Sex differences in ET‐1 levels in primary human endothelial cells

Abstract

Endothelin‐1 (ET‐1) is produced and secreted primarily by endothelial cells and acts as a potent vasoconstrictor. Our laboratory has previously shown that ET‐1 contributes to impaired vascular endothelial function in postmenopausal women (PMW). However, compared to PMW, ET‐1 mediated vasoconstriction is greater in men of a similar age. Although sex differences in ET‐1 receptor expression and/or responses may partially explain these differences, it is unknown whether ET‐1 levels in endothelial cells differ between PMW and men of similar age. The purpose of this study was to test the hypothesis that PMW have lower endothelial cell ET‐1 levels compared to men of similar age. Endothelial cells were harvested from the antecubital vein of healthy PMW (n=9, 59±3 yrs) and age‐matched men (n=7, 58±6 yrs) by threading a guide wire through an 18g IV catheter. The guide wire was rinsed in dissociation buffer to remove endothelial cells, which were then fixed in 4% paraformaldehyde, washed in phosphate buffered saline, pipetted onto coverglass, dried at 60°C, and stored at −80°C until staining. Immunocytochemistry was used to stain for ET‐1 (Endothelin‐1 Monoclonal Antibody (TR.ET.48.5), Thermo Fisher Scientific, Cat# MA3‐005), vascular endothelial cadherin (VE‐Cadherin polyclonal antibody, Thermo Fisher Scientific, Cat# 36‐1900) to identify endothelial cells, and 4′,6‐diamidino‐2‐phenylindole (DAPI, nuclear stain). Fluorescence intensity was measured in 30 cells on a Zeiss Axio imager using consistent laser intensity and exposure times. ET‐1 intensity was normalized to human umbilical vein endothelial cells, consistent with published literature. Results are reported as mean ± standard deviation. PMW and men were well match based on BMI (PMW; 25±5 kg/m2 vs. Men; 25±3), mean arterial blood pressure (PMW; 88±8 vs. Men; 93±9 mmHg), total cholesterol (PMW; 193±45 vs. Men; 192±40 mg/dL), LDL cholesterol (PMW; 103±39 vs. Men; 111±34 mg/dL), and fasting plasma glucose (PMW; 88±4 vs. Men; 92±6 mg/dL) all P>0.05. ET‐1 intensity levels were significantly lower in PMW (0.57±0.12 a.u) compared to Men (0.98±0.27 a.u, P =0.001). These preliminary findings suggest differences in endothelial ET‐1 levels between PMW and age‐matched men. Moreover, these data provide further evidence of the sexual dimorphisms in the ET‐1 system, and add insight into mechanisms underlying sex differences in vascular function.Support or Funding InformationNIH R01 HL 146558, HL‐113514, P20 GM 113125, AHA 16SDG30700015, and University of Delaware Research Foundation.