Long-term progestin-only contraceptives (LTPOCs) are ideally suited for women with limited access to healthcare. Unfortunately, abnormal uterine bleeding causes high rates of discontinuation. Endometria of LTPOC-treated patients display over-expression of an array of angiogenic factors and cytokines which lead to aberrant angiogenesis and inflammation. LTPOC-exposed endometria also show evidence of extensive oxidative stress and reduced endometrial blood flow. Our central hypothesis is that LTPOC reduces uterine blood flow to cause hypoxia/re-perfusion which triggers the generation of reactive oxygen species (ROS). The latter induce the expression of angiogenic and inflammatory modulators that produce abnormal, fragile endometrial vessels. The resultant focal bleeding facilitates tissue factor-generated thrombin which exacerbates these angiogenic and inflammatory stimuli. Hence we propose the following Specific Aims to test this hypothesis: 1) Laser Doppler flow fluxmetry will measure endometrial perfusion before and after LTPOC administration while endometrial biopsies will be collected for measurement of ROS and angiogenic endpoints; 2) molecular pathways by which ROS induce aberrant angiogenesis and inflammation will be studied in endometrial stromal cell and endothelial cell cultures and co-cultures exposed to progestins with or without hypoxia, ROS or thrombin. Putative intermediate transcription factors will be discovered by electrophoretic mobility shift assays and micorarray analyses while paracrine mediators of aberrant angiogenesis will be identified by proteomic analysis; and 3) effects of synthetic progestins on uterine artery basal tone and response to pressors will be assessed in guinea pig and human uterine artery vascular ring bioassays. Autocrine mediators of these steroid effects will be analyzed by microarray studies. These novel studies will define the precise uterine vasoconstrictive effects of LTPOCs, identify the underlying vascular regulatory mechanisms, and delineate the molecular pathways whereby hypoxia, ROS and thrombin mediate endometrial angiogenesis and inflammation. Moreover, a model will be established to test innovative LTPOC formulations that minimize uterine and endometrial vasoconstriction and ROS-generation, thus preventing abnormal bleeding to improve the acceptability of LTPOC therapies. ? ? ?
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