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C3 Deficiency Leads to Increased Angiogenesis and Elevated Pro-Angiogenic Leukocyte Recruitment in Ischemic Muscle Tissue.

ABSTRACT: 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.

SUBMITTER: Gotz P

PROVIDER: S-EPMC8198161 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Mitophagy, the elimination of damaged mitochondria through autophagy, promotes neuronal survival in cerebral ischemia. Previous studies found deficient mitophagy in ischemic neurons, but the mechanisms are still largely unknown. We determined that BNIP3L/NIX, a mitophagy receptor, was degraded by proteasomes, which led to mitophagy deficiency in both ischemic neurons and brains. BNIP3L exists as a monomer and homodimer in mammalian cells, but the effects of homodimer and monomer on mitophagy are unclear. Site-specific mutations in the transmembrane domain of BNIP3L (S195A and G203A) only formed the BNIP3L monomer and failed to induce mitophagy. Moreover, overexpression of wild-type BNIP3L, in contrast to the monomeric BNIP3L, rescued the mitophagy deficiency and protected against cerebral ischemic injury. The macroautophagy/autophagy inhibitor 3-MA and the proteasome inhibitor MG132 were used in cerebral ischemic brains to identify how BNIP3L was reduced. We found that MG132 blocked the loss of BNIP3L and subsequently promoted mitophagy in ischemic brains. In addition, the dimeric form of BNIP3L was more prone to be degraded than its monomeric form. Carfilzomib, a drug for multiple myeloma therapy that inhibits proteasomes, reversed the BNIP3L degradation and restored mitophagy in ischemic brains. This treatment protected against either acute or chronic ischemic brain injury. Remarkably, these effects of carfilzomib were abolished in bnip3l-/- mice. Taken together, the present study linked BNIP3L degradation by proteasomes with mitophagy deficiency in cerebral ischemia. We propose carfilzomib as a novel therapy to rescue ischemic brain injury by preventing BNIP3L degradation.Abbreviations: 3-MA: 3-methyladenine; AAV: adeno-associated virus; ATG7: autophagy related 7; BCL2L13: BCL2-like 13 (apoptosis facilitator); BNIP3L/NIX: BCL2/adenovirus E1B interacting protein 3-like; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; CFZ: carfilzomib; COX4I1: cytochrome c oxidase subunit 4I1; CQ: chloroquine; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; I-R: ischemia-reperfusion; MAP1LC3A/LC3A: microtube-associated protein 1 light chain 3 alpha; MAP1LC3B/LC3B: microtube-associated protein 1 light chain 3 beta; O-R: oxygen and glucose deprivation-reperfusion; OGD: oxygen and glucose deprivation; PHB2: prohibitin 2; pMCAO: permanent middle cerebral artery occlusion; PRKN/PARK2: parkin RBR E3 ubiquitin protein ligase; PT: photothrombosis; SQSTM1: sequestosome 1; tMCAO: transient middle cerebral artery occlusion; TOMM20: translocase of outer mitochondrial membrane 20; TTC: 2,3,5-triphenyltetrazolium hydrochloride.

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C3 Deficiency Leads to Increased Angiogenesis and Elevated Pro-Angiogenic Leukocyte Recruitment in Ischemic Muscle Tissue.

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