Project description:AimsInflammation and possibly hypoxia largely affect glucose utilization in atherosclerotic arteries, which could alter many metabolic systems. However, metabolic changes in atherosclerotic plaques remain unknown. The present study aims to identify changes in metabolic systems relative to glucose uptake and hypoxia in rabbit atherosclerotic arteries and cultured macrophages.MethodsMacrophage-rich or smooth muscle cell (SMC)-rich neointima was created by balloon injury in the iliac-femoral arteries of rabbits fed with a 0.5% cholesterol diet or a conventional diet. THP-1 macrophages stimulated with lipopolysaccharides (LPS) and interferon-γ (INFγ) were cultured under normoxic and hypoxic conditions. We evaluated comprehensive arterial and macrophage metabolism by performing metabolomic analyses using capillary electrophoresis-time of flight mass spectrometry. We evaluated glucose uptake and its relationship to vascular hypoxia using (18)F-fluorodeoxyglucose ((18)F-FDG) and pimonidazole, a marker of hypoxia.ResultsThe levels of many metabolites increased in the iliac-femoral arteries with macrophage-rich neointima, compared with those that were not injured and those with SMC-rich neointima (glycolysis, 4 of 9; pentose phosphate pathway, 4 of 6; tricarboxylic acid cycle, 4 of 6; nucleotides, 10 of 20). The uptake of (18)F-FDG in arterial walls measured by autoradiography positively correlated with macrophage- and pimonidazole-immunopositive areas (r = 0.76, and r = 0.59 respectively; n = 69 for both; p<0.0001). Pimonidazole immunoreactivity was closely localized with the nuclear translocation of hypoxia inducible factor-1α and hexokinase II expression in macrophage-rich neointima. The levels of glycolytic (8 of 8) and pentose phosphate pathway (4 of 6) metabolites increased in LPS and INFγ stimulated macrophages under hypoxic but not normoxic condition. Plasminogen activator inhibitor-1 protein levels in the supernatant were closely associated with metabolic pathways in the macrophages.ConclusionInfiltrative macrophages in atherosclerotic arteries might affect metabolic systems, and hypoxia but not classical activation might augment glycolytic and pentose phosphate pathways in macrophages.

Project description:The molecular mechanisms whereby placental growth factor (PlGF) mediates its effects in nonproliferative diabetic retinopathy (DR) are unknown. To better understand the role of PlGF in DR, we used tandem mass tags (TMT)-labeled quantitative proteomics to human retinal endothelial cells (HRECs), treated anti-PlGF antibody, and PBS as a control. Functional annotation and pathway enrichments were performed, which suggested that the differentially expressed proteins (DEPs) were involved in key metabolic processes, protein binding, and membrane, pentose phosphate pathway (PPP) and adherens junction. We conducted integrated gene profiles of our previously published transcriptomic data to the TMT-labeled proteomics data. The results showed the sixty genes were found to be changed at the proteome level. The functional annotation conducted for the sixty proteins suggested that 58.3% of proteins were involved in PPP, 25% of proteins were in interleukin-12 singling and 16.7% of proteins were involved in glycolysis and gluconeogenesis pathway. Mass spectrometry results were validated by transendothelial electrical resistance measurement by an electrical cell-impedance sensing (ECIS) and western blot analysis of VE-cadherin, G6PD. These findings suggest that the PPP proteins and antioxidants may act as a downstream target of PlGF and may play a decisive role in HREC biological functions in DR. SIGNIFICANCE: PlGF (Placental growth factor) is known to play a pivotal role in pathological angiogenesis and inflammation by stimulating endothelial cell migration and by recruiting pericytes and inflammatory cells such as microglia and macrophages. Despite the well-defined pathophysiological roles of PlGF, the underlying molecular and cellular mechanisms are not completely understood, especially the exact relationships between biochemical events and molecular pathways regulated by PlGF, whose inhibition exhibits a protective role in DR. This study provides new insights into protein expression patterns and enables the identification of many attractive candidates for investigation of PPP pathway role in the activation of the antioxidant defense system in DR. Our findings suggest that the PPP proteins and antioxidants (PRDX6, HMOX1, NQO1 and YES1) may act as downstream targets of PlGF and may play a decisive role in HREC biological functions in DR.

Project description:Oxygen fluctuation during tissue remodeling imposes a major metabolic challenge in human tumors. Stem-like tumor cells in glioblastomas are believed to possess extraordinary metabolic flexibility, enabling them to initiate growth even under non-permissive conditions. To identify adaptive response mechanism, we compared the effects of acute and chronic hypoxia versus oxygenation on stem-like glioblastoma (GS) cells. GS cell lines were established and propagated either under normoxia (21% O2) or hypoxia (1% O2) and subsequently exposed to acute normoxia or hypoxia, respectively. Gene expression profiling revealed that acute hypoxia predominantly induced metabolic pathways and cell cycle arrest, whereas chronic hypoxia activated neurodevelopmental processes. In particular, we found increased expression of glycolytic enzymes, especially of the preparatory phase of glycolysis, under acute hypoxia, whereas pentose phosphate pathway (PPP) enzymes were downregulated. The opposite was found for acute oxygenation of hypoxic GS cells. Findings were confirmed by qPCR and immunoblot analyses. Despite downregulation by hypoxia, expression of PPP enzymes is increased in GBMs compared to normal brain, whereas expression of hypoxia-inducible enzymes of the parallel preparatory phase of glycolysis is decreased. Immunohistochemistry revealed strong staining for PPP enzymes in the bulk of GBM tissue, especially in highly proliferative areas, but not in pseudopalisading (hypoxic) cells. Glycolytic enzymes displayed an inverse pattern. Mass spectrometric analysis using [1,2-13C2]-D-glucose showed reduced glucose flux through the PPP under hypoxia in favor of flux through glycolysis. Acute and chronic hypoxia increased cell migration but reduced proliferation, whereas normoxia had opposite effects. Our findings extend Warburg’s observations by showing that in most tumor cells the PPP, which supplies metabolites for biomass production, is favored over the parallel preparatory phase of glycolysis, but is suppressed under acute severe hypoxia, causing a switch to direct glycolysis to protect against hypoxic stress.

Dataset Information

Metabolic effect of TAp63?: enhanced glycolysis and pentose phosphate pathway, resulting in increased antioxidant defense.

Publications

Metabolic effect of TAp63α: enhanced glycolysis and pentose phosphate pathway, resulting in increased antioxidant defense.

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