γδ T cells, a small subset of T cells in blood, play a substantial role in influencing immunoregulatory and inflammatory processes. The functional impact of γδ T cells on angiogenesis in ischemic muscle tissue has never been reported and is the topic of the present work. Femoral artery ligation (FAL) was used to induce angiogenesis in the lower leg of γδ T cell depleted mice and wildtype and isotype antibody-treated control groups. Gastrocnemius muscle tissue was harvested 3 and 7 days after FAL and assessed using (immuno-)histological analyses. Hematoxylin and Eosin staining showed an increased area of tissue damage in γδ T cell depleted mice 7 days after FAL. Impaired angiogenesis was demonstrated by lower capillary to muscle fiber ratio and decreased number of proliferating endothelial cells (CD31⁺/BrdU⁺). γδ T cell depleted mice showed an increased number of total leukocytes (CD45⁺), neutrophils (MPO⁺) and neutrophil extracellular traps (NETs) (MPO⁺/CitH3⁺), without changes in the neutrophils to NETs ratio. Moreover, the depletion resulted in a higher macrophage count (DAPI/CD68⁺) caused by an increase in inflammatory M1-like macrophages (CD68⁺/MRC1⁻). Altogether, we show that depletion of γδ T cells leads to increased accumulation of leukocytes and M1-like macrophages, along with impaired angiogenesis.

Cold-inducible RNA-binding protein (CIRP) is an intracellular RNA-chaperone and extracellular promoter of inflammation, which is increasingly expressed and released under conditions of hypoxia and cold stress. The functional relevance of CIRP for angiogenesis and regeneration of ischemic muscle tissue has never been investigated and is the topic of the present study. We investigated the role of CIRP employing CIRP deficient mice along with a hindlimb model of ischemia-induced angiogenesis. 1 and 7 days after femoral artery ligation or sham operation, gastrocnemius muscles of CIRP-deficient and wildtype mice were isolated and processed for (immuno-) histological analyses. CIRP deficient mice showed decreased ischemic tissue damage as evidenced by Hematoxylin and Eosin staining, whereas angiogenesis was enhanced as demonstrated by increased capillary/muscle fiber ratio and number of proliferating endothelial (CD31⁺/BrdU⁺) cells on day 7 after surgery. Moreover, CIRP deficiency resulted in a reduction of total leukocyte count (CD45⁺), neutrophils (myeloperoxidase, MPO⁺), neutrophil extracellular traps (NETs) (MPO⁺/CitH3⁺), and inflammatory M1-like polarized macrophages (CD68⁺/MRC1^-), whereas the number of tissue regenerating M2-like polarized macrophages (CD68⁺/MRC1^-) was increased in ischemic tissue samples. In summary, we show that the absence of CIRP ameliorates angiogenesis and regeneration of ischemic muscle tissue, most likely by influencing macrophage polarization in direction to regenerative M2-like macrophages.

Collateral artery growth (arteriogenesis) involves the proliferation of vascular endothelial cells (ECs) and smooth muscle cells (SMCs). Whereas the proliferation of ECs is directly related to shear stress, the driving force for arteriogenesis, little is known about the mechanisms of SMC proliferation. Here we investigated the functional relevance of the potassium channels K_V1.3 and K_Ca3.1 for SMC proliferation in arteriogenesis. Employing a murine hindlimb model of arteriogenesis, we found that blocking K_V1.3 with PAP-1 or K_Ca3.1. with TRAM-34, both interfered with reperfusion recovery after femoral artery ligation as shown by Laser-Doppler Imaging. However, only treatment with PAP-1 resulted in a reduced SMC proliferation. qRT-PCR results revealed an impaired downregulation of α smooth muscle-actin (αSM-actin) and a repressed expression of fibroblast growth factor receptor 1 (Fgfr1) and platelet derived growth factor receptor b (Pdgfrb) in growing collaterals in vivo and in primary murine arterial SMCs in vitro under K_V1.3. blockade, but not when K_Ca3.1 was blocked. Moreover, treatment with PAP-1 impaired the mRNA expression of the cell cycle regulator early growth response-1 (Egr1) in vivo and in vitro. Together, these data indicate that K_V1.3 but not K_Ca3.1 contributes to SMC proliferation in arteriogenesis.

The complement system is a potent inflammatory trigger, activator, and chemoattractant for leukocytes, which play a crucial role in promoting angiogenesis. However, little information is available about the influence of the complement system on angiogenesis in ischemic muscle tissue. To address this topic and analyze the impact of the complement system on angiogenesis, we induced muscle ischemia in complement factor C3 deficient (C3−/−) and wildtype control mice by femoral artery ligation (FAL). At 24 h and 7 days after FAL, we isolated the ischemic gastrocnemius muscles and investigated them by means of (immuno-)histological analyses. C3−/− mice showed elevated ischemic damage 7 days after FAL, as evidenced by H&E staining. In addition, angiogenesis was increased in C3−/− mice, as demonstrated by increased capillary/muscle fiber ratio and increased proliferating endothelial cells (CD31⁺/BrdU⁺). Moreover, our results showed that the total number of leukocytes (CD45⁺) was increased in C3−/− mice, which was based on an increased number of neutrophils (MPO⁺), neutrophil extracellular trap formation (MPO⁺/CitH3⁺), and macrophages (CD68⁺) displaying a shift toward an anti-inflammatory and pro-angiogenic M2-like polarized phenotype (CD68⁺/MRC1⁺). In summary, we show that the deficiency of complement factor C3 increased neutrophil and M2-like polarized macrophage accumulation in ischemic muscle tissue, contributing to angiogenesis.

Arteriogenesis, the growth of a natural bypass from pre-existing arteriolar collaterals, is an endogenous mechanism to compensate for the loss of an artery. Mechanistically, this process relies on a locally and temporally restricted perivascular infiltration of leukocyte subpopulations, which mediate arteriogenesis by supplying growth factors and cytokines. Currently, the state-of-the-art method to identify and quantify these leukocyte subpopulations in mouse models is immunohistology. However, this is a time consuming procedure. Here, we aimed to develop an optimized protocol to identify and quantify leukocyte subpopulations by means of flow cytometry in adductor muscles containing growing collateral arteries. For that purpose, adductor muscles of murine hindlimbs were isolated at day one and three after induction of arteriogenesis, enzymatically digested, and infiltrated leukocyte subpopulations were identified and quantified by flow cytometry, as exemplary shown for neutrophils and macrophages (defined as CD45⁺/CD11b⁺/Ly6G⁺ and CD45⁺/CD11b⁺/F4/80⁺ cells, respectively). In summary, we show that flow cytometry is a suitable method to identify and quantify leukocyte subpopulations in muscle tissue, and provide a detailed protocol. Flow cytometry constitutes a timesaving tool compared to histology, which might be used in addition for precise localization of leukocytes in tissue samples.

Increasing evidence suggests that lymphocytes play distinct roles in inflammation-induced tissue remodeling and tissue damage. Arteriogenesis describes the growth of natural bypasses from pre-existing collateral arteries. This process compensates for the loss of artery function in occlusive arterial diseases. The role of innate immune cells is widely understood in the process of arteriogenesis, whereas the role of lymphocytes remains unclear and is the subject of the present study. To analyze the role of lymphocytes, we induced arteriogenesis in recombination activating gene-1 (Rag1) knockout (KO) mice by unilateral ligation of the femoral artery. The lack of functional lymphocytes in Rag1 KO mice resulted in reduced perfusion recovery as shown by laser Doppler imaging. Additionally, immunofluorescence staining revealed a reduced vascular cell proliferation along with a smaller inner luminal diameter in Rag1 KO mice. The perivascular macrophage polarization around the growing collateral arteries was shifted to more pro-inflammatory M1-like polarized macrophages. Together, these data suggest that lymphocytes are crucial for arteriogenesis by modulating perivascular macrophage polarization.