Evidence of synergistic/additive effects of sildenafil and erythropoietin in enhancing survival and migration of hypoxic endothelial cells

Endothelial cell dysfunction is a common event to several pathologies including pulmonary hypertension, which is often associated with hypoxia. As the endothelium plays an essential role in regulating the dynamic interaction between pulmonary vasodilatation and vasoconstriction, this cell type is fundamental in the development of vascular remodeling and increased vascular resistance. We investigated the protective effects of sildenafil, a phosphodiesterase type 5 inhibitor, given in combination with erythropoietin (Epo), as it has been demonstrated that both drugs have antiapoptotic effects on several cell types. Specifically, we examined the viability and angiogenic properties of rat pulmonary artery endothelial cells upon exposure to either 21% or 1% oxygen, in presence of sildenafil (1 and 100 nM) and Epo (5 and 20 U/ml) alone or in combination (1 nM and 20 U/ml). Cell proliferation and viability were analyzed by Trypan blue staining, MTT assay, and Annexin V/propidium iodide stainings. In all assays, the ability of the combination treatment in improving cell viability was superior to that of either drug alone. The angiogenic properties were studied using a migration and a 3D collagen assay, and the results revealed increases in the migration potential of endothelial cells as well as the ability to form tube-like structures in response to sildenafil and the combination treatment. We therefore conclude that both drugs exert protective effects on endothelial cells on hypoxia and that sildenafil enhances the migratory and angiogenic properties, especially in hypoxic conditions. Furthermore, we present evidence of possible additive or synergistic effects of both drugs.