Interaction of Estrogen and Tumor Necrosis Factor in Endothelial Cell Migration and Early Stage of Angiogenesis

The role of estrogen replacement therapy in postmenopausal women remains controversial. The authors hypothesized that contradictory results with estrogen therapy may be explained by estrogen's potent proangiogenic property, which could be protective in women without atherosclerotic disease but in the presence of chronic inflammation, could lead to destabilization of atherosclerotic plaques. The authors thus examined the interaction between 17-estradiol (E2) and the inflammatory cytokine tumor necrosis factor (TNF) in an early stage of angiogenesis. Human umbilical endothelial cells were grown to confluence. Migration was assessed with a wound assay and proliferation was assessed with 5-bromo-2'-deoxyuridine (BrDU). Cells were treated with medium alone, TNF at 0.3, 1, or 20 ng/ml, E2 at 20 nM, or the combination of E2 and TNF. The authors used real-time polymerase chain reaction (PCR) to measure changes in expression of the angiogenesis genes angiopoeitin-2 (Ang-2), vacular endothelial growth factor (VEGF)-A and -C, and interleukin (IL)-8. A large dose of TNF (20 ng/ml) inhibited healing at 24 to 48 h and the addition of E2 preserved some healing. E2 by itself doubled migration, with only a minimal effect on proliferation. A low dose of TNF (0.3 ng/ml) had no effect on migration, 1.0 ng/ml moderately increased it, but the addition of E2 to both doses of TNF increased migration. There was no change in migration when cells were pretreated with E2 and given TNF after wounding, whereas pretreatment with TNF followed by E2 significantly increased wound healing. The nitric oxide synthase (NOS) inhibitor N-nitro-l-arginine-methyl ester (l-NAME) completely blocked the E2 effect on migration. TNF (0.3 and 1.0 ng/ml) increased expression of VEGF-C (2.8 0.1- and 2.5 0.2-fold, respectively) and IL-8 (32.8 1.2- and 42.7 3.6-fold, respectively) mRNA, but E2 had no significant effect on these molecules. E2 increases the angiogenic activity of TNF. This could potentially worsen the stability of complex atherosclerotic plaques and increase cardiovascular events.