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.

Sterile inflammation after injury is important for tissue restoration. In injured human and mouse tissues, macrophages were recently found to accumulate perivascularly. This study investigates if macrophages adopt a mural cell phenotype important for restoration after ischemic injury. Single-cell RNA sequencing of fate-mapped macrophages from ischemic mouse muscles demonstrates a macrophage-toward-mural cell switch of a subpopulation of macrophages with downregulated myeloid cell genes and upregulated mural cell genes, including PDGFRβ. This observation was further strengthened when including unspliced transcripts in the analysis. The macrophage switch was proven functionally relevant, as induction of macrophage-specific PDGFRβ deficiency prevented their perivascular macrophage phenotype, impaired vessel maturation and increased vessel leakiness, which ultimately reduced limb function. In conclusion, macrophages in adult ischemic tissue were demonstrated to undergo a cellular program to morphologically, transcriptomically and functionally resemble mural cells while weakening their macrophage identity. The macrophage-to-mural cell-like phenotypic switch is crucial for restoring tissue function and warrants further exploration as a potential target for immunotherapies to enhance healing.

Carboxyxanthones containing carboxylic acid groups linked to lipophilic aromatic rings resemble the key pharmacophoric features of many nonsteroidal anti-inflammatory drugs (NSAIDs). This structural similarity makes them attractive scaffolds for the development of new anti-inflammatory agents. This study describes the production, cytocompatibility, and anti-inflammatory potential of ten carboxyxanthones (1–10) and two intermediates (11–12) by evaluating their effects on key pro-inflammatory mediators, namely interleukin 6 (IL-6) and prostaglandin E2 (PGE₂). As these compounds are produced by distinct mechanisms, their multi-target potential will be evaluated. Carboxyxanthones were obtained by multi-step pathways using different synthetic approaches through classical benzophenone or diaryl ether intermediates synthesis followed by intramolecular acylation. To the best of our knowledge, the synthesis of carboxyxanthones 3 and 5 is described herein for the first time. All tested compounds were cytocompatible with lipopolysaccharide (LPS)-stimulated macrophages. The most notable carboxyxanthones were 3, 4, 7, and 8, which were able to significantly reduce IL-6 production by approximately 60%. Molecular docking simulations between compounds 1–12 and cyclooxygenase-2 were conducted to characterize the structural features underlying molecular recognition, and to identify the most promising candidates for subsequent PGE₂ assays. Carboxyxanthones 3, 5, and 6, as well as intermediate 12, were predicted to be the best. In the human in vitro inflammation model used, carboxyxanthone 6 exhibited the most potent and consistent inhibitory effect on PGE₂ production. At the highest concentration tested (100 µM), it presented an efficacy comparable to that of celecoxib. Carboxyxanthones 3 and 5 demonstrated a biphasic effect, decreasing and increasing PGE₂ production at lower (5, 12.5, and 25 µM) and higher (50 and 100 µM) concentrations, respectively. These results highlight the potential of carboxyxanthones as promising modulators of inflammatory pathways, paving the way for further studies aimed at elucidating their mechanisms of action, optimizing structural features, and assessing their safety and therapeutic potential in relevant disease models.

Background: Neutrophils accumulate at sites of ischemic injury, where a distinct subset characterized by CD49d ⁺ CXCR4 ^hi VEGFR1 ^hi expression promotes angiogenesis and contributes to blood flow recovery. This study investigated the function, origin, and recruitment mechanisms of these pro-angiogenic neutrophils (PANs) in a murine model of hindlimb ischemia and explored whether a corresponding population responds to acute ischemia in humans. Methods: Using spectral flow cytometry and immunohistochemistry, PANs and classical neutrophils were characterized in ischemic gastrocnemius muscle following hindlimb ischemia in mice. Blood flow recovery was assessed by laser speckle flowmetry. To determine the origin and mobilization mechanisms of PANs, splenectomy, chemical sympathectomy with 6-hydroxydopamine (6-OHDA), and surgical splenic denervation were performed. Translational studies examined blood samples collected during elective abdominal aortic aneurysm repair to assess PAN mobilization following acute lower limb ischemia induced by aortic cross-clamping. Results: PANs rapidly accumulated in ischemic muscle through a CD49d-dependent mechanism, and blockade of CD49d impaired blood flow recovery. During homeostasis, PANs were maintained within a splenic reservoir and were rapidly released into circulation following ischemia. Splenectomy and disruption of splenic sympathetic signaling significantly reduced PAN recruitment to ischemic tissue and impaired functional recovery. Mobilization was driven by ischemia-induced sympathetic activation, which decreased CXCL12 levels in the splenic red pulp, disrupting CXCR4-mediated PAN retention. In humans, a phenotypically similar PAN population was identified in peripheral blood, which increased rapidly following lower limb ischemia during aortic cross-clamping. The speed of this response is consistent with release from an extramedullary reservoir, although its anatomical source remains unknown. Conclusion: These findings identify the spleen as a reservoir of pro-angiogenic neutrophils in mice and reveal a neuroimmune mechanism regulating their mobilization and recruitment after ischemic injury. The rapid appearance of PAN in human circulation after acute ischemia suggests that comparable extramedullary neutrophil reservoirs may also exist in humans.

Sterile inflammation following injury is important for tissue restoration. In injured human and mouse tissues, macrophages were recently found to accumulate perivascularly. This study investigates if macrophages adopt a mural cell identity important for restoration following ischemic injury. Single-cell RNA-sequencing of fate-mapped macrophages from ischemic mouse muscles demonstrates an identity switch of a subpopulation of macrophages with downregulated myeloid cell genes and upregulated mural cell genes. This macrophage-to-mural cell switch was further strengthened when including unspliced transcripts in the analysis. Induction of macrophage-specific PDGFRβ-deficiency prevented the perivascular macrophage phenotype, impaired vessel maturation and increased vessel leakiness, which ultimately reduced limb function. In conclusion, macrophages in adult ischemic tissue were demonstrated to undergo a transdifferentiation program to morphologically, transcriptomically and functionally resemble mural cells while losing their macrophage identity. The macrophage-to-mural cell switch is crucial for restored tissue function, and warrants exploration for future immunotherapies to enhance healing following injury.