Mobilization of Endothelial Progenitor Cells in Autologous and Allogeneic Peripheral Blood Stem Cell Grafts.

Abstract

Endothelial-like progenitor cells circulate in the peripheral blood and can be enumerated using cell culture progenitor assays. These circulating vascular progenitor cells (VPCs) are implicated in new vessel formation and regenerative potential in several animal and human models of tissue injury. A small proportion likely arise from rare marrow-derived hemangioblasts, a greater number from vessel-wall endothelial precursors, and the majority derive from circulating monocytic progenitors under angiogenic stimulation. Given the emerging role of VPCs in regenerative processes, we sought to characterize the mobilization of VPCs in peripheral blood stem cell grafts used in autologous and allogeneic hematopoietic transplantation. Mononuclear cells were separated from peripheral blood (PB) or peripheral blood stem cell (PBSC) samples using Ficoll density centrifugation and plated on fibronectin-coated plastic dishes in RPMI supplemented with 20% fetal calf serum, 50 μg/ml endothelial cell growth factor, and 10 IU/ml heparin. After 48 h, the more immature non-adherent cells were collected, washed and re-plated in fibronectin-coated dishes under similar angiogenic conditions. Media and growth factors were replaced every 2 – 3 days and putative VPC clusters were enumerated 7 days after replating. Immunohistochemistry revealed that VPC clusters from PB and PBSCs were CD45+ and acquired endothelial features (CD31 and VE-cadherin) in vitro under angiogenic stimulation and gradually lost monocytic surface markers (CD14) suggesting the important contribution of monocyte-derived VPCs. VPCs were then enumerated in transplant candidates with hematologic malignancies (multiple myeloma = 4 pts, and lymphoma = 3 pts, having received a median of 2 previous chemotherapy regimens (range: 1–4) and 7 previous treatment cycles (range: 4–14)), and compared to healthy controls (n=21). Before mobilization, VPCs were markedly reduced in patients with hematologic malignancies as compared to normal donors with 16±10 vs 99±21 VPCs per ml (mean±SD), respectively (p=0.006). In 5 patients undergoing mobilization with cyclophosphamide and G-CSF, VPCs in the PB increased from 7±2 on day 0 to 51±9 by day 7 of mobilization (p=0.05), representing a median fold-increase of 8.9 (range, 3.0 – 29.8). Levels of VPCs in PB on day 7 of mobilization correlated with the peripheral blood CD34 counts measured on the same day (r=0.94). In an effort to compare the regenerative capacity and blood-forming potential of autologously mobilized PBSCs (n=5) and PBSCs from normal donors (n=14), graft VPCs were compared with levels of CD34+ cells, total CFU and CFU-GM. None of these hematopoietic progenitors correlated with VPC numbers in a significant manner (r < 0.5 in all tests of linearity). Using standard mobilization strategies, we have observed that VPCs were higher in allogeneic (7.5±1.6 x103/kg) than autologous (2.7±1.7 x103/kg) mobilized peripheral blood grafts (p=0.05). Our analysis of PBSC grafts indicates that VPC content in patients undergoing autologous SCT is low both before and after standard mobilisation strategies. In addition, VPC mobilization may occur independently of hematopoietic mobilization. In view of the potential role of VPCs in recovery from transplant related tissue injury, it may be useful to devise angiogenic mobilization strategies that complement hematopoietic mobilization.