Mitochondrial Turnover Regulates Ischemic Endothelial Angiogenic Capacity in Peripheral Artery Disease

Background: Atherosclerotic occlusions in lower limbs cause ischemia that results in Peripheral artery disease. Despite functional mitochondria, normal endothelial cells use Glycolysis as the primary metabolic pathway for cellular maintenance. While mitochondrial anomalies are extensively studied in skeletal muscle myocytes in PAD, the role of mitochondria in regulating ischemic endothelial (EC) function is largely unknown. Rationale: Based on a recent study that showed Mitochondrial OxPhos is required for EC migration, we aimed to understand the role of EC Mitochondrial OxPhos in regulating ischemic EC angiogenic capacity in PAD. Methods: To study the role of EC-Mitochondrial OxPhos, we developed an EC-specific (using Cdh5-Cre) mitochondrial Transcription factor-A gene (TFAM, shown to be a critical regulator of mitochondrial OxPhos) deficient mice. Since complete deletion of TFAM in ECs resulted in embryonic lethality, male and female EC-TFAM +/− mice were used to study the role of mitochondrial metabolism in regulating EC angiogenic capacity and perfusion recovery in preclinical PAD model (hind limb ischemia (HLI) by femoral artery ligation and resection). Results: HLI significantly decreased perfusion recovery and significantly increased necrosis in both male and female EC-TFAM +/− mice compared to WT controls. Consistent with impaired perfusion recovery, microvascular density was also significantly decreased in EC-TFAM +/− mice vs. WT controls. To understand the mechanism by which EC-TFAM deficiency results in impaired perfusion recovery, we isolated ECs from EC-TFAM +/− and WT mice ischemic muscle at day-7 post HLI, and performed bulk RNA-Seq. GeneOntology Pathway analysis of RNA Seq data showed that Autophagy/mitophagy and OxPhos as the top 2 pathways that were significantly inhibited in EC-TFAM +/− ischemic ECs vs. WT ischemic ECs. Hypothesizing 'accumulation of non-functional mitochondria due to impaired mitophagy inhibits the angiogenic capacity of ischemic ECs in PAD' we systematically examined the expression of key mitophagy gene expression in EC-TFAM +/− ischemic ECs vs. WT ischemic ECs. This mitophagy gene screening showed a significant decrease in Parkin expression in EC-TFAM +/− ischemic ECs vs. WT ischemic ECs. Despite higher mitochondrial DNA content in hypoxia serum starved (HSS, in vitro model for PAD), TFAM +/− ECs vs. WT, seahorse Mitostress assays showed a significant decrease in the OxPhos of HSS-TFAM +/− ECs vs. WT. Accordingly, flow cytometry analysis showed a significant decrease in mitochondrial membrane potential (using Mito-ID) in HSS-TFAM +/− ECs vs. WT. Loss of mitochondrial membrane potential and OxPhos in HSS-TFAM +/− ECs resulted in a significant loss of HSS-TFAM +/− EC angiogenic capacity on growth factor reduced matrigel compared to HSS-WT ECs. Conclusion: These data presented that 'impaired mitophagy results in the accumulation of non-functional mitochondria that inhibits the angiogenic capacity of ischemic ECs resulting in impaired perfusion recovery in PAD. Ongoing experiments are aimed at understanding the causal role of Parkin-Mitophagy in regulating ischemic EC angiogenic capacity and perfusion recovery in PAD. R01HL146673-02. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.