinolytic pathway. A feature of this disorder is the shallow invasion of trophoblast cells into the uterus. The processes of normal embryo implantation and placentation are dependent upon the regulated expression of proteolytic enzymes that degrade the uterine matrix. There is substantial evidence indicating that the alpha2-macroglobulin receptor (alpha2MR) plays an essential role in mediating trophoblast invasive activity, possibly by facilitating the removal of urokinase type plasminogen activator (u-PA) complexed to plasminogen inhibitor complex (PAI-1) from cell surface receptors. We propose that trophoblast invasion requires a constant turnover of inhibited proteinases on the cell surface. We also hypothesize that expression or function of the alpha2MR is abnormal in pregnancies complicated by preeclampsia where trophoblast invasion is shallow. The goals of the proposed research project are to explore the role of the alpha2MR and the 39 kDa receptor-associated protein (RAP) in the implantation process. The first specific aim is to define the role of alpha2MR in removing uPA:PAI-1 complexes from the cell surface, and to determine if the alpha2MR mediates catabolism of metalloproteinase complexed to their inhibitor, tissue inhibitor of metalloproteinases (TIMP). The second specific alm is designed to explore the role of RAP in trophoblast invasion. This molecule has been observed to block binding of all ligands to alpha2MR. RAP function will be examined by using established assays which quantitates cell movement through a layer of Matrigel on a porous membrane and assays that measure the extracellular matrix degradation by cells from mouse blastocyst outgrowths. The third specific alm is to define the expression of the alpha2MR in human placenta at different gestational ages and in placental bed biopsies. The receptor and RAP will be localized with specific antibodies to determine the spatial and temporal patterns of expression. The expression of alpha2MR and RAP in placental tissue and placental bed biopsies of pregnancies complicated by preeclampsia will also be examined. Immunocytochemistry, Western blotting and mRNA analyses will be employed. The proposed studies will provide a comprehensive description of the alpha2MR and its endogenous ligand, RAP, in trophoblast invasion, and will test the hypothesis that abnormal receptor expression or function is associated with preeclampsia. If validated, we will have contributed a model for the molecular basis of this important disorder.
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