EPC cluster formation, enhanced proliferation, and elevated levels of VEGF protein in hypoxic conditions. (A) A magnified view of the central ischemic zone with EPC cluster formation reminiscent of embryologic blood island formation. (B) In contrast, a decrease of such cluster formation was seen along the nonischemic wounds. (C) (D) EPC cluster formation typically appeared at 1 week following ischemia and costained with proliferative marker Ki67 (panel D); positive staining (DAB) appears brown, and arrows point to costaining cells. (E) Exposure of human EPCs to varying O₂ concentrations in vitro demonstrated enhanced proliferation at reduced O₂ levels; representative pictures of DAPI-stained EPCs are shown on the right. (F) In comparison to EPCs, HUVECs showed significantly decreased proliferation at 0.5% O₂. (G) The increase in circulating EPC levels also correlated with an increase in VEGF protein levels. (H) Circulating mouse MNCs were isolated at the time of killing and analyzed with FACS for flk1⁺/CD11b^–, markers previously demonstrated to represent circulating EPCs. A significant increase in cell number was noted at 7 days following ischemic surgery.