Aim: 

There is few data regarding the RhoA pathway and its relationship with the estradiol effects on reendothelialization. Our aim was to evaluate if RhoA is involved in the proliferative and migratory-estradiol effects on cultured human umbilical vein endothelial cells (HUVEC).

Methods: 

HUVEC were exposed to different combinations of 10 nM estradiol, 1 mM ICI182780 (an estrogen receptor antagonist) and 100 mM Y27632 (Rho kinase inhibitor). Cell proliferation was evaluated using BrdU incorporation assay. Gene expression as well as the protein levels of cyclin A, cyclin B1, cyclin D1, cyclin dependent protein kinase (CDK) 2, CDK4, and p27^Kip1 (key elements of the cell cycle) were measured by quantitative RT-PCR and immunoblotting, respectively. Migration was measured in HUVEC cultures previously injured, with UTHCSA programme. RhoA, cyclin D1, CDK2 and actin filaments were visualized either by immunocytochemistry.

Results: 

Estradiol increased RhoA, cyclin D1, CDK2 and stress fibres expression, as well as, cell proliferation and migration, whereas ICI182780 and Y27632 blocked estradiol-induced effects. Immunoblotting detected an increased expression of RhoA, cyclins A, B1 and D1 and CDK1, showing a decreasing trend for p27^Kip1. RhoA, cyclin A and B1 gene expressions were also enhanced in response to estradiol, without changes for p27^Kip1 mRNA, and decreased after the use of ICI182780.

Conclusion: 

RhoA pathway is involved in the proliferative and migratory-estradiol effect on HUVEC in a estrogen receptor-dependent manner.

Supported by Ministerio Ciencia e Innovación, ISCIII (FIS 06/0589, RED HERACLES RD06/0009), Consellería. Sanidad (AP 10/2007, AP 121/08) and Consellería de Educación, Generalitat Valenciana (GVPRE/2008/276). PJO holds a post-doc position, and AS is a FPI fellow (BFPI 06/145), both from Conselleria de Educación, Generalitat Valenciana, Spain.