Estrogen Attenuates Phenotypic Switch of Human Arteriolar Smooth Muscle Cells: Role of p53 and pRb

Abstract

Cardiovascular diseases (CVD) remains the leading cause of mortality worldwide. Vascular inflammation is a major contributor to the onset and the pathogenesis of this disease. In response to inflammatory stimuli, vascular smooth muscle cells (VSMCs) switch from a contractile to a dedifferentiated synthetic phenotype, characterized by decreased expression of differentiation markers and increased proliferative and migratory abilities. By virtue of its ability to attenuate these processes, estrogen (E2) is known to play a vasculoprotective role. While the vast majority of studies investigating the role of estrogen on phenotypic modulation were conducted on VSMCs isolated from large vessels, the effect of estrogen on VSMCs extracted from microvessels (microVSMCs) remains largely obscure. Thus, we sought assess the effect of estrogen on phenotypic switch of microVSMCs, and characterize the underlying molecular mechanism.We were able to extract microVSMCs from human dermal arterioles by enzymatic sprouting method. The purity of the cells was identified by their morphology and by immunostaining of two VSMC markers: smooth muscle alpha‐actin and calponin. In addition, we previously assessed the concentration‐dependent effect of estrogen on the VSMC response, with 10−10 M being the optimal physiologic concentration. Results showed that E2 (10−10 M) inhibited FBS‐induced cell proliferation and migration. This estrogen‐induced inhibition of was accompanied by decreased activation of mitogenic extracellular signal–regulated kinase (ERK1/2) and focal adhesion kinase (FAK), involved in cell proliferation and migration, respectively. Furthermore, E2 (10−10 M) increased 5′ AMP‐activated protein kinase (AMPK) phosphorylation of. In addition, E2 blocked the cells in G0/G1 phase of cell cycle. These estrogenic effects were not inhibited by ICI 280,780 (5 μM, estrogen receptor (ERα/β) antagonist). Surprisingly, ICI 280,780 mimicked estrogen, acting as an ER agonist. Furthermore, estrogen‐induced cell cycle arrest was concomitant with increased expression of p53 and decreased phosphorylation of retinoblastoma protein (pRb). The regulation of the aforementioned tumor suppressor proteins, p53 and pRb, was not mirrored by apoptosis evident by the absence of E2‐induced caspase‐3 cleavage. Alternatively, SA‐β‐gal staining showed that E2 induced senescence of VSMC. Moreover, E2 increased the expression of contractile differentiation markers: calponin and caldesmon.Taken together, this is the first study to report the effect of estrogen on phenotypic switch of VSMCs extracted from human arterioles. Our results indicate that estrogen attenuated VSMC proliferation by inducing VSMC senescence, via an ERα/β‐independent p53/pRb‐mediated mechanism. These results potentially implicate estrogen membrane receptor, GPR30, as the mediator of rapid estrogenic response in microVSMCs. In addition, due to the potential of estrogen to favorably modulate VSMC phenotype, our results may explain the lower incidence of CVDs in estrogen‐replete premenopausal females.Support or Funding InformationMPP # 320133 to A.E. and CNRS to M.F.Effect of estrogen on VSMC proliferationCells were treated with 10% FBS in the presence or absence of E2 (10‐10 M) for 1,3,5,7, and 9 days. E2 was added 30 minutes before and during treatment with FBS.Figure 1Effect of estrogen on cell senescence in VSMCs. Cells were treated with FBS, in the presence or absence of E2 (10‐10 M) or ICI 280,780 (5 μM). E2 or ICI 280,780 were added 30 minutes before and during treatment of FBS. Photography image (magnification 5 X) of SA‐β‐gal stained cells were obtained.Figure 2