-2-Methoxyestradiol (2-ME), a metabolite of estradiol with little affinity for estrogen receptors, inhibits proliferation of vascular smooth muscle cells; however, the molecular mechanisms underlying this effect are incompletely understood. Our previous work shows that 2-ME inhibits initiation (blocks phosphorylation of ERK and Akt) and progression (reduces cyclin expression and increases expression of cyclin inhibitors) of the mitogenic pathway and interferes with mitosis (disrupts tubulin organization). Because the RhoA/ROCK1 pathway (RhoA ¡ ROCK1 ¡ myosin phosphatase targeting subunit ¡ myosin light chain) is involved in cytokinesis, herein we tested the concept that 2-ME also blocks the RhoA/ROCK1 pathway. Because of the potential importance of 2-ME for preventing/treating vascular diseases, experiments were conducted in female human aortic vascular smooth muscle cells. Microarray transcriptional profiling suggested an effect of 2-ME on the RhoA/ROCK1 pathway. Indeed, 2-ME blocked mitogen-induced GTP-bound RhoABC expression and membrane-bound RhoA, suggesting interference with the activation of RhoA. 2-ME also reduced ROCK1 expression, suggesting reduced production of the primary downstream signaling kinase of the RhoA pathway. Moreover, 2-ME inhibited RhoA/ROCK1 pathway downstream signaling, including phosphorylated myosin phosphatase targeting subunit and myosin light chain; the ROCK1 inhibitor H-1152 mimicked these effects of 2-ME; both 2-ME and H-1152 blocked cytokinesis. 2-ME also reduced the expression of tissue factor, yet another downstream signaling component of the RhoA/ROCK1 pathway. We conclude that 2-ME inhibits the pathway RhoA ¡ ROCK1 ¡ myosin phosphatase targeting subunit ¡ myosin light chain, and this likely contributes to the reduced cytokinesis in 2-ME treated HASMCs.2-methoxyestradiol; RhoA; ROCK1; myosin phosphatase targeting subunit; myosin light chain; cytokinesis 2-METHOXYESTRADIOL (2-ME) is an endogenous metabolite of estradiol that attenuates vascular smooth muscle cell (VSMC) proliferation, migration, and extracellular matrix synthesis (5,8,9) and reduces injury-induced neointima formation (4), cholesterol-induced atherosclerosis (9), monocrotaline-induced vascular thickening in pulmonary hypertension (9), and injuryinduced glomerosclerosis (9). Although 2-ME inhibits VSMC proliferation and is thus effective against multiple proliferative disorders (4, 9, 10), the mechanisms via which 2-ME mediates these actions remain incompletely understood.Our previously published work shows that in human aortic vascular smooth muscle cells (HASMCs) 2-ME inhibits cell proliferation in part by blocking initiation of the mitogenic pathway (4). Specifically, 2-ME inhibits phosphorylation of both ERK1/2 and Akt (Fig. 1), which turns off the mitogenic pathway by decreasing the expression and activity of cyclins. Indeed, we (4) found that 2-ME 1) blocks cell-cycle progression in both G0/G1 and in G2/M phases, 2) reduces cyclin D1 and cyclin B1 expression, 3) inhibits Cdk-1 and Cdk-2 activity, 4) reduc...

Estradiol (E2) induces vascular repair by promoting endothelial growth and capillary formation. Based on our previous findings that the capillary stimulating effects of E2 are mimicked by its non-permeable analog, BSA-tagged E2, we hypothesize that the stimulatory effects are potentially mediated via the newly discovered membrane bound G-protein coupled estrogen receptor (GPER). To investigate this, we assessed capillary formation by endothelial cells in response to E2, and GPER agonist (G1)and antagonist (G15) in a 2-D matrigel based assay. Capillary formation was assessed microscopically by quantifying junction/sprout formation and capillary length. E2 (10nM) increased capillary formation and this effect was mimicked by G1 (10nM; stimulated from 100% to 517±46% and 210±41%, respectively; p<.05 vs control). The effects of E2 and G1 were significantly abrogated by G15 (100nM; p<.05), suggesting a role for GPER in mediating the capillary stimulatory effects of E2. Because G-protein coupled mechanisms, Akt, ALK1 and SMAD1/5/8 are involved in capillary formation, we investigated their role in GPER induced capillary formation. ECs expressed GPER, ALK1 and SMAD1/5/8. Treatment with G1 (10-100nM) up-regulated the expression of ALK1 from 100% to 168±21% (p<.05 vs control) and phosphorylated SMAD1/5/8 from 100% to 208±36% (p<.05 vs control). Similar to capillary formation the stimulatory effects of G1 on SMAD1/5/8 were blocked significantly (p<.05) by G15 (100nM), Pertussis Toxin (0,1ng/ml; G protein pathway inhibitor), LY294002 (5μM; Akt/PI3K inhibitor) and ALK1Fc (100ng/ml; specific ALK-1 antagonist). Silencing of GPER and SMAD1 by siRNA (50nM) abrogated the stimulatory effects of E2 and G1 on capillary formation, and SMAD1/5/8 and Akt phosphorylation. Moreover, treatment with G1(100nM) upregulated ID-1 expression from 100% to 179±26% (p<.05 vs control), a downstream target of ALK1/pSMAD1/5/8. We conclude that E2 via GPER promotes EC-mediated capillary formation by a mechanism that involves non-genomic activation of ALK1→ pSMAD1/5/8 ↔ pAkt → ID-1. GPER agonists in general may promote healing of injured vascular beds by promoting EC activity leading to more rapid endothelial recovery and capillary formation following injury.

MicroRNAs play a key role in vascular remodeling associated with cardiovascular disease. MiR-221 actively contributes to injury-induced neointima formation by inhibiting endothelial cell (EC) growth and promoting smooth muscle cell (SMC) growth. Since estradiol (E2) prevents neointimal thickening, we hypothesize that E2 mediates its vasoprotective actions by downregulating miR-221 expression and abrogating its effects on SMC and EC growth. RT-qPCR confirmed that both Human Umbilical Vein ECs (HUVECs) and Human Coronary Artery SMCs (HCASMCs) produce miR-221. Treatment of HCASMCs with PDGF-BB (20ng/ml) induced miR-221 levels from 100±8% to 189±9% (p<.05) and treatment with E2 (100nM) inhibited this to 126±4% (p<.05 vs PDGF). PDGF-BB stimulated DNA synthesis (BrdU incorporation), CyclinD1 expression (Western Blot) and migration (Scratch assay) in HCASMCs and these effects were mimicked by miR-221 (25nM) overexpression; and abrogated in HCASMCs transfected with miR-221 antimiR (25nM). E2 inhibited PDGF-induced HCASMC numbers by 30±4% and these effects were reversed by miR-221 (p<0.05). Inhibitory effects of E2 on PDGF-induced miR-221 production in HCASMCs were mimicked by estrogen receptor (ER) α agonist PPT, but not by ERβ and GPER agonists; and blocked by ERα antagonist MPP, suggesting these effects are ERα mediated. In contrast to SMCs, transfection of HUVECs with miR-221 mimic inhibited capillary formation and wound healing by 39±8% and 27±6%, respectively (p<.05). Neutralization of miR-221 with antimiR induced capillary formation and wound closure by 26±3 % and 51±15%, respectively (p<.05). E2 (10nM) inhibited miR-221 levels in HUVECs from 100 to 73±6% (p<.05). Moreover, transfection of HUVECs with miR-221 mimic inhibited E2-induced capillary formation (from137±9% to 85±13%; p<.05 vs E2) and wound closure (from 125±5% to 82±12%; p<.05 vs E2). Our findings provide the first evidence that E2 inhibits miR-221 production in HCASMCs and HUVECs and these effects contribute to its antimitogenic effects on HCASMCs and capillary promoting effects in HUVECs. Modulation of miR-221 by E2 represents a novel mechanism by which E2 may mediate its differential effects on SMC and EC growth, and confer vascular protection.

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