Shear Stress Regulates ABCA1-Dependent Membrane Cholesterol Content in Endothelial Cells Facilitating H2S-Dependent Vasodilation

Abstract

Endothelial cells (ECs) express an array of integral membrane proteins, including ion channels and transporters that contribute to blood flow regulation and cell-cell communication. Many of these membrane proteins are regulated by plasma membrane cholesterol content. The ATP-binding cassette family A1 (ABCA1) transporter is a regulator of membrane cholesterol content. We have shown increased ABCA1 mRNA expression and reduced EC membrane cholesterol in resistance mesenteric arteries compared to conduit arteries. Previous studies suggest shear stress (SS) can increase or decrease ABCA1 expression in a cell-type-dependent manner.

Hypothesis: SS sustains lower EC membrane cholesterol concentration through ABCA1-mediated cholesterol transport, facilitating H₂S-mediated vasodilation.

Methods: The effect of SS on ABCA1 and membrane cholesterol content was assessed in pressurized mesenteric arteries from male Sprague-Dawley rats and cultured human aortic endothelial cells. Pressure myography was used to assess the effects of ABCA1 inhibition on H₂S-mediated vasodilation. Filipin was used to assess EC membrane cholesterol content.

Results: SS increased ABCA1 expression in the endothelium of mesenteric arteries and cultured human aortic endothelial cells and markedly reduced EC membrane cholesterol. Inhibition of ABCA1 increased EC membrane cholesterol content and abolished H₂S-induced vasodilation.

Conclusion: SS facilitation of EC-dependent vasodilation appears to be mediated by membrane cholesterol content.

Keywords: Sprague–Dawley rat; hydrogen sulfide; mesenteric; shear stress; vasodilation.

FIGURE 1

Shear stress increases ABCA1 expression. (A) Representative images of ABCA1 expression (yellow) in response to static, 6 dynes/cm², or 20 dynes/cm² in human aortic endothelial cells. (B) Quantification of mean fluorescence intensity for ABCA1 expression. 8–10 images were acquired per condition (n = 5/group) with 6–7 cells per image. (C) Abca1 mRNA expression normalized to GAPDH in response to static conditions, 6 dynes/cm², or 20 dynes/cm² in HAoECs. (D) Abca1 mRNA in response to cholesterol manipulation in HAoECs. One‐way ANOVA with Tukey's Post hoc test was used for statistical analysis.

FIGURE 2

Shear stress decreases membrane cholesterol. (A) Representative images of membrane cholesterol expression in response to static, 6 dynes/cm², or 20 dynes/cm² in human aortic endothelial cells. (B) Quantification of mean fluorescence intensity for filipin III staining (orange) for the plasma membrane and (C) internal cholesterol content. 8–10 images per condition (n = 3/group) with 6–7 cells per image were acquired. One‐way ANOVA with Tukey's Post hoc test was used for statistical analysis.

FIGURE 3

Increasing shear stress increases ABCA1 expression. (A) Representative images of ABCA1 expression in response to increasing shear stress in intact large mesenteric arteries. (B) Quantification of mean fluorescence intensity expressed as fold change for ABCA1 expression. Five images per artery were acquired per condition (n = 3/group). Nuclear stain DAPI (magenta), ABCA1 (green). Student's t‐test was used for statistical analysis.

FIGURE 4

Inhibition of ABCA1 increases membrane cholesterol and reduces cholesterol efflux. (A) Representative images of membrane cholesterol in response to vehicle or probucol in endothelial tubes isolated from small mesenteric arteries (< 150 μm). (B) Quantification of mean fluorescence intensity for filipin III staining (yellow). 4 tubes/artery from 5 animals. (C) Quantification of cholesterol efflux from HAoECs. (D) Quantification of cholesterol efflux from HAoECs in response to shear stress. Student's t‐test or one‐way ANOVA with Tukey's Post hoc test was used for statistical analysis. ****p < 0.0001, ***p < 0.0007.

FIGURE 5

Inhibition of ABCA1 attenuates H₂S‐mediated vasodilation. (A) Representative trace of vehicle treated artery, The red arrow indicates pre‐constrictor U46619, black arrows indicate H₂S dosage, and the blue arrow indicates Ca²⁺ free PSS. (B) representative trace of probucol treated artery. (C) Summary responses of NaHS‐dependent dilation in arteries treated with vehicle (n = 5) or the ABCa1 inhibitor probucol (100 μM, n = 5). Unpaired Student's t test was used for statistical analysis.

FIGURE 6

The liver X receptor agonist (T0901317) increases ABCA1 expression in HAoECs. (A) Representative images of ABCA1 expression in response to vehicle (DMSO) or T09 (10 μM and 100 μM). (B) Quantification of mean fluorescence intensity for ABCA1 expression. 8–10 images per condition (n = 3) with 6–7 cells per image. One‐way ANOVA with Tukey's post hoc test was used for statistical analysis.