Differentiation of bone marrow–derived GFP⁺cells into functional endothelial cells with a schematic of ischemia-induced vasculogenesis. A second bone marrow transplantation model was employed, in which mice were reconstituted with bone marrow cells ubiquitously expressing GFP⁺ (ie, not restricted to tie-2⁺ cells). Similar findings of bone marrow progenitor involvement during ischemic neovascularization were noted in these mice. (A) GFP⁺ (green) bone marrow–derived cells were recruited to ischemic tissue and formed vascular-like structures by day 14; blue staining (DAPI) represents all cell nuclei in the tissue. (B) Lectin (red) staining highlighted functional neovessels within the tissue oriented in the plane of ischemia. (C) Analysis of the tissue sections under multiple fluorescent windows identified vessels lined by bone marrow–derived EPCs. (D) Tissue sections were also taken perpendicular to the plane of ischemia, which similarly identified regions of costaining of lectin-perfused blood vessels (red) and BM-derived EPCs (green); arrows point to costained vessels, which appear orange. (E) (F) (G) GFP⁺ cells that lined blood vessels exhibited triple staining for CD31 (blue), lectin (red), and GFP (green). (H) An overlay of the triple staining is shown. (I) Schematic representation of the proposed mechanism by which EPCs contribute to neovascularization within ischemic tissue. Tissue ischemia causes the release of growth factors/cytokines (including VEGF), leading to a systemic response and the mobilization of BM-derived EPCs. Hypoxic conditions alter the vascular endothelium, causing circulating EPCs' arrest and egress into the tissue. Once in the interstitium, EPCs form cellular clusters and proliferate to increase the pool of cells available for neovascularization. Gradients of ischemia drive the formation of vascular cords in the direction of relatively hypoxic regions. Vascular cords tubulize and unite with existing vasculature, leading to increased tissue perfusion.