EXCEED THE SPACE PROVIDED. The clinical utility of long-term, progestin-only contraceptives (LTPOC) is limited by their high incidence of abnormal uterine bleeding. Endometrial hemostasis is mediated in part by progesterone-induced decidualization of estradiol (E2)-primed human endometrial stromal cells (HESCs) which elevates tissue factor (TF). By acting as a transmembrane receptor for plasma-derived factor Vila, TF ultimately mediates hemostasis via thrombin generation. Paradoxically, Norplant-associated endometrial bleeding persists after 6 months despite high levels of stromal cell-expressed TF and rising endogenous E2 levels. Moreover, bleeding sites contain a higher density of enlarged, fragile vessels and increased immunostaining for HESC TF than non-bleeding sites. Moreover, Norplant-exposed endometria display increased expression of the potent angiogenic factors: vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2). This proposal hypothesizes that LTPOCs enhance endometrial stromal cell-expressed TF. The resulting TF/factor Vila complex promotes stromal cell synthesis of VEGF and Ang-1/2; and generates thrombin, which acts as a paracrine mediator of angiogenesis in adjacent endothelial cells and as an autocrine enhancer of VEGF and Ang-1 expression in the stromal cells. A potent angiogenic feedback loop is thus created resulting in aberrant angiogenesis, endothelial cell """"""""leakiness"""""""", vascular damage and bleeding. To test this hypothesis, three Specific Aims will ascertain: 1) Mechanisms by which TF/VlIa or thrombin modulate human endometrial stromal cell (HESC) VEGF and Ang expression. 2) Mechanism(s) by which VEGF, Ang and thrombin mediate aberrant angiogenesis in human endometrial endothelial cells (HEECs).3) Whether TF/VIIa and thrombin modulates angiogenesis in HESC-HEEC co-cultures. The information derived from the proposed studies is expected to elucidate the mechanisms of abnormal uterine bleeding which will lead to the development of new contraceptives agents in which abnormal uterine bleeding is minimized or eliminated.
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