ALBERT

Description: Introduction Ischemic cardiovascular diseases are a leading cause of mortality in the industrialized world. Cell-based therapies have been tested to promote revascularization, mainly through the release and actions of paracrine factors that may prevent apoptosis and support migration and proliferation of resident endothelial cells (EC) and circulating endothelial progenitors (EPC). The mononuclear cell fraction of umbilical cord blood (CB-MNC) is rich in pluripotent progenitors and represents an attractive cell source for therapeutic angiogenesis. In order to demonstrate the paracrine action of CB-MNC we tested the angiogenic potential of conditioned medium (CM) derived from apoptotic CB-MNC evaluating the effects exerted over mature EC and peripheral blood (PB)-derived EPC in terms of proliferation, migration and capillary-network formation in vitro. Methods Human Umbilical Vein Endothelial Cells (HUVEC, Lonza) were cultured in Endothelial Basal Medium (EBM2) supplemented with SingleQuotes (EGM2). EPCs were obtained from PB as “Endothelial Colony Forming Cells” (ECFC). Both HUVEC and ECFC were used for subsequent experiments from passage 2 to passage 6. MNC were isolated from CB by density gradient centrifugation, gamma-irradiated (70Gy) to induce apoptosis and incubated in EBM2 for 72h to obtain the release of CM. CM was recovered from culture, centrifuged (4000 rpm 10 min) to remove cell debris and stored at -80°C. MNC viability after gamma-irradiation was evaluated by flow cytometry (7-AAd staining). RayBio Human Angiogenesis Array and ELISA assays (to quantify PDGF-AB, VEGF165, EGF, IGF-I) were used to assess CM soluble factors content. The angiogenic potential of soluble factors released by gamma-irradiated MNC was assessed by Matrigel capillary-like network formation assay using the Transwell Permeable Supports and confirmed using the sole CM in the Matrigel assay. CM capacity to support HUVEC and ECFC viability was assessed by the MTT assay and the capacity to induce HUVEC and ECFC migration by the Chemicon Cell Migration Assay. EBM2 was used as control medium. All experiments were repeated at least 4 times. Results Gamma-irradiation induced a significant time-dependent reduction in MNC viability (p