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Abstract: Migration and proliferation of endothelial cells are involved in re-endothelialization and angiogenesis, two important cardiovascular processes that are increased in response to estrogens. RhoA, a small GTPase which controls multiple cellular processes, is involved in the control of cell migration and proliferation. Our aim was to study the role of RhoA on estradiol-induced migration and proliferation and its dependence on estrogen receptors activity. Human umbilical vein endothelial cells were stimulated with estradiol, in the presence or absence of ICI 182780 (estrogen receptors antagonist) and Y-27632 (Rho kinase inhibitor). Estradiol increased Rho GEF-1 gene expression and RhoA (gene and protein expression and activity) in an estrogen receptor-dependent manner. Cell migration, stress fiber formation and cell proliferation were increased in response to estradiol and were also dependent on the estrogen receptors and RhoA activation. Estradiol decreased p27 levels, and significantly raised the expression of cyclins and CDK. These effects were counteracted by the use of either ICI 182780 or Y-27632. In conclusion, estradiol enhances the RhoA/ROCK pathway and increases cell cycle-related protein expression by acting through estrogen receptors. This results in an enhanced migration and proliferation of endothelial cells. [Copyright &y& Elsevier]

Objective: To examine the proliferative effect of of raloxifene on human umbilical-vein endothelial cells (HUVECs), and to investigate whether there is an associated increased expression of some key regulators of the cell cycle. Design: Cell culture for different incubation times. Setting: University research laboratory. Patient(s): Sources of HUVECs. Intervention(s): Measurement of cell proliferation, of protein levels of cyclin A, cyclin B1, cyclin D1, cyclin-dependent protein kinase (CDK) 2, CDK4, and p27Kip1, and of messenger RNA expression of cyclin A, cyclin B1, and p27Kip1. Main Outcome Measure(s): Cell proliferation was measured by the 3-(4.5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, the bromo-2′-deoxyuridine assay, and the sodium 3′-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis (4-methoxy-6-nitro) benzene sulfonic acid hydrate assay. Changes in protein expression were measured by immunoblotting, and modifications in gene expression were measured by quantitative real-time polymerase chain reaction. Result(s): Both 1 nM and 10 nM of either E2 or raloxifene achieved a similar increase in cell proliferation. The pure antiestrogen ICI 182780 only blocked the E2-induced proliferative effect. Western blot experiments detected an increase in the expression of only cyclin A and B1, and a decreasing trend for p27Kip1. Enhancements in gene expression were observed in response to E2 and raloxifene for cyclin A and B1. No significant changes were found for p27Kip1. The ICI 182780 effectively abrogated the increased gene expression associated with E2 for cyclin B1, but not for cyclin A. In contrast, ICI 182780 was ineffective in the case of raloxifene. Conclusion(s): Raloxifene increased the proliferation of HUVECs in association with enhanced gene expression of cyclins A and B1. [Copyright &y& Elsevier]

Objective: To examine the effects of raloxifene on prostacyclin production by human umbilical vein endothelial cells (HUVEC) and to shed light on the molecular details of that action. Design: Cell culture for 4, 8, 16, 24, and 48 hours. Setting: University research laboratory. Patient(s): Source of HUVEC. Intervention(s): Measurement of prostacyclin production and of protein levels and mRNA expression of cyclooxygenase (COX)-1 and -2. Main Outcome Measure(s): Prostacyclin production was measured by enzyme immunoassay, the mRNA expression of COX-1 was measured by quantitative real time-polymerase chain reaction, and the protein levels of COX-1 and -2 were measured by immunoblotting. Result(s): Raloxifene significantly increased prostacyclin release in a time- and dose-dependent manner, being higher than control after 24 hours. Raloxifene, at 0.1–10 nM, increased the mRNA expression of COX-1 and the protein content of both COX-1 as well as COX-2. All of these effects were independent of the classical pathway for estrogen receptor (ER) activation because the treatment of cells with the ER antagonist ICI 182780 did not eliminate any of the effects. Although treatment with either the selective COX-1 inhibitor SC-560 or the selective COX-2 inhibitor NS-398 significantly diminished prostacyclin release (20% ± 5% and 24% ± 7%, respectively), co-treatment with raloxifene and either SC-560 or NS-398 was followed by a smaller increase than that achieved by raloxifene alone. The nonselective COX inhibitor indomethacin, however, reduced prostacyclin production to 37% ± 11% of control values. Conclusion(s): Raloxifene increased HUVEC prostacyclin release through a mechanism possibly distinct from the classical ER pathway and involving enhanced COX-1 and COX-2 expression and activity. [Copyright &y& Elsevier]