Purpose: Endothelial progenitor cells (EPCs) contribute to ischemic tissue repair by secretion of paracrine factors. Our aim was to analyze the effect of hypoxic stress, the patho-physiological status of ischemic tissue, on EPC viability and paracrine secretion and to assess the effect of EPC conditioned medium (EPC-CM) on in vitro and in vivo angiogenesis. Methods: EPCs were obtained from peripheral blood of healthy donors. To obtain EPC-CM, EPCs were cultured for 24 h in growth factor- and serum-free medium under hypoxia (1%O2). EPC-CM was pooled from 6 different donors before storage at –80°C. EPC-CM obtained in normoxia was used as a control. EPC-CM was analyzed by both proteomics and cytokine evaluation. EPC-CM activity was evaluated on human umbilical vein endothelial cells (HUVECs) by WST-1 and angiogenesis assay. For cell viability, EPC-CM at different dilutions (100-1.5%) was incubated for 24 h. For angiogenesis assay, HUVECs seeded on Matrigel were incubated in EPC-CM for 8 h before evaluation of capillary-like structures. Cytotoxicity test and assessment of the presence of anti-HLA antibody in EPC-CM were also performed. To test hypoxic EPC-CM in vivo, unilateral hindlimb ischemia was induced in adult male Sprague-Dawley rats (n=36). 3 intramuscular injections were performed. Blood flow was monitored at 3 time points, up to 21 d. Explants were also evaluated by histology and immunohistochemistry. Results: EPC viability was not significantly affected by hypoxia. 298 proteins were identified by proteomics, comprising factors potentially involved in angiogenesis (protease inhibitors, apolipoprotein E, protein S100 family), with 17 proteins significantly upregulated and 12 downregulated as compared to normoxia. Hypoxic upregulation of 6 factors involved in cell recruitment to ischemic tissues (IL-8, VEGF, PDGF-BB, MCP- 1, MIF, HGF) was also observed. WST-1 assay showed a significant positive effect of EPC-CM on HUVEC viability (p<0.01), with a dose-related effect. In vitro angiogenesis assay showed that EPC-CM significantly promoted capillary-like structures formation. EPC-CM was not cytotoxic for humans, with no presence of anti-HLA antibodies. EPC-CM promoted blood flow in ischemic hindlimbs, enhancing angiogenesis. Conclusions: EPC-CM obtained after hypoxic stress may represent an alternative for regenerative therapy of ischemic tissue. Novel therapeutic strategies based on EPC paracrine factors may replace cell transplantation, as "cell-free" therapy could overcome the risk of adverse immunological reactions and the problem of heterologous rejection.

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

FELICE, FRANCESCA;BARSOTTI, MARIA CHIARA;SANTONI, TATIANA;CECCHETTINI, ANTONELLA;PIRAS, ANNA MARIA;CHIELLINI, FEDERICA;DI STEFANO, ROSSELLA
2014-01-01

Abstract

Purpose: Endothelial progenitor cells (EPCs) contribute to ischemic tissue repair by secretion of paracrine factors. Our aim was to analyze the effect of hypoxic stress, the patho-physiological status of ischemic tissue, on EPC viability and paracrine secretion and to assess the effect of EPC conditioned medium (EPC-CM) on in vitro and in vivo angiogenesis. Methods: EPCs were obtained from peripheral blood of healthy donors. To obtain EPC-CM, EPCs were cultured for 24 h in growth factor- and serum-free medium under hypoxia (1%O2). EPC-CM was pooled from 6 different donors before storage at –80°C. EPC-CM obtained in normoxia was used as a control. EPC-CM was analyzed by both proteomics and cytokine evaluation. EPC-CM activity was evaluated on human umbilical vein endothelial cells (HUVECs) by WST-1 and angiogenesis assay. For cell viability, EPC-CM at different dilutions (100-1.5%) was incubated for 24 h. For angiogenesis assay, HUVECs seeded on Matrigel were incubated in EPC-CM for 8 h before evaluation of capillary-like structures. Cytotoxicity test and assessment of the presence of anti-HLA antibody in EPC-CM were also performed. To test hypoxic EPC-CM in vivo, unilateral hindlimb ischemia was induced in adult male Sprague-Dawley rats (n=36). 3 intramuscular injections were performed. Blood flow was monitored at 3 time points, up to 21 d. Explants were also evaluated by histology and immunohistochemistry. Results: EPC viability was not significantly affected by hypoxia. 298 proteins were identified by proteomics, comprising factors potentially involved in angiogenesis (protease inhibitors, apolipoprotein E, protein S100 family), with 17 proteins significantly upregulated and 12 downregulated as compared to normoxia. Hypoxic upregulation of 6 factors involved in cell recruitment to ischemic tissues (IL-8, VEGF, PDGF-BB, MCP- 1, MIF, HGF) was also observed. WST-1 assay showed a significant positive effect of EPC-CM on HUVEC viability (p<0.01), with a dose-related effect. In vitro angiogenesis assay showed that EPC-CM significantly promoted capillary-like structures formation. EPC-CM was not cytotoxic for humans, with no presence of anti-HLA antibodies. EPC-CM promoted blood flow in ischemic hindlimbs, enhancing angiogenesis. Conclusions: EPC-CM obtained after hypoxic stress may represent an alternative for regenerative therapy of ischemic tissue. Novel therapeutic strategies based on EPC paracrine factors may replace cell transplantation, as "cell-free" therapy could overcome the risk of adverse immunological reactions and the problem of heterologous rejection.
2014
https://academic.oup.com/eurheartj/issue/35/suppl_1
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11568/566267
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