Role of Cholesterol in the Regulation of Hydrogen Sulfide Signaling within the Vascular Endothelium

Abstract

H₂S is a gaseous signaling molecule enzymatically produced in mammals and H₂S-producing enzymes are expressed throughout the vascular wall. We previously reported that H₂S-induced vasodilation is mediated through transient receptor potential cation channel subfamily V member 4 (TRPV4) and large conductance (BK_Ca) potassium channels; however, regulators of this pathway have not been defined. Previous reports have shown that membrane cholesterol limits activity of TRPV4 and BK_Ca potassium channels. The current study examined the ability of endothelial cell (EC) plasma membrane (PM) cholesterol to regulate H₂S-induced vasodilation. We hypothesized that EC PM cholesterol hinders H₂S-mediated vasodilation in large mesenteric arteries. In pressurized, U46619 pre-constricted mesenteric arteries, decreasing EC PM cholesterol in large arteries using methyl-β-cyclodextrin (MBCD, 100 µM) increased H₂S-induced dilation (NaHS 10, 100 µM) but MBCD treatment had no effect in small arteries. Enface fluorescence showed EC PM cholesterol content is higher in large mesenteric arteries than in smaller arteries. The NaHS-induced vasodilation following MBCD treatment in large arteries was blocked by TRPV4 and BK_Ca channel inhibitors (GSK219384A, 300 nM and iberiotoxin, 100 nM, respectively). Immunohistochemistry of mesenteric artery cross-sections show that TRPV4 and BK_Ca are both present in EC of large and small arteries. Cholesterol supplementation into EC PM of small arteries abolished NaHS-induced vasodilation but the cholesterol enantiomer, epicholesterol, had no effect. Proximity ligation assay studies did not show a correlation between EC PM cholesterol content and the association of TRPV4 and BK. Collectively, these results demonstrate that EC PM cholesterol limits H₂S-induced vasodilation through effects on EC TRPV4 and BK_Ca channels.

Keywords: endothelial cell; methyl-β-cyclodextrin; microdomains.

Figure 1

H₂S-induced vasodilation in rat and mouse mesenteric arteries. Representative traces and data summary of H₂S-induced vasodilation following cholesterol reduction with MβCD (100 μM) in rat large arteries (A–C). Mean ± S.D. * p = 0.026 vs. vehicle; **** p < 0.0001 vs. vehicle and rat small arteries (D–F). Mean ± S.D. (G–H). H₂S-induced vasodilation in mouse large and small mesenteric arteries. Mean ± S.D. * p = 0.01, ** p = 0.0011 vs. large, two-way ANOVA with Sidak’s multiple comparison test.

Figure 2

Cholesterol content in large (>200 μm) and small (<200 μm) mesenteric arteries. (A). Representative en face images of cholesterol marker, filipin III (green, 20 μg/mL) and endothelial glycoprotein marker Lectin (red, 20 μg/mL) acquired at 63x with additional digital zoom of small artery for visualization of cells. Nuclei are labeled with SYTOX (blue). (B). Mean ± S.D. *** p = 0.0004, unpaired t test.

Figure 3

Representative traces and data summary of H₂S-induced vasodilation under (A). vehicle (Veh) condition or following (B). epicholesterol (Epi, 20 μM) or (C). cholesterol (Chol, 28 μM) supplementation into EC PM of small mesenteric arteries. Summary data of H₂S dilation in cholesterol and epicholesterol treated small arteries (D) Mean ± S.D. * p = 0.04, ** p = 0.0012, **** p < 0.001, one-way ANOVA with Sidak’s multiple comparison test.

Figure 4

H₂S-induced vasodilation following cholesterol reduction in large arteries is mediated by activation of TRPV4 and BK channels. (A). Representative images of EC PM TRPV4 and BK (green), endothelial glycoprotein marker Lectin (red) in cross-sections of large and small mesenteric arteries. Nuclei are labeled with SYTOX (blue). “NO 1°” shows the no primary Ab negative controls. Representative traces and data summary of H₂S-mediated vasodilation following cholesterol reduction (B) in large arteries in the presence of BK inhibitor iberiotoxin ((C), Ibtx, 100 nM) or TRPV4 inhibitor GSK 2193874 ((D), GSK, 300 nM) Summary data of H₂S-induced dilation in cholesterol depleted arteries (E). Mean ± S.D. **** p < 0.0001 vs. control, one-way ANOVA with Sidak’s multiple comparison test.

Figure 5

Endothelial TRPV4 and BK association in large and small mesenteric arteries following sterol manipulation. (A). Representative images of mesenteric artery cross-sections with endothelial glycoprotein marker Lectin (green) and PLA puncta for TRPV4/BK association (red). Nuclei are labeled with SYTOX (blue). (B). Number (#) of puncta count /total area and (C). area of puncta from 23 arteries/size group per animal. Mean ± S.D. n = 4 animals/group. (D). Primary antibodies without ligation or amplification (+Prim/No Amp); inclusion of BK but not TRPV4 primary antibody with ligation and amplification (+BK/-TRPV4/+Amp); and No primary No amplification as negative controls.