Abstract
Female hormones and sex-specific factors are established determinants of endothelial function, yet their relative contribution to human endothelium phenotypes has not been defined. Using human umbilical vein endothelial cells (HUVECs) genotyped by donor's sex, we investigated the influence of sex and estrogenic agents on the main steps of the angiogenic process and on key proteins governing HUVEC metabolism and migratory properties. HUVECs from female donors (fHUVECs) showed increased viability (p < 0.01) and growth rate (p < 0.01) compared with those from males (mHUVECs). Despite higher levels of G-protein coupled estrogen receptor (GPER) in fHUVECs (p < 0.001), treatment with 17β-estradiol (E2) and the selective GPER agonist G1 (both 1–100 nM) did not affect HUVEC viability. Migration and tubularization in vitro under physiological conditions were higher in fHUVECs than in mHUVECs (p < 0.05). E2 treatment (1–100 nM) upregulated the glycolytic activator PFKFB3 with higher potency in fHUVECs than in mHUVECs, despite comparable baseline levels. Moreover, Y576/577 phosphorylation of focal adhesion kinase (FAK) was markedly enhanced in fHUVECs (p < 0.001), despite comparable Src activation levels. While the PI3K inhibitor LY294002 (25 µM) inhibited HUVEC migration (p < 0.05), Akt phosphorylation levels in fHUVECs and mHUVECs were comparable. Finally, digitoxin treatment, which inhibits Y576/577 FAK phosphorylation, abolished sexual dimorphism in HUVEC migration. These findings unravel complementary modulation of HUVEC functional phenotypes and signaling molecules involved in angiogenesis by hormone microenvironment and sex-specific factors, and highlight the need for sex-oriented pharmacological targeting of endothelial function.
Highlights
The endothelium is essential in maintaining vascular homeostasis (Cines et al, 1998; Vanhoutte et al, 2017)
This was observed when FBS supplementation was reduced to 5% (Figure 1B, middle panel), a condition slowing down cell proliferation that was selected to test the effects of estrogenic agents. fHUVEC viability remained higher than that of mHUVECs at lower baseline cell density (2.5 × 103 cells/well; Figure 1B, right panel), which allowed longer culture for up to 6 days
Based on the knowledge that neovascularization is a physiological feature in the female reproductive tract and is regulated by estrogenic agents (Rubanyi et al, 2002; Trenti et al, 2017a), we further explored potential gender differences using a number of additional in vitro experimental approaches that mimic the major steps of the angiogenic process (Simons et al, 2015)
Summary
The endothelium is essential in maintaining vascular homeostasis (Cines et al, 1998; Vanhoutte et al, 2017). While the endothelium represents a key target of vascular estrogen action (Bolego et al, 2006a; Bolego et al, 2006b; Pinna et al, 2008; Arnal et al, 2010), endothelial biology is governed by sex-specific factors (Mudrovcic et al, 2017; Stanhewicz et al, 2018). Sex hormones exert direct effects and interact with the effects of sex chromosomes during lifetime (Arnold et al, 2017; Ventura-Clapier et al, 2017), suggesting that sex hormones in the local microenvironment as well as intrinsic differences at the cellular level account for gender differences in endothelial function. Due to easy access and high purity, human umbilical vein endothelial cells (HUVECs) are a valuable model for the study of physiopathological processes.
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