Angiogenesis by Implantation of Peripheral Blood Mononuclear Cells and Platelets Into Ischemic Limbs

Abstract

Background— Peripheral blood mononuclear cells (PBMNCs), platelets, and polymorphonuclear leukocytes (PMNs) contain various angiogenic factors and cytokines. Methods and Results— Unilateral hindlimb ischemia was surgically induced in athymic nude rats, and fluorescence-labeled human blood cells (PBMNCs [10 7 cells]+platelets [10 9 ] or PBMNCs [10 7 ]+platelets [10 9 ]+PMNs [10 7 ]) were intramuscularly implanted into the ischemic limbs. Laser Doppler imaging revealed markedly increased blood perfusion in PBMNC+platelet-implanted limbs (44% increase, P <0.001) compared with control implantation of human umbilical vein vascular endothelial cells. The addition of PMNs to PBMNCs+platelets attenuated blood perfusion (27% decrease, P <0.01). Neocapillary densities were increased by implantation of PBMNCs+platelets or platelets alone (3.5-fold and 2.4-fold, respectively; P <0.001), whereas PMNs inhibited (32%, P <0.05) PBMNC+ platelet-mediated capillary formation. There was no incorporation of implanted PBMNCs into neocapillaries, whereas PBMNCs and platelets accumulated around arterioles after implantation. Cellular extract from PBMNCs+platelets, in which vascular endothelial growth factor (VEGF), basic fibroblast growth factor, platelet-derived growth factor-AB, and transforming growth factor-β were detected, markedly stimulated tubule formation of human umbilical vein vascular endothelial cells. Anti-VEGF neutralizing antibody markedly inhibited tubule formation and in vivo vessel formation. Neutrophil elastase inhibitor blocked the antiangiogenic action of PMNs, whereas inhibitors of oxygen metabolites had no effect. Conclusions— This study demonstrated that implantation of PBMNCs and platelets into ischemic limbs effectively induces collateral vessel formation by supplying angiogenic factors (mainly VEGF) and cytokines, suggesting that this cell therapy is useful as a novel strategy for therapeutic angiogenesis.