Activation of GPER Induces Differentiation and Inhibition of Coronary Artery Smooth Muscle Cell Proliferation

BACKGROUND: Vascular pathology and dysfunction are direct life-threatening outcomes resulting from atherosclerosis or vascular injury, which are primarily attributed to contractile smooth muscle cells (SMCs) dedifferentiation and proliferation by re-entering cell cycle. Increasing evidence suggests potent protective effects of G-protein coupled estrogen receptor 1 (GPER) activation against cardiovascular diseases. However, the mechanism underlying GPER function remains poorly understood, especially if it plays a potential role in modulating coronary artery smooth muscle cells (CASMCs). METHODOLOGY/PRINCIPAL FINDINGS: The objective of our study was to understand the functional role of GPER in CASMC proliferation and differentiation in coronary arteries using from humans and swine models. We found that the GPER agonist, G-1, inhibited both human and porcine CASMC proliferation in a concentration- (10(−8) to 10(−5) M) and time-dependent manner. Flow cytometry revealed that treatment with G-1 significantly decreased the proportion of S-phase and G2/M cells in the growing cell population, suggesting that G-1 inhibits cell proliferation by slowing progression of the cell cycle. Further, G-1-induced cell cycle retardation was associated with decreased expression of cyclin B, up-regulation of cyclin D1, and concomitant induction of p21, and partially mediated by suppressed ERK1/2 and Akt pathways. In addition, G-1 induces SMC differentiation evidenced by increased α-smooth muscle actin (α-actin) and smooth muscle protein 22α (SM22α) protein expressions and inhibits CASMC migration induced by growth medium. CONCLUSION: GPER activation inhibits CASMC proliferation by suppressing cell cycle progression via inhibition of ERK1/2 and Akt phosphorylation. GPER may constitute a novel mechanism to suppress intimal migration and/or synthetic phenotype of VSMC

Effect of GPER stimulation on CASMC migration.

<p>(A) Representative of three experiments of porcine CASMC migration study. Cellular migration was assessed by using scratch-wound assay. Cells were cultured in ibidi μ-dish culture insert until they reached confluence. Inserts (500 µm; dashed lines) were then removed, and cells were exposed to control media or media supplemented with either 1 µM G-1 or 1 µM G-1+5 µM G15. Five images per treatment were collected immediately and then 48 h following insert removal. Within a specific image five different distances were measured from the edge of the dashed-line. (B) Each bar represents the mean distance traveled from the edge +/− SEM. Comparison between the different treatments was done with One-way ANOVA followed by Tukey's multiple comparison test, and treatment groups identified by different letter (a,b, or c) indicate significant difference (p<0.05) between each of the three experimental groups.</p

<b>Table 3. </b>The effect of G-1 on porcine CASMCs after 48 hour treatment.

<p>The numbers in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064771#pone-0064771-t001" target="_blank">Tables 1</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064771#pone-0064771-t003" target="_blank">3</a> are from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064771#pone-0064771-g001" target="_blank">Figure 1 B–H</a>.</p

Effect of G-1 treatment on cell morphology in cultured CASMCs.

<p>Human and porcine CASMCs were incubated in the presence of either a SMDS or G-1 (1 µM and 10 nM) for 1 and 2 day followed by immunostaining using anti-α-smooth muscle antibody and FITC-conjugated anti-rabbit IgG secondary antibody (green).</p

<b>Table 1.</b> The effect of G-1 treatment on human CASMCs for 48 hours.

<p><b>Table 1.</b> The effect of G-1 treatment on human CASMCs for 48 hours.</p

G-1 inhibits the phosphorylation of Akt and ERK1/2 in CASMCs.

<p>A and B: HCASMCs were cultured in serum and phenol red free medium for two days followed by PDGF-BB (10 ng/ml) (A) or G-1 (1 µM) treatment (B) for indicated time in the presence of 10% FCS. C: PCASMCs were cultured in the same medium for two days followed by G-1(10⁻⁷–10⁻⁵M); PDGF-BB (10 ng/ml); and G-1(10⁻⁷–10⁻⁵M) plus PDGF-BB (10 ng/ml) treatments for indicated time in the presence of 10% FCS. Total cell extracts (1×10⁶) were subjected to Western-blot analyses for Phospho-Akt and ERK1/2 level. Under the Western blot panels, a quantitative representation of the expression analysis from 3 independent experiments is shown. Vehicle-treated CASMCs cells were used as control. Data are normalized by β-Actin and expressed as means ±SD (n = 3). A significant difference is indicated by either **** p<0.0001, *** p<0.001, ** p<0.01 or * p<0.05(one or two-way ANOVA); C, G and P represent control, G-1 and PDGF-BB treatment sample respectively, ns indicates no significant difference.</p

Effects of G-1 treatment on the protein level of cyclinB1, cyclinD1 and p21 in CASMCs.

<p>A and C: Western blot results of cyclinB1, cyclinD1 and p21 protein levels in human CASMCs (A) and porcine CASMCs (C). CASMCs were synchronized by 3 days of cultivation in serum-free and phenol red free medium followed by vehicle treatment as control (C); G-1(G, 10⁻⁷–10⁻⁵ M); 10 ng/ml PDGF-BB (P); and G-1(10⁻⁷–10⁻⁵ M) plus 10 ng/ml PDGF treatment (GP or P+G) for 24 or 48 hours in the presence 10% FCS. Total cell extracts (1×10⁶) were subjected to Western-blot analyses for cyclinB1, cyclinD1 and p21 level. B and D: Quantitative densitometric analyses of band intensities from 3 independent experiments. Data are normalized by GAPDH or β-Actin, expressed as the mean±SD (n = 3). A significant difference is indicated by either *(p<0.05); **(p<0.01) or *** (p<0.001) (one-way ANOVA). Representative histograms are shown.</p