NF κB activation in embryonic endothelial progenitor cells enhances neovascularization via PSGL-1 mediated recruitment: Novel role for LL37

Embryonal endothelial progenitor cells (eEPCs) are capable of inducing therapeutic angiogenesis in a chronic hind limb model. However, the proportion of eEPCs recruited to the ischemic tissue appears to be a limiting step for the induction of cell-based therapeutic neovascularization. In the present study, we primed eEPCs with the human cathelicidin LL37 (hCAP-18) ex vivo to selectively enhance the eEPC-dependent gain of perfusion in vivo and elucidated the mechanism of action of LL37 on eEPCs. Seven days after femoral artery excision, 5 × 10⁶ eEPCs (wt, wild type; p65t, transiently p65 transient; p65s, stable p65-transfected; LL37-eEPCs, LL37 peptide preincubated) were retroinfused into the anterior tibial vein. Recruitment of diI-labeled eEPCs in the ischemic gastrocnemic muscle was investigated 2 days later, whereas collateral growth and perfusion score (obtained by fluorescent microspheres) were assessed at day 7 and day 35 and are given as percentage of day 7 level. Capillary/muscle fiber ratio in the ischemic lower limb was obtained at day 35. Embryonic EPC recruitment in vitro and in vivo was found elevated after LL37 and p65t pretreatment, but not in p65s-eEPCs displaying increased IκBα or after LL37 in IκB-DN overexpressing eEPCs. Using LL37- and p65-teEPCs, collateral growth (181 ± 10% and 165 ± 8%, respectively) surpassed that of wt-eEPCs (135 ± 7%), increasing perfusion ratio (208 ± 20% and 210 ± 17% vs. 142 ± 12% in wt-eEPCs, respectively), whereas p65s-eEPCs exerted no additive effect (collateral growth 130 ± 8%; perfusion ratio 155 ± 15%). Moreover, p65t-eEPC-induced neovascularization was abrogated by blocking antibodies against E-selectin and P-selectin glycoprotein ligand-1 (PSGL-1). We conclude that NF κB activation by LL37 or transient p65-transfection increases functionally relevant eEPC recruitment to ischemic muscle tissue via induction of PSGL-1 and E-selectin.