Abstract Body: Background: Role of mitochondrial anomalies including impaired mitochondrial bioenergetics, accumulation of mitochondrial DNA/proteins, loss of membrane potential, and mitochondrial turnover in regulating ischemic endothelial (EC) angiogenic capacity and perfusion recovery in PAD are not known.
Methods: Hind limb ischemia (HLI) was used as the Preclinical-PAD model. Hypoxia serum starvation (HSS) was used as an in vitro PAD model. EC-specific TFAM (Mitochondrial Transcription factor-A) deficient mice were developed by crossing TFAM^Floxed mice with constitutive Cdh5^Cre (Vec-Cre) mice. Parkin was overexpressed by electroporating skeletal muscle. Urolithin-A was delivered intra-peritoneally. Transmission electron microscopy, hanging drop Matrigel assay, Seahorse metabolic assays were performed in in vitro HSS-ECs and fresh isolated ischemic-ECs from skeletal muscle post-HLI.
Results: HLI significantly decreased perfusion recovery and increased necrosis in both male and female EC-TFAM^Het-KO mice vs. EC-TFAM^WT. Gene Ontology Pathway analysis of ischemic-ECs isolated from ischemic-muscle at day-7 post-HLI showed Lysosomal pathway as the top differentially regulated pathway between TFAM^Het-KO ECs vs. TFAM^WT. Systematic analysis of the key genes that regulate mitochondrial turn-over showed that TFAM-deficiency specifically decreases the expression of Parkin, a key regulator of mitophagy in ischemic-ECs. Overexpressing Parkin significantly increased ischemic-EC angiogenic capacity and perfusion recovery by facilitating functional mitochondrial-biogenesis which is reflected in a significant increase in mitochondrial potential and bioenergetics. Pharmacologically, Urolithin-A, a postbiotic derivative of Pomegranates that has been shown to induce mitophagy, also significantly induced ischemic-EC angiogenic capacity and perfusion recovery by promoting functional mitochondrial-biogenesis in Preclinical-PAD. P<0.05 considered significant in all experiments.
Conclusion: Our data shows that impaired mitophagy results in the accumulation of non-functional mitochondria that inhibit ischemic-EC angiogenic capacity and perfusion recovery in PAD. Targeting mitophagy genetically by Parkin-overexpression or pharmacologically by Urolithin-A treatment facilitates the removal of dysfunctional mitochondria that allows functional mitochondrial-biogenesis and induces perfusion recovery in PAD.