The Role of The G Protein Coupled Estrogen Receptor in Endothelial Progenitor Cell Number and Function

Abstract

Microvascular dysfunction is a key component of cardiovascular disease and plasma estrogen levels have been shown to impact vascular function. Endothelial progenitor cells (EPCs) are a stem cell population that support the integrity of the microvasculature and show increased proliferation in response to estradiol exposure when assessed in vitro. The plasma concentration of EPCs negatively correlates with incidence of stroke and coronary artery disease. In vivo studies of the effect of estradiol exposure on EPC number are complicated by the presence of other estrogens and complex estrogen signaling pathways. In EPCs, pathways that are activated via nuclear estrogen receptors are well investigated and pro‐angiogenic. Recent evidence suggests that other estrogens may be biologically active through a G protein coupled estrogen receptor, GPER, and that activation of GPER may have a modulatory relationship with nuclear estrogen receptor pathways. Investigation of GPER in EPCs is crucial to understanding the effects of estrogens on EPC health and vascular function.Here, we present the first evidence that GPER is present in human EPCs via immunohistochemistry and PCR analysis. We also investigated the role of agonism and antagonism of GPER in human EPC proliferation, migration, adhesion and paracrine function. Human EPCs were defined as cells that are isolated from bone marrow or peripheral blood mononuclear cell fractions, display typical endothelial cell (EC) morphology and are positive for cell surface proteins: VEGFR2, CD133, cKIT, CD34, and Dil‐ac‐LDL uptake. We assessed GPER expression via qPCR and immunocytochemistry on EPCs without hormone treatment. The proliferation assays were performed over a 24 hour exposure to 100 nM estradiol in combination with one of the following: 0.1, 1 and 10 μM concentrations of GPER agonist, G1 or GPER antagonist, G36. In our migration, adhesion and proliferation assays, EPCs were exposed to 100 nM estradiol in combination with one of the GPER modulator concentrations (as described above) for 24 hours and assessed thereafter. Migration was assessed via Transwell migration assay. To assay adhesion 1000 EPCs were seeded onto a 96 well plate, incubated for 4 hours and rinsed. Wells were then imaged and adherent cells were quantified. EPC paracrine function was assessed by tube formation assay. In this assay, treated EPCs were added to an endothelial cell tube formation assay to assess their ability to augment endothelial cell tubulogenesis.Our results show that GPER is expressed on human EPCs. In vitro EPC proliferation, migration, adhesion and paracrine function was enhanced by antagonism of GPER in a dose dependent manner. In vitro EPC proliferation, migration, adhesion and paracrine function was attenuated by agonism of GPER in a dose dependent manner. To investigate candidates for activators of this modulatory pathway, further studies will investigate estrone, the most prominent estrogen in males and postmenopausal females, and its action at GPER.