Pentose Pathway Activation Is Superior to Increased Glycolysis for Therapeutic Angiogenesis in Peripheral Arterial Disease

Abstract

Background In endothelial cells (ECs), glycolysis, regulated by PFKFB3 (6‐phosphofructo‐2‐kinase/fructose‐2,6‐biphosphatase, isoform‐3), is the major metabolic pathway for ATP generation. In preclinical peripheral artery disease models, VEGF 165 a (vascular endothelial growth factor 165 a) and microRNA‐93 both promote angiogenesis. Methods and Results Mice following hind‐limb ischemia (HLI) and ECs with, and without, hypoxia and serum starvation were examined with, and without, microRNA‐93 and VEGF 165 a. Post‐HLI perfusion recovery was monitored. EC metabolism was studied using seahorse assay, and the expression and activity of major metabolism genes were assessed. Reactive oxygen species levels and EC permeability were evaluated. C57Bl/6J mice generated a robust angiogenic response to HLI, with ECs from ischemic versus nonischemic muscle demonstrating no increase in glycolysis. Balb/CJ mice generated a poor angiogenic response post‐HLI; ischemic versus nonischemic ECs demonstrated significant increase in glycolysis. MicroRNA‐93‐treated Balb/CJ mice post‐HLI showed better perfusion recovery, with ischemic versus nonischemic ECs showing no increase in glycolysis. VEGF 165 a‐treated Balb/CJ mice post‐HLI showed no improvement in perfusion recovery with ischemic versus nonischemic ECs showing significant increase in glycolysis. ECs under hypoxia and serum starvation upregulated PFKFB3. In ECs under hypoxia and serum starvation, VEGF 165 a versus control significantly upregulated PFKFB3 and glycolysis, whereas miR‐93 versus control demonstrated no increase in PFKFB3 or glycolysis. MicroRNA‐93 versus VEGF 165 a upregulated glucose‐6‐phosphate dehydrogenase expression and activity, activating the pentose phosphate pathway. MicroRNA‐93 versus control increased reduced nicotinamide adenine dinucleotide phosphate and virtually eliminated the increase in reactive oxygen species. In ECs under hypoxia and serum starvation, VEGF 165 a significantly increased and miR‐93 decreased EC permeability. Conclusions In peripheral artery disease, activation of the pentose phosphate pathway to promote angiogenesis may offer potential therapeutic advantages.