Estrogen Replacement Therapy in Ovariectomized Nonpregnant Ewes Stimulates Uterine Artery Hydrogen Sulfide Biosynthesis by Selectively Up-Regulating Cystathionine β-Synthase Expression

Abstract

Estrogens dramatically dilate numerous vascular beds with the greatest response in the uterus. Endogenous hydrogen sulfide (H2S) is a potent vasodilator and proangiogenic second messenger, which is synthesized from L-cysteine by cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE). We hypothesized that estrogen replacement therapy (ERT) selectively stimulates H2S biosynthesis in uterine artery (UA) and other systemic arteries. Intact and endothelium-denuded UA, mesenteric artery (MA), and carotid artery (CA) were obtained from ovariectomized nonpregnant ewes (n = 5/group) receiving vehicle or estradiol-17β replacement therapy (ERT). Total RNA and protein were extracted for measuring CBS and CSE, and H2S production was determined by the methylene blue assay. Paraffin-embedded UA rings were used to localize CBS and CSE proteins by immunofluorescence microscopy. ERT significantly stimulated CBS mRNA and protein without altering CSE mRNA or protein in intact and denuded UA. Quantitative immunofluorescence microscopic analyses showed CBS and CSE protein localization in endothelium and smooth muscle and confirmed that ERT stimulated CBS but not CSE protein expression in UA endothelium and smooth muscle. ERT also stimulated CBS, but not CSE, mRNA and protein expression in intact and denuded MA but not CA in ovariectomized ewes. Concomitantly, ERT stimulated UA and MA but not CA H2S production. ERT-stimulated UA H2S production was completely blocked by a specific CBS but not CSE inhibitor. Thus, ERT selectively stimulates UA and MA but not CA H2S biosynthesis by specifically up-regulating CBS expression, implicating a role of H2S in estrogen-induced vasodilation and postmenopausal women's health.

Figure 1.

CBS and CSE mRNA and protein expression in UAs from OVX ewes receiving Veh and E₂β replacement therapy (ERT) ewes. CBS and CSE mRNAs in intact (top, left) and denuded (top, right) UA. CBS and CSE protein analysis in intact (bottom, left) and denuded (bottom, right) UA. Data (mean ± SEM) are from 3–5 ewes/group. **, P < .01; ***, P < .001 compared with Veh.

Figure 2.

Immunofluorescence localization of CBS and CSE protein in UA from OVX ewes receiving Veh and E₂β replacement therapy (ERT). Sections were labeled with specific primary antibodies against CBS (green) or CSE and endothelial cell marker CD31 (red) with nuclei stained with DAPI (blue). Negative control slides were treated similarly but without primary antibodies or with antimouse IgG; no positive staining was detected on these slides (inset of upper left image). Images were analyzed for relative fluorescence intensity (RFI). Representative outlines of cell bodies used for analysis are outlined (left panels) and border of CD31-positive endothelial cells was also indicated (center panels). Data (mean ± SEM) are from 3–5 different ewes/group. *, P < .05; **, P < .01. Scale bar, 50 μm.

Figure 3.

CBS and CSE mRNA and protein expression in MA from OVX ewes receiving Veh and E₂β replacement therapy (ERT). Relative mRNA levels of CBS and CSE in intact (top, left) and denuded (top, right) MA. CBS and CSE proteins in intact (bottom, left) and denuded (bottom, right) MA. Data (mean ± SEM) are from 3–5 different ewes/group. *, P < .05; **, P < .01; ***, P < .001.

Figure 4.

CBS and CSE mRNA and protein expression in CA from OVX ewes receiving Veh and E₂β replacement therapy (ERT). Relative mRNA levels of CBS and CSE mRNAs in intact (top, left) and denuded (top, right) CA. CBS and CSE proteins from intact and denuded CA (bottom). ERT did not change CBS or CSE expression. Data (mean ± SEM) are from 3–5 different ewes/group; n.s., not statistically different.

Figure 5.

Comparisons of the effects of E₂β replacement therapy (ERT) on CBS and CSE mRNA (A) and protein (B) expression in uterine and systemic arteries from OVX ewes. Data (mean ± SEM) were summed from 3–5 different ewes/group. Different letters differ significantly; n.s., no statistical difference.

Figure 6.

Effects of E₂β replacement therapy (ERT) on H2S production in UA (A), MA (B), and CA (C) from OVX ewes. Proteins from OVX ewes were used to measure H2S production in the presence or absence of the specific inhibitors of CBS (CHH), CSE (BCA), or their combination. Data (mean ± SEM) are from 3 different ewes per group. ***, P < .001; bars with different letters differ significantly; n.s., not statistically different.