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.

N-Methyl-D-aspartate receptor activation, novel mechanism of homocysteine-induced blood-retinal barrier dysfunction.

Elevated levels of amino acid homocysteine (Hcy) recognized as hyperhomocysteinemia (HHcy) was reported in several human visual disorders, such as diabetic retinopathy (DR) and age-related macular degeneration (AMD). Breakdown of blood-retinal barrier (BRB) is concomitant with vision loss in DR and AMD. We previously reported that HHcy alters BRB. Here, we tested the hypothesis that HHcy alters BRB via activation of N-methyl-D-aspartate receptor (NMDAR). Human retinal endothelial cells subjected to high level of Hcy and mouse model of HHcy were used. We injected Hcy intravitreal and used a mouse model of HHcy that lacks cystathionine-ß-synthase (CBS). RT-PCR, western blot, and immunofluorescence showed that retinal endothelial cells (RECs) express NMDAR at the gene and protein levels both in vitro and in vivo and this was increased by HHcy. We assessed BRB function and retinal morphology using fluorescein angiogram and optical coherence tomography (OCT) under HHcy with and without pharmacological inhibition of NMDAR by (MK801) or in mice lacking endothelial NMDAR (NMDARE-/- mouse). Additionally, retinal albumin leakage and tight junction proteins ZO-1 and occludin were assessed by western blotting analysis. Inhibition or elimination of NMDAR was able to improve the altered retinal hyperpermeability and morphology under HHcy as indicated by significant decrease in retinal albumin leakage and restoration of tight junction proteins ZO-1 and occludin. Our findings underscore a potential role for endothelial NMDAR in mediating Hcy-induced breakdown of BRB and subsequently as a potential therapeutic target in retinal diseases associated with HHcy such as DR and AMD. KEY MESSAGES: • Elevated levels of homocysteine (Hcy) are defined as hyperhomocysteinemia (HHcy). • HHcy is implicated in diabetic retinopathy and age-related macular degeneration. • HHcy alters BRB via activation of N-methyl-D-aspartate receptor.

Homocysteine Induces Inflammation in Retina and Brain.

Homocysteine (Hcy) is an amino acid that requires vitamins B12 and folic acid for its metabolism. Vitamins B12 and folic acid deficiencies lead to hyperhomocysteinemia (HHcy, elevated Hcy), which is linked to the development of diabetic retinopathy (DR), age-related macular degeneration (AMD), and Alzheimer's disease (AD). The goal of the current study was to explore inflammation as an underlying mechanism of HHcy-induced pathology in age related diseases such as AMD, DR, and AD. Mice with HHcy due to a lack of the enzyme cystathionine-ß-synthase (CBS) and wild-type mice were evaluated for microglia activation and inflammatory markers using immuno-fluorescence (IF). Tissue lysates isolated from the brain hippocampal area from mice with HHcy were evaluated for inflammatory cytokines using the multiplex assay. Human retinal endothelial cells, retinal pigment epithelial cells, and monocyte cell lines treated with/without Hcy were evaluated for inflammatory cytokines and NFκB activation using the multiplex assay, western blot analysis, and IF. HHcy induced inflammatory responses in mouse brain, retina, cultured retinal, and microglial cells. NFκB was activated and cytokine array analysis showed marked increase in pro-inflammatory cytokines and downregulation of anti-inflammatory cytokines. Therefore, elimination of excess Hcy or reduction of inflammation is a promising intervention for mitigating damage associated with HHcy in aging diseases such as DR, AMD, and AD.