18 Hydrogen sulfide stimulates human myometrial angiogenesis: Influence of endogenous estrogens

Abstract

Introduction Angiogenesis regulates myometrial spiral artery expansion and remodeling that are key mechanisms for upregulating uterine blood flow during pregnancy. Hydrogen sulfide (H 2 S), mainly synthesized from l -cysteine by cystathionine-beta synthase (CBS) and cystathionine-gamma lyase (CSE), is a potent vasodilator and proangiogenic factor. However, it is unknown if pregnancy regulates H 2 S biosynthesis and if H 2 S plays a role in myometrial angiogenesis. Hypothesis Correlating with endogenous estrogens during the menstrual cycle and pregnancy, H 2 S biosynthesis is augmented to stimulate myometrial angiogenesis in vivo and in vitro. Methods Myometrial tissues were obtained from hysterectomies from women with different endogenous estrogens ( n = 5–7/group), including late pregnant (very high estrogen), premenopausal proliferative (high estrogen) and secretory (low estrogen) of the menstrual cycle, and postmenopausal (very low estrogen). Tissue levels of CBS and CSE mRNA and protein were determined by real-time qPCR and immunoblotting, respectively. Tissue sections were analyzed by immunofluorescence microscopy with antibodies of CBS or CSE with an endothelial marker protein CD31. Images were analyzed for quantifying cellular CBS and CSE protein expression and angiogenesis index. Primary myocytes and microvascular endothelial cells (hMMEC) were enzymatically isolated for in vitro angiogenesis assays. Results Myometrial H 2 S levels and angiogenesis index were greatest in association with significant upregulation of CBS but not CSE mRNA and protein and elevated estrogens in pregnant vs. non-pregnant women. CBS protein was localized in myocytes and microvessels. A slow releasing H 2 S donor significantly stimulated hMMEC proliferation, migration and tube formation in vitro (in vivo angiogenesis). Co-culture with myocytes also stimulated hMMEC migration in vitro, which was blocked by a specific inhibitor of CBS. Conclusion Pregnancy augmented H 2 S biosynthesis due to enhanced CBS expression in myocytes and microvessels stimulates myometrial angiogenesis in the presence of elevated estrogens during the menstrual cycle and pregnancy (Funded by NIH RO1 HL70562 and R21 HL98746).