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, VEGF165a (vascular endothelial growth factor165a) 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 VEGF165a. 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. VEGF165a-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, VEGF165a versus control significantly upregulated PFKFB3 and glycolysis, whereas miR-93 versus control demonstrated no increase in PFKFB3 or glycolysis. MicroRNA-93 versus VEGF165a 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, VEGF165a 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.

Elevated Cytokine Levels in Plasma of Patients with SARS-CoV-2 Do Not Contribute to Pulmonary Microvascular Endothelial Permeability.

The vascular endothelial injury occurs in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections, but the mechanisms are poorly understood. We sought to determine the frequency and type of cytokine elevations and their relationship to endothelial injury induced by plasma from patients with SARS-CoV-2 versus controls. Plasma from eight consecutively enrolled patients hospitalized with acute SARS-CoV-2 infection was compared to controls. Endothelial cell (EC) barrier integrity was evaluated using ECIS (electric cell-substrate impedance sensing) on human lung microvascular EC. Plasma from all SARS-CoV-2 but none from controls decreased transendothelial resistance to a greater degree than that produced by tumor necrosis factor-alpha (TNF-α), the positive control for the assay. Thrombin, angiopoietin 2 (Ang2), and vascular endothelial growth factor (VEGF), complement factor C3a and C5a, and spike protein increased endothelial permeability, but to a lesser extent and a shorter duration when compared to SARS-CoV-2 plasma. Analysis of Ang2, VEGF, and 15 cytokines measured in plasma revealed striking patient-to-patient variability within the SARS-CoV-2 patients. Pretreatment with thrombin inhibitors, single, or combinations of neutralizing antibodies against cytokines, Ca3 and C5a receptor antagonists, or with ACE2 antibody failed to lessen the SARS-CoV-2 plasma-induced EC permeability. The EC barrier destructive effects of plasma from patients with SARS-CoV-2 were susceptible to heat inactivation. Plasma from patients hospitalized with acute SARS-CoV-2 infection uniformly disrupts lung microvascular integrity. No predicted single, or set of, cytokine(s) accounted for the enhanced vascular permeability, although the factor(s) were heat-labile. A still unidentified but potent circulating factor(s) appears to cause the EC disruption in SARS-CoV-2 infected patients. IMPORTANCE Lung vascular endothelial injury in SARS-CoV-2 patients is one of the most important causes of morbidity and mortality and has been linked to more severe complications including acute respiratory distress syndrome (ARDS) and subsequent death due to multiorgan failure. We have demonstrated that in eight consecutive patients with SARS-CoV-2, who were not selected for evidence of endothelial injury, the diluted plasma-induced intense lung microvascular damage, in vitro. Known endothelial barrier-disruptive agents and proposed mediators of increased endothelial permeability in SARS-CoV-2, induced changes in permeability that were smaller in magnitude and shorter in duration than plasma from patients with SARS-CoV-2. The effect on endothelial cell permeability of plasma from patients with SARS-CoV-2 was heat-labile. The main plasma factor that causes the increased endothelial permeability remains to be identified. Our study provides a possible approach for future studies to understand the underlying mechanisms leading to vascular injury in SARS-CoV-2 infections.