Project Summary New vessel formation from existing vessels termed angiogenesis is a key mechanism for endometrial turnover and regeneration during the menstrual cycle and normal pregnancy during which estrogens play a critical role. Angiogenesis in endometrium, like it occurs in any other organs, is initiated by enhanced local production of angiogenic factors. Hydrogen sulfide (H2S) is a gaseous signaling molecule that participates into the regulation of numerous physiological and pathophysiological processes. Endogenous H2S is mainly produced from L-cysteine by cystathionine-?-synthase (CBS) and cystathionine-?-lyase (CSE). H2S is a potent proangiogenic vasodilator because H2S donors promote endothelial cell (EC) angiogenesis in vitro and in vivo. CBS and CSE have been found in many organs but their expression is tissue/cell-specific; both are needed to generate H2S in some tissues while one enzyme is sufficient in others. Recent studies have implicated a role of H2S in regulating endometrial function during the menstrual cycle and pregnancy; however, hitherto it has reported whether endometrial H2S generating system is regulated during the menstrual cycle and pregnancy and whether H2S plays a role in endometrial angiogenesis. The overall hypothesis of this RO3 is that elevated estrogens stimulate estrogen receptor (ER?/?) dependent upregulation of stromal CBS-H2S production, which in turn stimulates EMEC angiogenesis during the proliferative phase and pregnancy in women. This hypothesis will be tested by using endometrial biopsies from Cesarean hysterectomies and primary endometrial stromal cells (ESC) and endometrial microvascular endothelial cells (EMEC) cell models derived from these tissues. Aim 1 will determine if endometrial H2S production correlates to endometrial angiogenesis index under the influence of endogenous estrogens in women ex vivo; Aim 2 To determine if estrogens stimulate ESC H2S production is mediated via ER (ER?/ER?) dependent CBS transcription and in vitro; and Aim 3 will determine if ESC-derived H2S mediates estrogen stimulation of EMEC angiogenesis. Accomplishing these important specific aims will establish a new paracrine physiological role of ESC-derived H2S in endometrial EMEC angiogenesis.