The Inflammatory Subset of Monocytes Stimulates Angiogenesis at Sites of Ischemia by Altering the Balance of Pro- and Anti-Inflammatory Cytokines.

Our laboratory has previously shown that early delivery of monocytes to ischemic tissue by mobilization from the bone marrow to blood or by adoptive transfer enhances revascularization after acute vascular injury. There is accumulating evidence that monocytes represent a heterogeneous population, containing subsets with distinct functional properties. Herein, we show that the inflammatory subset of monocytes mediates the angiogenic response in a murine model of hindlimb ischemia induced by femoral artery excision (FAE). Inflammatory (CX3CR1^loGr-1⁺) and resident (CX3CR1^hiGr-1⁻) monocytes were sorted from the bone marrow of CX3CR1^+/GFP mice and injected intravenously into recipient mice 24 after FAE. Revascularization was monitored by laser Doppler imaging. A marked improvement in blood flow was observed after adoptive transfer of inflammatory but not resident monocytes [ratio of blood flow in ischemic to nonischemic limb 7 days after surgery: 0.75±0.05 (inflammatory monocytes); 0.40±0.07 (resident monocytes); 0.40±0.03 (saline control); P <0.01]. Consistent with this finding, inflammatory monocytes are selectively recruited to sites of ischemia. Finally, we show that the bone marrow is a rich reservoir of inflammatory monocytes that is readily mobilized into the blood by ischemia. To begin to elucidate the mechanisms by which inflammatory monocytes stimulate angiogenesis, we determined the effect of the adoptive transfer of monocytes on the local production of key cytokines/chemokines in the ischemic tissue. Specifically, a non-biased multiplex immunoassay-based screen for soluble factors present in the cell-free muscle homogenates was performed. Data revealed decreased levels of the pro-inflammatory cytokines IL-6, KC, and mip-1beta and increased levels of the anti-inflammatory cytokine IL-10 in mice receiving an adoptive transfer of monocytes. In keeping with this observation, levels of free myoglobin were dramatically decreased in mice receiving an adoptive transfer of monocytes, indicating a reduction in inflammation-mediated tissue damage [myoglobin levels at 28 hours post-ischemia: 18,580±879 ng/mL (no adoptive transfer); 158±112 ng/mL (adoptive transfer)]. Interestingly, known regulators of angiogenesis, MCP-1, MMP-9, VEGF, and FGF-basic were also significantly increased. Taken together, these data indicate that adoptive transfer of a small number of monocytes shifts the balance of pro- and anti-inflammatory signals in the ischemic environment, which limits tissue damage and enhances revascularization. This finding has important therapeutic implications, since deliver of inflammatory monocytes to sites of ischemia may polarize the microenviroment towards tissue regeneration and angiogenesis.