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

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, VEGF₁₆₅ a (vascular endothelial growth factor₁₆₅ 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 VEGF₁₆₅ 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. VEGF₁₆₅ a-treated Balb/CJ mice post-HLI showed no improvement in perfusion recovery with ischemic versus noni-schemic ECs showing significant increase in glycolysis. ECs under hypoxia and serum starvation upregulated PFKFB3. In ECs under hypoxia and serum starvation, VEGF₁₆₅ a versus control significantly upregulated PFKFB3 and glycolysis, whereas miR-93 versus control demonstrated no increase in PFKFB3 or glycolysis. MicroRNA-93 versus VEGF₁₆₅ 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 spe-cies. In ECs under hypoxia and serum starvation, VEGF₁₆₅ 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.