The Pro-neovascularization Activity And Therapeutic Potential Of Peripheral Blood Stem Cells In Limb Ischemia

Background: Autologous transplantation of mobilized peripheral blood mononuclear cells (M-PBMNCs) is a novel approach to improve critical limb ischemia (CLI) in diabetes. The M-PBMNCs obtained after G-CSF mobilization contain much more endothelial progenitor cells (EPCs), and they also secrete various angiogenic factors for therapeutic neovascularization. However, EPCs from diabetes are dysfunctional and impaired in ischemia-induced neovascularization.Objective: This study aimed to confirm the compromised efficiency of diabetic M-PBMNCs in therapeutic neovascularization, and to determine the underlying mechanisms of this impairment.Methods: 10~6 diabetic M-PBMNCs from 17 diabetic patients or healthy controls, or PBS were injected into the ischemic limbs of streptozotocin-indcued diabetic nude mice. The limb blood perfusion was detected by laser Doppler blood perfusion imaging between these 3 groups in the following weeks. Ambulatory score and ischemia damage were also evaluated in the following 4 weeks. Capillary/fiber ratio was detected by CD31 or BS-1 lectin, and arteriole density by a-smooth muscle actin (a-SMactin), collateral vessel formation by microangiopathy, and pericytes recruitment by myosin, calponin and a-SMactin. Non-invasive real time image and histopathology were used to detect the in vivo role of transplanted M-PBMNCs. EPCs were produced by culture of M-PBMNCs in endothelial growth medium-2 (EGM-2 MV) for 7days, then characterized and counted by uptake of Dil-acLDL and staining of UEA-1. Proliferation and adhesion ability of EPCs were compared between diabetic M-PBMNCs and controls. In vitro vascular network incorporation based on matrigel and subcutaneous matrigel plug assay were used to test the pro-neovascularization role of M-PBMNCs.