Pannexin 1 modulates angiogenic activities of human endothelial colony-forming cells through IGF-1 mechanism and is a marker of senescence

Abstract

BACKGROUNDWe examined the role of pannexins in human endothelial progenitor cell (EPC) senescence. METHODSYoung and replication-induced senescent endothelial colony-forming cells (ECFCs) derived from human circulating EPCs were used to examine cellular activities and senescence-associated indicators after transfection of siRNA specific to Panx1 or lentivirus-mediated Panx1 overexpression. Hindlimb ischemia mice were used as in vivo angiogenesis model. Protein and phospho-kinase arrays were used to determine underlying mechanisms. RESULTSPanx1 was the predominant pannexin isoform in human ECFCs and up-regulated in both replication-induced senescent ECFCs and circulating EPCs from aged mice and humans. Cellular activities of the young ECFCs were enhanced by Panx1 down-regulation, but attenuated by its up-regulation. In addition, reduction of Panx1 in the senescent ECFCs could rejuvenate cellular activities with reduced senescence-associated indicators, including senescence-associated {beta}-galactosidase activity, p16INK4a, p21, acetyl-p53, and phospho-Histone H2A.X. In mouse ischemic hindlimbs injected senescent ECFCs, blood perfusion ratio, salvaged limb outcome, and capillary density were all improved by Panx1 knockdown. Insulin-like growth factor 1 (IGF-1) was significantly increased in the supernatant from senescent ECFCs after Panx1 knockdown. The enhanced activities and paracrine effects of Panx1 knockdown senescent ECFCs were completely inhibited by anti-IGF-1 antibodies. FAK, ERK and STAT3 were activated in senescent ECFCs with Panx1 knockdown, in which the intracellular calcium level was reduced, and the activation was inhibited by supplemented calcium. The increased IGF-1 in Panx1-knockdown ECFCs was abrogated respectively by inhibitors of FAK (PF562271), ERK (U0126), and STAT3 (NSC74859), and supplemented calcium. CONCLUSIONSPanx1 expression is up-regulated in human ECFCs/EPCs with replication-induced senescence and during aging. Angiogenic potential of senescent ECFCs is improved by Panx1 reduction through increased IGF-1 production via activation of FAK-ERK axis following calcium influx reduction. Our findings provide new strategies to evaluate EPC activities and rejuvenate senescent EPCs for therapeutic angiogenesis.