Endothelial progenitor cell conditioned medium as a new possible alternative to cell therapy in tissue regeneration

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Purpose: Endothelial progenitor cells (EPCs) contribute to ischemic tissue repair by secretion of \nparacrine factors. Our aim was to analyze the effect of hypoxic stress, the patho-physiological status \nof ischemic tissue, on EPC viability and paracrine secretion and to assess the effect of EPC \nconditioned medium (EPC-CM) on in vitro and in vivo angiogenesis. Methods: EPCs were obtained \nfrom peripheral blood of healthy donors. To obtain EPC-CM, EPCs were cultured for 24 h in growth \nfactor- and serum-free medium under hypoxia (1%O2). EPC-CM was pooled from 6 different donors \nbefore storage at –80°C. EPC-CM obtained in normoxia was used as a control. EPC-CM was analyzed \nby both proteomics and cytokine evaluation. EPC-CM activity was evaluated on human umbilical vein \nendothelial cells (HUVECs) by WST-1 and angiogenesis assay. For cell viability, EPC-CM at different \ndilutions (100-1.5%) was incubated for 24 h. For angiogenesis assay, HUVECs seeded on Matrigel \nwere incubated in EPC-CM for 8 h before evaluation of capillary-like structures. Cytotoxicity test and \nassessment of the presence of anti-HLA antibody in EPC-CM were also performed. To test hypoxic \nEPC-CM in vivo, unilateral hindlimb ischemia was induced in adult male Sprague-Dawley rats (n=36). \n3 intramuscular injections were performed. Blood flow was monitored at 3 time points, up to 21 d. \nExplants were also evaluated by histology and immunohistochemistry. Results: EPC viability was not \nsignificantly affected by hypoxia. 298 proteins were identified by proteomics, comprising factors \npotentially involved in angiogenesis (protease inhibitors, apolipoprotein E, protein S100 family), with \n17 proteins significantly upregulated and 12 downregulated as compared to normoxia. Hypoxic \nupregulation of 6 factors involved in cell recruitment to ischemic tissues (IL-8, VEGF, PDGF-BB, MCP- \n1, MIF, HGF) was also observed. WST-1 assay showed a significant positive effect of EPC-CM on \nHUVEC viability (p<0.01), with a dose-related effect. In vitro angiogenesis assay showed that EPC-CM \nsignificantly promoted capillary-like structures formation. EPC-CM was not cytotoxic for humans, with \nno presence of anti-HLA antibodies. EPC-CM promoted blood flow in ischemic hindlimbs, enhancing \nangiogenesis. Conclusions: EPC-CM obtained after hypoxic stress may represent an alternative for \nregenerative therapy of ischemic tissue. Novel therapeutic strategies based on EPC paracrine factors \nmay replace cell transplantation, as "cell-free" therapy could overcome the risk of adverse \nimmunological reactions and the problem of heterologous rejection.

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