Arteriogenesis is an inflammatory driven mechanism, describing the growth of a natural bypass from pre-existing collateral arteries to compensate for an occluded artery. The complement system component C3 is a potent natural inflammatory activator. Here, we investigated its impact on the process of collateral artery growth using C3-deficient (C3 −/−) and wildtype control mice in a murine hindlimb model of arteriogenesis. Induction of arteriogenesis by unilateral femoral artery ligation resulted in decreased perfusion recovery in C3 −/− mice on day 7 as shown by Laser Doppler imaging. Immunofluorescence staining revealed a reduced vascular cell proliferation in C3 −/− mice. Gene expression analysis displayed a significant reduction in monocyte chemoattractant protein-1 (MCP-1) expression in C3 −/− mice. Interestingly, 3 days after induction of arteriogenesis, the number of macrophages (CD68⁺) recruited to growing collaterals was not affected by C3 deficiency. However, a significant reduction in inflammatory M1-like polarized macrophages (CD68⁺/MRC1⁻) was noted. Forced mast cell activation by Compound 48/80 as well as exogenous MCP-1 application rescued the number of M1-like polarized macrophages along with perfusion recovery in C3 −/− mice. In summary, this study demonstrates that complement C3 influences arteriogenesis by mediating MCP-1 expression, which is essential for the induction and enhancement of sterile inflammation.

Peripheral artery disease is a common manifestation of atherosclerosis, characterized by insufficient tissue perfusion and chronic ischemia. Arteriogenesis and angiogenesis are essential endogenous mechanisms to restore blood flow and limit ischemic injury. The metalloprotease ADAMTS13, known for cleaving ultra-large von Willebrand factor, has been implicated in thrombotic and inflammatory regulation. However, its role in ischemic vascular remodeling remains unclear. Using a murine hind limb ischemia model, we investigated the effect of ADAMTS13 deficiency on arteriogenesis and angiogenesis by comparing male ADAMTS13^−/− and wild-type control mice. Perfusion recovery, vascular cell proliferation, immune cell infiltration, and thrombotic activity were evaluated using laser Doppler measurements, immunohistochemical analysis of adductor and gastrocnemius muscle tissues, and in vivo microscopy. ADAMTS13 deficiency did not impair perfusion recovery, collateral artery growth, or capillarization. While platelet adhesion was slightly increased in ADAMTS13^−/− mice, no thrombotic occlusions were observed. Inflammatory responses, including macrophage and neutrophil infiltration as well as macrophage polarization, were largely unaffected. Despite previous in vitro evidence indicating an angiogenic role for ADAMTS13, its absence did not compromise angiogenesis in vivo. Our findings suggest that ADAMTS13 does not play a critical role in ischemia-related angiogenesis and arteriogenesis under sterile conditions and may be relevant only in contexts involving acute and sufficiently strong thromboinflammatory stimuli.

Arteriogenesis, the growth of pre-existing collateral arteries in response to arterial occlusion, is critically regulated by perivascular immune cells. While innate immune contributions are well-established, the role of B lymphocytes remains poorly understood. We investigated the impact of B cell deficiency on arteriogenesis using B cell-deficient J_HT mice in a murine hindlimb model of femoral artery ligation (FAL). Laser-Doppler perfusion imaging, immunofluorescence staining, Giemsa staining, flow cytometry, and differential blood counts were performed at defined time points after FAL. B cell-deficient mice exhibited significantly impaired perfusion recovery on days 3 and 7 post-FAL, accompanied by reduced collateral artery diameter growth and diminished vascular cell proliferation. Early immune analysis revealed elevated platelet–neutrophil aggregate (PNA) formation and increased perivascular mast cell degranulation in B cell-deficient mice despite unchanged circulating cell counts. On day 7, perivascular M2-like macrophage numbers were selectively reduced. Furthermore, B cell deficiency disrupted the temporal reprogramming of γδ T cell subsets, impairing the shift from pro-inflammatory IFN-γ-associated to anti-inflammatory IL-10-associated subpopulations. These findings demonstrate for the first time that B cells are essential coordinators of the sequential innate and regenerative immune response driving productive collateral artery growth, positioning them as potential targets for therapeutic arteriogenesis strategies.

B cells are crucial mediators of immune homeostasis. Their interaction with and functional relevance for angiogenesis in muscle ischemia, however, have remained unclear and are the focus of the present study. We investigated their role by inducing muscle ischemia in B cell-deficient J_HT and wildtype control mice via femoral artery ligation (FAL). Ischemic gastrocnemius muscles were harvested 7 days after FAL and analyzed by (immuno-)histology. B cell-deficient mice displayed aggravated ischemic tissue damage, as evidenced by H&E staining. In addition, angiogenesis was impaired in B cell-deficient mice, as demonstrated by a reduced capillary-to-muscle fiber ratio, and decreased endothelial cell proliferation (CD31⁺/BrdU⁺). Moreover, B cell-deficient mice showed increased macrophage (CD68⁺) infiltration with a predominance of pro-inflammatory M1-like polarized macrophages (CD68⁺MRC1⁻) and a corresponding reduction in regenerative anti-inflammatory M2-like polarized macrophages (CD68⁺MRC1⁺). In summary, our data on B cell deficiency identifies B cells as potential regulators of ischemia-induced angiogenesis, most likely by influencing macrophage infiltration and polarization toward a regenerative M2-like polarized phenotype in muscle tissue ischemia.