Bone marrow subpopulations contain distinct types of endothelial progenitor cells and angiogenic cytokine-producing cells

Therapeutic angiogenesis can be induced by the implantation of bone marrow cells (BMCs). However, the mechanism of BMC-mediated neovascularization remains to be clarified. We investigated the differential activities of bone marrow subpopulations in angiogenesis and cytokine production. BMCs were separated into positive and negative fractions by surface expression of Mac-1, Gr-1, CD19, and c-kit, respectively. After 7 days of culture in the presence of vascular endothelial growth factor (VEGF), the cells produced adherent cells which incorporate acetylated low-density lipoprotein (acLDL). Mac-1(+) and Mac-1(−) cells produced almost equal numbers of acLDL(+) cells, but only Mac-1(−) cells expressed endothelial markers, including Flk-1, vWF, and CD31. Similarly, the expression of endothelial markers was detected in Gr-1(−), CD19(−), and c-kit(+) BMC fractions at 7-day cultures, but not in Gr-1(+), CD19(+), or c-kit(−) cells. In contrast, freshly isolated Mac-1(+) and Gr-1(+) BMCs expressed higher levels of mRNAs for angiogenic cytokines (including VEGF-A, FGF-2, and HGF) than Mac-1(−) and Gr-1(−) cells, respectively. Moreover, Mac-1(+)/c-kit(+) BMC subpopulation expressed higher levels of VEGF-A and SDF-1 mRNAs than other subpopulations. These data demonstrate that a relatively small proportion of VEGF-cultured adherent cells are true endothelial cells with a Flk-1(+)/vWF(+)/CD31(+) phenotype. Moreover, endothelial stem/progenitor cells (EPCs) are limited primarily to Mac-1(−), Gr-1(−), and c-kit(+) BMC populations. In contrast, angiogenic cytokine mRNAs were also produced by Mac-1(+), Gr-1(+), and c-kit(−) BMCs, suggesting the heterogeneity of effector cell types for neovasculatization therapy.