Endothelial cells stimulate differentiation of circulating endothelial precursors through soluble factors.

Objectives: Several studies have demonstrated that endothelial progenitor cells (EPCs) from either the bone marrow or circulating blood are essential for functional recovery of ischemic tissue after cell therapy. Although promising preliminary results, the mechanism by which EPCs interact with vascular wall cells and ischemic tissues remains unclear. We have previously reported that human coronary artery endothelial cells (HCAECs) co-cultured with peripheral blood mononuclear cell (PBMC) can stimulate their early differentiation toward a pre-endothelial phenotype. We investigated possible soluble factors, released from the co-culture, involved in EPC differentiation. Materials and methods: PBMC isolated from healthy donors were co-cultures with human coronary endothelial cells (HCAEC) by means of 0.4 µm pore sized membranes transwell. EPC differentiation was evaluated by means flow cytometry. Milliplex assay was used to measure soluble factors. Results: Co-coltures released IL-6, IL-8, EGF and CCL-2 which levels were significantly higher than that found in HCAEC or in PBMC alone. In order to check their involvement in PBMC differentiation, blocking experiments with neutralizing antibodies were performed. Flow cytometry analysis confirmed an impairment of PBMC differentiation toward a pre-endothelial phenotype when IL-6, IL-8 and with a lesser extent CCL-2 were blocked. Conclusions: These data add a new insight into the mechanisms by which endothelial precursors interact with vascular wall, thus suggesting future directions in understanding and treating ischemic injury.