Introduction: Switching from fatty acid oxidation to less efficient but more expeditious glycolysis is associated with oncometabolism and more recently cardiac hypertrophy. Hypothesis: We hypothesized that such a switch drives HFpEF in two diverse mouse models. Methods: WT/129J mice at age 4-weeks were subjected to biweekly p407 i.p. injections and a single i.v. injection of AAV9-cTnT-LDLR. A separate group of injected mice were fed a diet supplemented with alpha-ketoglutarate (2% αKG) at onset. 4-week-old Alport mice received either αKG diet or normal chow. Glucose uptake was measured by whole-body PET-CT imaging of 18F-FDG in four groups: (1) untreated (n=5), (2) LDLR/P407 at 1, 2, 4, 7 and 8-week treatment (n=3-5), (3) LDLR/P407 + αKG mice at 4-week treatment (n=5), and (4) 9-12-week Alport mice (n=5). Echocardiography was performed on all the groups (n=4-17). Mechanical and energetic measurements on skinned cardiac pupillary muscles were performed on late-stage Alport , 8-week-treated LDLR/P407 , 4-week-treated LDR/P407 + αKG mice, and untreated controls (n=4-9). Survival of LDLR/P407, LDLR/P407/αKG and control groups (n=8-13) were recorded. Results: PET-CT scans showed increased cardiac glucose uptake in Alport (FC=3, p< 0.05) and LDLR/P407 mice at 1, 2, 4 (FC = 2, p<0.05), and 8 weeks (FC=3.2, p<0.001). αKG diet increased cardiac glucose uptake in LDLR/P407 at 4 weeks compared to normal chow (FC=2.15, p<0.05). Doppler and echocardiography showed worse diastolic dysfunction (E/E’) (FC = 1.8, P<.05) in LDLR/P407 αKG mice at 4-weeks versus control chow and reduced heart size (volume) (FC = 0.41, P<0.05) and worse diastolic dysfunction in Alport mice fed αKG diet. Alport hearts favored a DRX state (FC= 1.52, p <.0001) while 8-week-LDLR/P407 hearts favored SRX (FC=1.1, p< 0.05). The SRX state was exacerbated by αKG diet in 4-week-LDLR/P407 relative to normal chow. Life spans of LDLR/P407 mice fed αKG diet were significantly shortened (p<0.001). Conclusions: The results support roles for oncometabolism in promoting HF and sudden death in Alport and LDLR/P407 mice, effects that were exacerbated by an αKG diet, and possibly caused by enhanced hypertrophic growth. The results suggest an αKGDH and phenotype-dependent redistribution of myosin energetic states
Introduction: The conserved miR17~92 cluster, is up-regulated in tumors, and its OE promotes tumor angiogenesis and growth. Recent studies indicate that miR92a is anti-angiogenic while the regulation of angiogenesis by miR17/20 is context-dependent. Hypothesis: miR92a action is also context and cell type-dependent and differentially regulates cell growth and angiogenesis in tumor vs endothelial cells. Methods: HepG2 and HUVECs were transfected with pre and anti miR92a molecules. Cell proliferation was assayed in both cell types using BrdU 48hr after transfection. HUVEC-Matrigel tube formation assays were implemented in parallel to assess angiogenic potential. VEGF was measured in spent media from HepG2 cells and the same media was used in HUVEC tube formation. Protein lysates were collected for WB 48hr after transfections of both cell types. Results: Pre-miR92a transfection inhibited proliferation of HUVECs while anti-miR92a, but not pre-miR92a inhibited proliferation of HepG2 cells (all, p<0.05). Pre-miR transfection blocked HUVEC tube formation (p<0.01). Tube formation was supported by spent medium from HepG2 cells ± transfection with pre-miR-92a but the ability of HepG2 spent medium to support tube formation was blocked by transfection with anti miR92a (all, p<0.05). Consistent with the latter result, VEGF levels in spent HepG2 media were decreased by transfection with anti-miR92a. In HepG2 cells transfection of anti-miR92a increased PTEN expression and decreased AKT-P-Thr308 (both p<0.05) PTEN and AKT-P-Thr-308 were not affected by miR92a transfection of HUVECs, however pre-miR-92a significantly decreased eNOS, ITG5α, and CCND1 protein levels (all, p<0.05). These results support our hypothesis that miR92a has opposite effect on the angiogenic properties of HepG2 vs HUVECs. Conclusions: MiR92a augments proliferation of HepG2 cells and promotes angiogenesis by inhibiting PTEN, activating Akt and increasing VEGF expression and secretion. Conversely, miR92a inhibits angiogenesis of HUVECs by targeting angiogenic regulators. The differential contextual functions of miR92a explain why this regulation supports tumor growth and angiogenesis while blocking EC function.
Neovascular age-related macular degeneration (nAMD) with choroidal neovascularization (CNV) is a leading cause of blindness in the elderly in developed countries. The disease is currently treated with anti-angiogenic biologics, including aflibercept, against vascular endothelial growth factor (VEGF) but with limited efficacy, treatment resistance and requirement for frequent intravitreal injections. Although anti-VEGF gene therapy may provide sustained therapy that obviates multiple injections, the efficacy and side effects related to VEGF pathway targeting remain, and alternative strategies to block angiogenesis independently of VEGF are needed. We recently reported that secretogranin III (Scg3) induces only pathological angiogenesis through VEGF-independent pathways, and Scg3-neutralizing antibodies selectively inhibit pathological but not physiological angiogenesis in mouse proliferative retinopathy models. Anti-Scg3 antibodies synergize dose-dependently with VEGF inhibitors in a CNV model. Here, we report that an adeno-associated virus-8 (AAV8) vector expressing anti-Scg3 Fab ameliorated CNV with an efficacy similar to that of AAV-aflibercept in a mouse model. This study is the first to test an anti-angiogenic gene therapy protocol that selectively targets pathological angiogenesis via a VEGF-independent mechanism. The findings support further safety/efficacy studies of anti-Scg3 gene therapy as monotherapy or combined with anti-VEGF to treat nAMD.
c-Jun NH(2)-terminal kinase (JNK), a member of the MAPK family of protein kinases, is a stress-response kinase that is activated by proinflammatory cytokines and growth factors coupled to membrane receptors or through nonreceptor pathways by stimuli such as heat shock, UV irradiation, protein synthesis inhibitors, and conditions that elevate the levels of reactive oxygen intermediates (ROI). Ischemia followed by reperfusion or hypoxia with reoxygenation represents a condition of high oxidative stress where JNK activation is associated with elevated ROI. We recently demonstrated that the activation of JNK by this condition is initiated by ROI generated by mitochondrial electron transport and involves sequential activation of the proline-rich kinase 2 and the small GTP-binding factors Rac-1 and Cdc42. Here we present evidence that protein kinase C (PKC) and transforming growth factor-beta-activated kinase-1 (TAK-1) are also components of this pathway. Inhibition of PKC with the broad-range inhibitor calphostin C, the PKC-alpha/beta-selective inhibitor Go9367, or adenovirus-expressing dominant-negative PKC-alpha blocked the phosphorylation of proline-rich kinase 2 and JNK. Reoxygenation activated the mitogen-activated protein kinase kinase kinase, TAK-1, and promoted the formation of a complex containing Rac-1, TAK-1, and JNK but not apoptosis-stimulating kinase-1 or p21-activated kinase-1, which was detected within the first 10 min of reoxygenation. These results identify two new components, PKC and TAK-1, that have not been previously described in this signaling pathway.
Background Diet and exercise promote cardiovascular health but their relative contributions to atherosclerosis are not fully known. The transition from a sedentary to active lifestyle requires increased caloric intake to achieve energy balance. Using atherosclerosis-prone ApoE-null mice we sought to determine whether the benefits of exercise for arterial disease are dependent on the food source of the additional calories. Methods and Results Mice were fed a high-fat diet (HF) for 4.5 months to initiate atherosclerosis after which time half were continued on HF while the other half were switched to a high protein/fish oil diet (HP). Half of each group underwent voluntary running. Food intake, running distance, body weight, lipids, inflammation markers, and atherosclerotic plaque were quantified. Two-way ANOVA tests were used to assess differences and interactions between groups. Exercised mice ran approximately 6-km per day with no difference between groups. Both groups increased food intake during exercise and there was a significant main effect of exercise F((1,44) = 9.86, p<0.01) without interaction. Diet or exercise produced significant independent effects on body weight (diet: F(1,52) = 6.85, p = 0.012; exercise: F(1,52) = 9.52, p<0.01) with no significant interaction. The combination of HP diet and exercise produced a greater decrease in total cholesterol (F(1, 46) = 7.9, p<0.01) and LDL (F(1, 46) = 7.33, p<0.01) with a large effect on the size of the interaction. HP diet and exercise independently reduced TGL and VLDL (p<0.05 and 0.001 respectively). Interleukin 6 and C-reactive protein were highest in the HF-sedentary group and were significantly reduced by exercise only in this group. Plaque accumulation in the aortic arch, a marker of cardiovascular events was reduced by the HP diet and the effect was significantly potentiated by exercise only in this group resulting in significant plaque regression (F1, 49 = 4.77, p<0.05). Conclusion In this model exercise is beneficial to combat dyslipidemia and protect from atherosclerosis only when combined with diet.
Cigarette smoking is a major risk factor for atherosclerosis and cardiovascular disease. CD36 mediates oxidized LDL (oxLDL) uptake and contributes to macrophage foam cell formation. We investigated a role for the CD36 pathway in nicotine-induced activation of macrophages and foam cell formation in vitro and in vivo. Nicotine in the same plasma concentration range found in smokers increased the CD36 + /CD14 + cell population in human peripheral blood mononuclear cells, increased CD36 expression of human THP1 macrophages, and increased macrophage production of reactive oxygen species, PKCδ phosphorylation, and peroxisome proliferator-activated receptor-γ (PPARγ) expression. Nicotine-induced CD36 expression was suppressed by antioxidants and by specific PKCδ and PPARγ inhibitors, implicating mechanistic roles for these intermediates. Nicotine synergized with oxLDL to increase macrophage expression of CD36 and cytokines TNF-α, monocyte chemoattractant protein-1, IL-6, and CXCL9, all of which were prevented by CD36 small interfering (si)RNA. Incubation with oxLDL (50 μg/ml) for 72 h resulted in lipid deposition in macrophages and foam cell formation. Preincubation with nicotine further increased oxLDL-induced lipid accumulation and foam cell formation, which was also prevented by CD36 siRNA. Treatment of apoE −/− mice with nicotine markedly exacerbated inflammatory monocyte levels and atherosclerotic plaque accumulation, effects that were not seen in CD36 −/− apoE −/− mice. Our results show that physiological levels of nicotine increase CD36 expression in macrophages, a pathway that may account at least in part for the known proinflammatory and proatherogenic properties of nicotine. These results identify such enhanced CD36 expression as a novel nicotine-mediated pathway that may constitute an independent risk factor for atherosclerosis in smokers. The results also suggest that exacerbated atherogenesis by this pathway may be an adverse side effect of extended use of high concentrations of nicotine independent of their mode of administration.
Abstract Changes in the mRNA levels during mammalian myogenesis were compared for seven polypeptides of mitochondrial respiration (the mitochondrial DNA‐encoded cytochrome oxidase subunit III, ATP synthase subunit 6, NADH dehydrogenase subunits 1 and 2, and 16S ribosomal RNA; the nuclear encoded ATP synthase β subunit and the adenine nucleotide translocase) and three polypeptides of glycolysis (glyceraldehyde‐3‐phosphate dehydrogenase, pyruvate kinase, and triose‐phosphate isomerase). Progressive changes during the conversion from myoblasts to myotubes were monitored under both atmospheric oxygen (nor‐moxic) and hypoxic environments. Northern analyses revealed coordinate, biphasic, and reciprocal expression of the respiratory and glycolytic mRNAs during myogenesis. In normoxic cells the mitochondrial respiratory enzymes were highest in myoblasts, declined 3‐ to 5‐fold during commitment and exit from the cell cycle, and increased progressively as the myotubes matured. By contrast, the glycolytic enzyme mRNAs rose 3‐ to 6‐fold on commitment and then progressively declined. When partially differentiated myotubes were switched to hypoxic conditions, the glycolytic enzyme mRNAs increased and the respiratory mRNAs declined. Hence, the developmental regulation of muscle bioenergetic metabolism appears to be regulated at the pretranslational level and is modulated by oxygen tension.
Redox-sensitive cysteine residues are present in the interaction domains of many protein complexes. There are examples in all of the major categories of transcription factors, including basic region, leucine zipper, helix-loop-helix, and zinc finger. Zinc finger structures require at least two zinc-coordinated cysteine sulfhydryl groups, and oxidation or alkylation of these can eliminate DNA-binding and transcriptional functions. We review here the evidence for oxidation of zinc finger cysteines, the pathways and reactive oxygen intermediates involved, and the functional and physiological consequences of these reactions. Despite skepticism that the strongly reducing intracellular environment would permit significant oxidation of cysteine residues within zinc finger transcription factors, there is compelling evidence that oxidation occurs both in vitro and in vivo. Early reports demonstrating reversible oxidation of zinc-coordinated cysteines with loss of binding function in vitro were shown to reflect accurately the changes in intact cells, and these in turn have been shown to correlate with physiological changes. In particular, the accumulation of oxidized Sp1 zinc fingers during aging, and estrogen receptors in tamoxifen-resistant breast cancers are dramatic examples of what may be a general sensitivity of zinc finger factors to changes in the redox state of the cell.
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