Rationale: Ischemic injuries remain a leading cause of mortality and morbidity worldwide, and restoration of functional blood perfusion is vital to limit tissue damage and support healing. Objective: To reveal a novel role of macrophages in reestablishment of functional tissue perfusion following ischemic injury that can be targeted to improve tissue restoration. Methods and Results: Using intravital microscopy of ischemic hindlimb muscle in mice, and confocal microscopy of human tissues from amputated legs, we found that macrophages accumulated perivascularly in ischemic muscles, where they expressed high levels of iNOS (inducible nitric oxide [NO] synthase). Genetic depletion of iNOS specifically in macrophages (Cx3cr1-CreERT2;Nos2 ^fl/fl or LysM-Cre;Nos2 ^fl/fl ) did not affect vascular architecture but highly compromised blood flow regulation in ischemic but not healthy muscle, which resulted in aggravated ischemic damage. Thus, the ability to upregulate blood flow was shifted from eNOS (endothelial)-dependence in healthy muscles to completely rely on macrophage-derived iNOS during ischemia. Macrophages in ischemic muscles expressed high levels of CXCR4 (C-X-C chemokine receptor type 4) and CCR2 (C-C chemokine receptor type 2), and local overexpression by DNA plasmids encoding the corresponding chemokines CXCL12 (stromal-derived factor 1) or CCL2 (chemokine [C-C motif] ligand 2) increased macrophage numbers, while CXCL12 but not CCL2 induced their perivascular positioning. As a result, CXCL12-overexpression increased the number of perfused blood vessels in the ischemic muscles, improved functional muscle perfusion in a macrophage-iNOS-dependent manner, and ultimately restored limb function. Conclusions: This study establishes a new function for macrophages during tissue repair, as they regulate blood flow through the release of iNOS-produced NO. Further, we demonstrate that macrophages can be therapeutically targeted to improve blood flow regulation and functional recovery of ischemic tissues.

Open aortic surgery evokes a systemic inflammatory response and is associated with high morbidity and mortality. Purinergic signaling has been shown to be crucial for maintaining vascular integrity and attenuating inflammation related to hypoxia. The involvement of purinergic signaling in cross clamping of major human arteries is unknown. Our aim was to compare systemic inflammatory responses and hypoxia-induced purinergic signaling in patients undergoing either open infra-renal abdominal aortic repair or infra-inguinal revascularization. Pre- and 24 h post-operative blood samples were gathered from 6 patients undergoing aortic clamping and 6 similar patients undergoing common femoral artery cross-clamping. Using Biorad Multipex™ 21- and 27-panels 48 different cytokines, chemokines and growth factors were analyzed, in addition to circulating levels of ATP, ADP, CD39, CD73 and HIF-1α, and compared between the groups. Several inflammatory cytokines were elevated from baseline levels after aortic clamping, but not after femoral cross clamping. Most pronoun rises were seen in IL-6 (667 %, P = 0.016) and HGF (760 %, P = 0.016). HIF-1α values showed a steady increase after clamping of either artery unless the subject underwent blood transfusion. Despite an adequate increase in HIF-1α CD39 and CD73 activity decreased significantly after aortic clamping (P = 0.047 and P = 0.016, respectively). Aortic clamping is associated with a clear and strong systemic inflammatory response and impaired repair mechanisms in terms of purinergic signaling. Patients undergoing open aorta repair could benefit from pre-operative medical therapy, which enhances CD73 expression.