Glycolytic PFKFB3 and Glycogenic UGP2 Axis Regulates Perfusion Recovery in Experimental Hind Limb Ischemia.

BACKGROUND: Despite being in an oxygen-rich environment, endothelial cells (ECs) use anaerobic glycolysis (Warburg effect) as the primary metabolic pathway for cellular energy needs. PFKFB (6-phosphofructo-2-kinase/fructose-2,6-biphosphatase)-3 regulates a critical enzymatic checkpoint in glycolysis and has been shown to induce angiogenesis. This study builds on our efforts to determine the metabolic regulation of ischemic angiogenesis and perfusion recovery in the ischemic muscle. METHODS: Hypoxia serum starvation (HSS) was used as an in vitro peripheral artery disease (PAD) model, and hind limb ischemia by femoral artery ligation and resection was used as a preclinical PAD model. RESULTS: Despite increasing PFKFB3-dependent glycolysis, HSS significantly decreased the angiogenic capacity of ischemic ECs. Interestingly, inhibiting PFKFB3 significantly induced the angiogenic capacity of HSS-ECs. Since ischemia induced a significant in PFKFB3 levels in hind limb ischemia muscle versus nonischemic, we wanted to determine whether glucose bioavailability (rather than PFKFB3 expression) in the ischemic muscle is a limiting factor behind impaired angiogenesis. However, treating the ischemic muscle with intramuscular delivery of D-glucose or L-glucose (osmolar control) showed no significant differences in the perfusion recovery, indicating that glucose bioavailability is not a limiting factor to induce ischemic angiogenesis in experimental PAD. Unexpectedly, we found that shRNA-mediated PFKFB3 inhibition in the ischemic muscle resulted in an increased perfusion recovery and higher vascular density compared with control shRNA (consistent with the increased angiogenic capacity of PFKFB3 silenced HSS-ECs). Based on these data, we hypothesized that inhibiting HSS-induced PFKFB3 expression/levels in ischemic ECs activates alternative metabolic pathways that revascularize the ischemic muscle in experimental PAD. A comprehensive glucose metabolic gene qPCR arrays in PFKFB3 silenced HSS-ECs, and PFKFB3-knock-down ischemic muscle versus respective controls identified UGP2 (uridine diphosphate-glucose pyrophosphorylase 2), a regulator of protein glycosylation and glycogen synthesis, is induced upon PFKFB3 inhibition in vitro and in vivo. Antibody-mediated inhibition of UGP2 in the ischemic muscle significantly impaired perfusion recovery versus IgG control. Mechanistically, supplementing uridine diphosphate-glucose, a metabolite of UGP2 activity, significantly induced HSS-EC angiogenic capacity in vitro and enhanced perfusion recovery in vivo by increasing protein glycosylation (but not glycogen synthesis). CONCLUSIONS: Our data present that inhibition of maladaptive PFKFB3-driven glycolysis in HSS-ECs is necessary to promote the UGP2-uridine diphosphate-glucose axis that enhances ischemic angiogenesis and perfusion recovery in experimental PAD.

Fecal total iron concentration is inversely associated with fecal Lactobacillus in preschool children.

BACKGROUND AND AIM: Iron deficiency is associated with stunting and poor performance in children. Oral iron supplementation is widely promoted to correct iron deficiency. However, excess iron may be toxic to beneficial luminal gut bacteria and could support growth of pathobionts. The aim of this study is to analyze the fecal total iron concentration and fecal Lactobacillus levels in a cohort of stunted and normal children. METHODS: The study was undertaken in two different locations. One of them is a rural area, and the other is a semi-urban-slum area; both areas are located in the Vellore district of Tamilnadu state. Twenty children (10 stunted and 10 normal growth) aged 2 to 5 years from each area were recruited. Both groups were nearly identical demographically. Fecal samples were collected. Fecal total iron was estimated, and fecal DNA was extracted and subjected to 16S rDNA-targeted real-time PCR to determine the relative predominance of Lactobacillus and Escherichia coli. RESULTS: The fecal total iron concentration in rural children (3656 µg/g wet wt. of feces) was significantly higher when compared with semi-urban-slum children (114.9 µg/g wet wt. of feces, P < 0.005). Inversely, fecal Lactobacillus in rural children (median 3.18 × 10-3 relative difference compared with total bacteria) was significantly lower when compared with semi-urban-slum children (median 59.33 × 10-3 , p < 0.005). There was no significant change observed between normal and stunted children. E. coli levels remained unaffected. CONCLUSION: The present study documents an inverse relationship between fecal iron concentration and fecal Lactobacillus concentration in children belonging to two different localities independent of their nutritional status.

The role of HDAC3 in inflammation: mechanisms and therapeutic implications.

Histone deacetylases (HDACs) are critical regulators of inflammatory gene expression, and the efficacy of pan-HDAC inhibitors has been implicated in various disease conditions. However, it remains largely unclear how HDACs precisely regulate inflammation. To this end, evaluating the isoform-specific function of HDACs is critical, and the isoform-specific targeting could also circumvent the off-target effects of pan-HDAC inhibitors. This review provides an overview of the roles of HDAC3, a class I HDAC isoform, in modulating inflammatory responses and discusses the molecular mechanisms by which HDAC3 regulates inflammation associated with brain pathology, arthritis, cardiovascular diseases, lung pathology, allergic conditions, and kidney disorders. The articles also identify knowledge gaps in the field for future studies. Despite some conflicting reports, the selective inhibition of HDAC3 has been demonstrated to play a beneficial role in various inflammatory pathologies. Exploring the potential of HDAC3 inhibition to improve disease prognosis is a promising avenue requiring further investigation.

Inhibiting anti-angiogenic VEGF165b activates a miR-17-20a-Calcipressin-3 pathway that revascularizes ischemic muscle in peripheral artery disease.

BACKGROUND: VEGF165a increases the expression of the microRNA-17-92 cluster, promoting developmental, retinal, and tumor angiogenesis. We have previously shown that VEGF165b, an alternatively spliced anti-angiogenic VEGF-A isoform, inhibits the VEGFR-STAT3 pathway in ischemic endothelial cells (ECs) to decrease their angiogenic capacity. In ischemic macrophages (Møs), VEGF165b inhibits VEGFR1 to induce S100A8/A9 expression, which drives M1-like polarization. Our current study aims to determine whether VEGF165b inhibition promotes perfusion recovery by regulating the microRNA(miR)-17-92 cluster in preclinical PAD. METHODS: Femoral artery ligation and resection was used as a preclinical PAD model. Hypoxia serum starvation (HSS) was used as an in vitro PAD model. VEGF165b was inhibited/neutralized by an isoform-specific VEGF165b antibody. RESULTS: Here, we show that VEGF165b-inhibition induces the expression of miR-17-20a (within miR-17-92 (miR-17-18a-19a-19b-20a-92) cluster) in HSS-ECs and HSS-Møs vs. respective normal and/or isotype-matched IgG controls to enhance perfusion recovery. Consistent with the bioinformatics analysis that revealed RCAN3 as a common target of miR-17 and miR-20a, Argonaute-2 pull-down assays showed decreased miR-17-20a expression and higher RCAN3 expression in the RNA-induced silencing complex of HSS-ECs and HSS-Møs vs. respective controls. Inhibiting miR-17-20a induced RCAN3 levels to decrease ischemic angiogenesis and promoted M1-like polarization to impair perfusion recovery. Finally, using STAT3 inhibitors, S100A8/A9 silencers, and VEGFR1-deficient ECs and Møs, we show that VEGF165b-inhibition activates the miR-17-20a-RCAN3 pathway independent of VEGFR1-STAT3 or VEGFR1-S100A8/A9 in ischemic-ECs and ischemic-Møs respectively. CONCLUSIONS: Our data revealed a hereunto unrecognized therapeutic 'miR-17-20a-RCAN3' pathway in the ischemic vasculature that is VEGFR1-STAT3/S100A8/A9 independent and is activated only upon VEGF165b-inhibition in PAD.

Therapies that can grow new blood vessels in the ischemic muscle are necessary to restore blood flow and provide relief to patients with peripheral artery disease (PAD). We have previously shown that blocking VEGF₁₆₅b, a small protein involved in the regulation of regenerating blood vessels, promotes the growth of new blood vessels in the ischemic muscle. However, the mechanism by which this occurs is not clear. Here, we build on this existing knowledge and show the complex processes driving the growth of new blood vessels, which will help to supply blood to the ischemic muscle and provide therapeutic relief from PAD.

Targeting Anti-Angiogenic VEGF165b-VEGFR1 Signaling Promotes Nitric Oxide Independent Therapeutic Angiogenesis in Preclinical Peripheral Artery Disease Models.

Nitric oxide (NO) is the critical regulator of VEGFR2-induced angiogenesis. Neither VEGF-A over-expression nor L-Arginine (NO-precursor) supplementation has been effective in helping patients with Peripheral Artery Disease (PAD) in clinical trials. One incompletely studied reason may be due to the presence of the less characterized anti-angiogenic VEGF-A (VEGF165b) isoform. We have recently shown that VEGF165b inhibits ischemic angiogenesis by blocking VEGFR1, not VEGFR2 activation. Here we wanted to determine whether VEGF165b inhibition using a monoclonal isoform-specific antibody against VEGF165b vs. control, improved perfusion recovery in preclinical PAD models that have impaired VEGFR2-NO signaling, including (1) type-2 diabetic model, (2) endothelial Nitric oxide synthase-knock out mice, and (3) Myoglobin transgenic mice that have impaired NO bioavailability. In all PAD models, VEGF165b inhibition vs. control enhanced perfusion recovery, increased microvascular density in the ischemic limb, and activated VEGFR1-STAT3 signaling. In vitro, VEGF165b inhibition vs. control enhanced a VEGFR1-dependent endothelial survival/proliferation and angiogenic capacity. These data demonstrate that VEGF165b inhibition induces VEGFR1-STAT3 activation, which does not require increased NO to induce therapeutic angiogenesis in PAD. These results may have implications for advancing therapies for patients with PAD where the VEGFR2-eNOS-NO pathway is impaired.