There is compelling evidence that circulating angiogenic cells exist in humans that are able to home to sites of vascular injury and stimulate angiogenesis. However, under normal conditions, the number of these cells in the blood is small, potentially limiting the angiogenic response. Certain cytokines, including G-CSF, are able to mobilize angiogenic cells into the blood, potentially circumventing this limitation. Previously, we reported that treatment with G-CSF and AMD3100, a novel CXCR4 antagonist, significantly improved revascularization in a mouse model of acute hindlimb ischemia. Interestingly, the kinetics of angiogenic cell mobilization were distinct, with maximal improvement in blood flow observed on days 5–7 in the AMD3100 treated cohort versus 10–14 in the G-CSF treated cohort. Accordingly, combination treatment with G-CSF and AMD3100 resulted in the earliest and most robust angiogenic response. This regimen may merit investigation in future clinical trials of circulating angiogenic cells for the treatment of ischemic vascular diseases.
In the present study, the mechanism by which these agents stimulate angiogenesis was explored. We first performed adoptive transfer studies. Blood mononuclear cells from G-CSF and AMD3100 treated mice were infused intravenously into recipient mice 24 hours after the induction of hindlimb ischemia. Perfusion in the ischemic hindlimb was significantly improved in mice receiving adoptively transferred cells compared to saline treated controls (overall effect of treatment, p<0.0001). Moreover, capillary density in the ischemic hindlimb was significantly increased in mice that had received adoptively transferred cells (p=0.0004). Interestingly, on a per cell basis, blood mononuclear cells from un-mobilized mice were equally effective as G-CSF and AMD3100 mobilized blood mononuclear cells in inducing revascularization. Together, these observations suggest that these agents improve angiogenesis by increasing the basal number of circulating angiogenic cells rather than mobilizing a unique angiogenic cell population.
The ability of circulating angiogenic cells to directly incorporate into neoendothelium is controversial. To address this issue, blood mononuclear cells from Tie2GFP transgenic mice were adoptively transferred into non-transgenic FVB mice following the induction of hindlimb ischemia. Importantly, GFP expression in Tie2GFP transgenic mice is mainly limited to endothelial cells. Thus, if adoptively transferred cells directly contribute to the neovasculature, GFP+ endothelial cells should be apparent in recipient mice. Endothelial cell chimerism in the adductor muscle from the ischemic limb was analyzed 2 weeks post surgery using a novel flow cytometry-based assay. Essentially no GFP+ cells were detected, showing that the great majority of neoendothelium was of recipient origin. These data were confirmed by histological assays showing that von Willibrand factor-positive endothelial cells in the ischemic muscle of these mice did not co-express GFP. Collectively, these data show that treatment with G-CSF or AMD3100 potently stimulates angiogenesis at sites of ischemia by mobilizing angiogenic cells into the blood. These cells are able to home to sites of injury where they stimulate angiogenesis in a paracrine fashion.