Rice bran enzymatic extract restores endothelial function and vascular contractility in obese rats by reducing vascular inflammation and oxidative stress

BackgroundRice bran enzymatic extract (RBEE) used in this study has shown beneficial activities against dyslipidemia, hyperinsulinemia and hypertension. Our aim was to investigate the effects of a diet supplemented with RBEE in vascular impairment developed in obese Zucker rats and to evaluate the main mechanisms mediating this action.Methods and resultsObese Zucker rats were fed a 1% and 5% RBEE-supplemented diet (O1% and O5%). Obese and their lean littermates fed a standard diet were used as controls (OC and LC, respectively). Vascular function was evaluated in aortic rings in organ baths. The role of nitric oxide (NO) was investigated by using NO synthase (NOS) inhibitors. Aortic expression of endothelial NOS (eNOS), inducible NOS (iNOS), tumor necrosis factor (TNF)-α and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits and superoxide production in arterial wall were determined. Endothelial dysfunction and vascular hyperreactivity to phenylephrine in obese rats were ameliorated by RBEE treatment, particularly with 1% RBEE. Up-regulation of eNOS protein expression in RBEE-treated aortas should contribute to this activity. RBEE attenuated vascular inflammation by reducing aortic iNOS and TNF-α expression. Aortas from RBEE-treated groups showed a significant decrease of superoxide production and down-regulation of NADPH oxidase subunits.ConclusionRBEE treatment restored endothelial function and vascular contractility in obese Zucker rats through a reduction of vascular inflammation and oxidative stress. These results show the nutraceutical potential of RBEE to prevent obesity-related vascular complications.     

Mitochondrial DNA Damage and Dysfunction,and Oxidative Stress Are Associated with Endoplasmic Reticulum Stress,Protein Degradation and Apoptosis in High Fat Diet-Induced Insulin Resistance Mice

Background

Recent studies showed a link between a high fat diet (HFD)-induced obesity and lipid accumulation in non-adipose tissues, such as skeletal muscle and liver, and insulin resistance (IR). Although the mechanisms responsible for IR in those tissues are different, oxidative stress and mitochondrial dysfunction have been implicated in the disease process. We tested the hypothesis that HFD induced mitochondrial DNA (mtDNA) damage and that this damage is associated with mitochondrial dysfunction, oxidative stress, and induction of markers of endoplasmic reticulum (ER) stress, protein degradation and apoptosis in skeletal muscle and liver in a mouse model of obesity-induced IR.

Methodology/Principal Findings

C57BL/6J male mice were fed either a HFD (60% fat) or normal chow (NC) (10% fat) for 16 weeks. We found that HFD-induced IR correlated with increased mtDNA damage, mitochondrial dysfunction and markers of oxidative stress in skeletal muscle and liver. Also, a HFD causes a change in the expression level of DNA repair enzymes in both nuclei and mitochondria in skeletal muscle and liver. Furthermore, a HFD leads to activation of ER stress, protein degradation and apoptosis in skeletal muscle and liver, and significantly reduced the content of two major proteins involved in insulin signaling, Akt and IRS-1 in skeletal muscle, and Akt in liver. Basal p-Akt level was not significantly influenced by HFD feeding in skeletal muscle and liver.

Conclusions/Significance

This study provides new evidence that HFD-induced mtDNA damage correlates with mitochondrial dysfunction and increased oxidative stress in skeletal muscle and liver, which is associated with the induction of markers of ER stress, protein degradation and apoptosis.     

Differential effects of short- and long-term high-fat diet feeding on hepatic fatty acid metabolism in rats

Imbalance in the supply and utilization of fatty acids (FA) is thought to contribute to intrahepatic lipid (IHL) accumulation in obesity. The aim of this study was to determine the time course of changes in the liver capacity to oxidize and store FA in response to high-fat diet (HFD). Adult male Wistar rats were fed either normal chow or HFD for 2.5weeks (short-term) and 25weeks (long-term). Short-term HFD feeding led to a 10% higher palmitoyl-l-carnitine-driven ADP-stimulated (state 3) oxygen consumption rate in isolated liver mitochondria indicating up-regulation of β-oxidation. This adaptation was insufficient to cope with the dietary FA overload, as indicated by accumulation of long-chain acylcarnitines, depletion of free carnitine and increase in FA content in the liver, reflecting IHL accumulation. The latter was confirmed by in vivo((1))H magnetic resonance spectroscopy and Oil Red O staining. Long-term HFD feeding caused further up-regulation of mitochondrial β-oxidation (24% higher oxygen consumption rate in state 3 with palmitoyl-l-carnitine as substrate) and stimulation of mitochondrial biogenesis as indicated by 62% higher mitochondrial DNA copy number compared to controls. These adaptations were paralleled by a partial restoration of free carnitine levels and a decrease in long-chain acylcarnitine content. Nevertheless, there was a further increase in IHL content, accompanied by accumulation of lipid peroxidation and protein oxidation products. In conclusion, partially effective adaption of hepatic FA metabolism to long-term HFD feeding came at a price of increased oxidative stress, caused by a combination of higher FA oxidation capacity and oversupply of FA.     

Effect of Vitamin E and Omega-3 Fatty Acids on Protecting Ambient PM2.5-Induced Inflammatory Response and Oxidative Stress in Vascular Endothelial Cells

Although the mechanisms linking cardiopulmonary diseases to ambient fine particles (PM_2.5) are still unclear, inflammation and oxidative stress play important roles in PM_2.5-induced injury. It is well known that inflammation and oxidative stress could be restricted by vitamin E (Ve) or omega-3 fatty acids (Ω-3 FA) consumption. This study investigated the effects of Ve and Ω-3 FA on PM_2.5-induced inflammation and oxidative stress in vascular endothelial cells. The underlying mechanisms linking PM_2.5 to vascular endothelial injury were also explored. Human umbilical vein endothelial cells (HUVECs) were treated with 50 μg/mL PM_2.5 in the presence or absence of different concentrations of Ve and Ω-3 FA. The inflammatory cytokines and oxidative stress markers were determined. The results showed that Ve induced a significant decrease in PM_2.5-induced inflammation and oxidative stress. Malondialdehyde (MDA) in supernatant and reactive oxygen species (ROS) in cytoplasm decreased by Ve, while the superoxide dismutase (SOD) activity elevated. The inflammatory cytokines interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) also reduced by Ve. Moreover, Ω-3 FA played the same role on decreasing the inflammation and oxidative stress. IL-6 and TNF-α expressions were significantly lower in combined Ve with Ω-3 FA than treatment with Ve or Ω-3 FA alone. The Ve and Ω-3 FA intervention might abolish the PM_2.5-induced oxidative stress and inflammation in vascular endothelial cells. There might be an additive effect of these two nutrients in mediating the PM_2.5-induced injury in vascular endothelial cells. The results suggested that inflammation and oxidative stress might be parts of the mechanisms linking PM_2.5 to vascular endothelial injury.     

Potential role of dietary flavonoids in reducing microvascular endothelium vulnerability to oxidative and inflammatory insults ( small star,filled)

Although antioxidant systems help control the level of reactive oxygen species they may be overwhelmed during periods of oxidative stress. Evidence suggests that oxidative stress components as well as inflammatory mediators may be involved in the pathogenesis of vascular disorders, where localized markers of oxidative damage have been found. In this regard we investigated the putative antioxidant and anti-inflammatory effects of blueberry and cranberry anthocyanins and hydroxycinnamic acids against H(2)O(2) and TNFalpha induced damage to human microvascular endothelial cells. Polyphenols from both berries were able to localize into endothelial cells subsequently reducing endothelial cells vulnerability to increased oxidative stress at both the membrane and cytosol level. Furthermore, berry polyphenols also reduced TNFalpha induced up-regulation of various inflammatory mediators (IL-8, MCP-1 and ICAM-1) involved in the recruitment of leukocytes to sites of damage or inflammation along the endothelium. In conclusion, polyphenols isolated from both blueberry and cranberry were able to afford protection to endothelial cells against stressor induced up-regulation of oxidative and inflammatory insults. This may have beneficial actions against the initiation and development of vascular diseases and be a contributing factor in the reduction of age-related deficits in neurological impairments previously reported by us.     

Vascular and Hepatic Impact of Short-Term Intermittent Hypoxia in a Mouse Model of Metabolic Syndrome

BackgroundExperimental models of intermittent hypoxia (IH) have been developed during the last decade to investigate the consequences of obstructive sleep apnea. IH is usually associated with detrimental metabolic and vascular outcomes. However, paradoxical protective effects have also been described depending of IH patterns and durations applied in studies. We evaluated the impact of short-term IH on vascular and metabolic function in a diet-induced model of metabolic syndrome (MS).MethodsMice were fed either a standard diet or a high fat diet (HFD) for 8 weeks. During the final 14 days of each diet, animals were exposed to either IH (1 min cycle, FiO₂ 5% for 30s, FiO₂ 21% for 30s; 8 h/day) or intermittent air (FiO₂ 21%). Ex-vivo vascular reactivity in response to acetylcholine was assessed in aorta rings by myography. Glucose, insulin and leptin levels were assessed, as well as serum lipid profile, hepatic mitochondrial activity and tissue nitric oxide (NO) release.ResultsMice fed with HFD developed moderate markers of dysmetabolism mimicking MS, including increased epididymal fat, dyslipidemia, hepatic steatosis and endothelial dysfunction. HFD decreased mitochondrial complex I, II and IV activities and increased lactate dehydrogenase (LDH) activity in liver. IH applied to HFD mice induced a major increase in insulin and leptin levels and prevented endothelial dysfunction by restoring NO production. IH also restored mitochondrial complex I and IV activities, moderated the increase in LDH activity and liver triglyceride accumulation in HFD mice.ConclusionIn a mouse model of MS, short-term IH increases insulin and leptin levels, restores endothelial function and mitochondrial activity and limits liver lipid accumulation.     

Defects in mitochondrial DNA replication and oxidative damage in muscle of mtDNA mutator mice

A causal role for mitochondrial dysfunction in mammalian aging is supported by recent studies of the mtDNA mutator mouse (“PolG” mouse), which harbors a defect in the proofreading-exonuclease activity of mitochondrial DNA polymerase gamma. These mice exhibit accelerated aging phenotypes characteristic of human aging, including systemic mitochondrial dysfunction, exercise intolerance, alopecia and graying of hair, curvature of the spine, and premature mortality. While mitochondrial dysfunction has been shown to cause increased oxidative stress in many systems, several groups have suggested that PolG mutator mice show no markers of oxidative damage. These mice have been presented as proof that mitochondrial dysfunction is sufficient to accelerate aging without oxidative stress. In this study, by normalizing to mitochondrial content in enriched fractions we detected increased oxidative modification of protein and DNA in PolG skeletal muscle mitochondria. We separately developed novel methods that allow simultaneous direct measurement of mtDNA replication defects and oxidative damage. Using this approach, we find evidence that suggests PolG muscle mtDNA is indeed oxidatively damaged. We also observed a significant decrease in antioxidants and expression of mitochondrial biogenesis pathway components and DNA repair enzymes in these mice, indicating an association of maladaptive gene expression with the phenotypes observed in PolG mice. Together, these findings demonstrate the presence of oxidative damage associated with the premature aging-like phenotypes induced by mitochondrial dysfunction.     

Cytotoxicity of Malondialdehyde and Cytoprotective Effects of Taurine <Emphasis Type="Italic">via</Emphasis> Oxidative Stress and PGC-1α Signal Pathway in C2C12 Cells

One of the end-products of ROS-induced peroxidation, malondialdehyde (MDA), induces the cross-links in proteins, which leads to perturbation of the physiological functions of cells and contributes to abnormal biological regulation and various disorders. Taurine (2-aminoethanesulfonic acid, Tau) aids in adjusting normal physiological functions to confer stress resistance. The protective effects of Tau against MDA stress in vitro or in vivo were reported previously. In this study, we had investigated the protective effects of taurine on viability, oxidative stress levels and mitochondrial biogenesis in mouse muscle C2C12 cells undergoing MDA induced stress. We show that the treatment with 100 μM MDA leads to increase in cell oxidative stress levels, inhibition of mitochondrial biogenesis and the reduction of the cell survival rates. The pretreatment with 0.1 μM taurine reduced MDA-induced death rate via inhibition of oxidative stress, restoration of mitochondrial functions of the mitochondrial membrane potential (MMP) and ATP production. In MDA stress, the pre-treatment with 0.1 μM taurine leads to upregulation of the factors of mitochondrial biogenesis. These observations suggest that the cytoprotective effects of taurine may be due to an induction of mitochondrial biogenesis.     

Antibiotic bedaquiline effectively targets growth,survival and tumor angiogenesis of lung cancer through suppressing energy metabolism

Tumor angiogenesis plays essential roles during lung cancer progression and metastasis. Therapeutic agent that targets both tumor cell and vascular endothelial cell may achieve additional anti-tumor efficacy. We demonstrate that bedaquiline, a FDA-approved antibiotic drug, effectively targets lung cancer cells and angiogenesis. Bedaquiline dose-dependently inhibits proliferation and induces apoptosis of a panel of lung cancer cell lines regardless of subtypes and molecular heterogeneity. Bedaquiline also inhibits capillary network formation of human lung tumor associated-endothelial cell (HLT-EC) on Matrigel and its multiple functions, such as spreading, proliferation and apoptosis, even in the presence of vascular endothelial growth factor (VEGF). We further demonstrate that bedaquiline acts on lung cancer cells and HLT-EC via inhibiting mitochondrial respiration and glycolysis, leading to ATP reduction and oxidative stress. Consistently, oxidative damage on DNA, protein and lipid were detected in cells exposed to bedaquiline. Importantly, the results obtained in in vitro cell culture are reproducible in in vivo xenograft lung cancer mouse model, confirming that bedaquiline suppresses lug tumor growth and angiogenesis, and increases oxidative stress. Our findings demonstrating that energy depletion is effectively against lung tumor cells and angiogenesis. Our work also provide pre-clinical evidence to repurpose antibiotic bedaquiline for lung cancer treatment.     

Cannabidiol Protects against Doxorubicin-Induced Cardiomyopathy by Modulating Mitochondrial Function and Biogenesis

Doxorubicin (DOX) is a widely used, potent chemotherapeutic agent; however, its clinical application is limited because of its dose-dependent cardiotoxicity. DOX’s cardiotoxicity involves increased oxidative/nitrative stress, impaired mitochondrial function in cardiomyocytes/endothelial cells and cell death. Cannabidiol (CBD) is a nonpsychotropic constituent of marijuana, which is well tolerated in humans, with antioxidant, antiinflammatory and recently discovered antitumor properties. We aimed to explore the effects of CBD in a well-established mouse model of DOX-induced cardiomyopathy. DOX-induced cardiomyopathy was characterized by increased myocardial injury (elevated serum creatine kinase and lactate dehydrogenase levels), myocardial oxidative and nitrative stress (decreased total glutathione content and glutathione peroxidase 1 activity, increased lipid peroxidation, 3-nitrotyrosine formation and expression of inducible nitric oxide synthase mRNA), myocardial cell death (apoptotic and poly[ADP]-ribose polymerase 1 [PARP]-dependent) and cardiac dysfunction (decline in ejection fraction and left ventricular fractional shortening). DOX also impaired myocardial mitochondrial biogenesis (decreased mitochondrial copy number, mRNA expression of peroxisome proliferator-activated receptor γ coactivator 1-alpha, peroxisome proliferator-activated receptor alpha, estrogen-related receptor alpha), reduced mitochondrial function (attenuated complex I and II activities) and decreased myocardial expression of uncoupling protein 2 and 3 and medium-chain acyl-CoA dehydrogenase mRNA. Treatment with CBD markedly improved DOX-induced cardiac dysfunction, oxidative/nitrative stress and cell death. CBD also enhanced the DOX-induced impaired cardiac mitochondrial function and biogenesis. These data suggest that CBD may represent a novel cardioprotective strategy against DOX-induced cardiotoxicity, and the above-described effects on mitochondrial function and biogenesis may contribute to its beneficial properties described in numerous other models of tissue injury.     

Improvement of mitochondrial energy and oxidative balance during intestinal differentiation

Mitochondria vary in their number and function, but how these variations are associated with intestinal cell differentiation remains elusive. The object of this study was to investigate the underlying mechanisms of inosine-mediated intestinal cell maturation, analysing the effects of this nutrient on metabolic functionality, mitochondrial biogenesis and mitochondrial function in human colonic cells. The role of oxidative stress in the control of intestinal cell growth was also explored. We report the novel finding that inosine-mediated differentiation improves aerobic metabolism through an increase in mitochondrial bioenergetics and biogenesis in colonic cells, which probably confers them greater resistance to cytotoxic oxidative stress.     

Exercise-Mediated Wall Shear Stress Increases Mitochondrial Biogenesis in Vascular Endothelium

Objective

Enhancing structural and functional integrity of mitochondria is an emerging therapeutic option against endothelial dysfunction. In this study, we sought to investigate the effect of fluid shear stress on mitochondrial biogenesis and mitochondrial respiratory function in endothelial cells (ECs) using in vitro and in vivo complementary studies.

Methods and Results

Human aortic- or umbilical vein-derived ECs were exposed to laminar shear stress (20 dyne/cm²) for various durations using a cone-and-plate shear apparatus. We observed significant increases in the expression of key genes related to mitochondrial biogenesis and mitochondrial quality control as well as mtDNA content and mitochondrial mass under the shear stress conditions. Mitochondrial respiratory function was enhanced when cells were intermittently exposed to laminar shear stress for 72 hrs. Also, shear-exposed cells showed diminished glycolysis and decreased mitochondrial membrane potential (ΔΨm). Likewise, in in vivo experiments, mice that were subjected to a voluntary wheel running exercise for 5 weeks showed significantly higher mitochondrial content determined by en face staining in the conduit (greater and lesser curvature of the aortic arch and thoracic aorta) and muscle feed (femoral artery) arteries compared to the sedentary control mice. Interestingly, however, the mitochondrial biogenesis was not observed in the mesenteric artery. This region-specific adaptation is likely due to the differential blood flow redistribution during exercise in the different vessel beds.

Conclusion

Taken together, our findings suggest that exercise enhances mitochondrial biogenesis in vascular endothelium through a shear stress-dependent mechanism. Our findings may suggest a novel mitochondrial pathway by which a chronic exercise may be beneficial for vascular function.     

PINK1 is necessary for long term survival and mitochondrial function in human dopaminergic neurons

Parkinson's disease (PD) is a common age-related neurodegenerative disease and it is critical to develop models which recapitulate the pathogenic process including the effect of the ageing process. Although the pathogenesis of sporadic PD is unknown, the identification of the mendelian genetic factor PINK1 has provided new mechanistic insights. In order to investigate the role of PINK1 in Parkinson's disease, we studied PINK1 loss of function in human and primary mouse neurons. Using RNAi, we created stable PINK1 knockdown in human dopaminergic neurons differentiated from foetal ventral mesencephalon stem cells, as well as in an immortalised human neuroblastoma cell line. We sought to validate our findings in primary neurons derived from a transgenic PINK1 knockout mouse. For the first time we demonstrate an age dependent neurodegenerative phenotype in human and mouse neurons. PINK1 deficiency leads to reduced long-term viability in human neurons, which die via the mitochondrial apoptosis pathway. Human neurons lacking PINK1 demonstrate features of marked oxidative stress with widespread mitochondrial dysfunction and abnormal mitochondrial morphology. We report that PINK1 plays a neuroprotective role in the mitochondria of mammalian neurons, especially against stress such as staurosporine. In addition we provide evidence that cellular compensatory mechanisms such as mitochondrial biogenesis and upregulation of lysosomal degradation pathways occur in PINK1 deficiency. The phenotypic effects of PINK1 loss-of-function described here in mammalian neurons provides mechanistic insight into the age-related degeneration of nigral dopaminergic neurons seen in PD.     

AMPK activation prevents prenatal stress-induced cognitive impairment: Modulation of mitochondrial content and oxidative stress

Prenatal stress induces cognitive functional impairment in offspring, an eventuality in which mitochondrial dysfunction and oxidative stress are believed to be closely involved. In this study, the involvement of the AMP-activated protein kinase (AMPK) pathway was investigated. A well-known activator, resveratrol (Res), was used to induce AMPK activation in SH-SY-5Y cells. Significant mitochondrial biogenesis and phase II enzyme activation, accompanied by decreased protein oxidation and GSSG content, were observed after Res treatment, and inhibition of AMPK with Compound c abolished the induction effects of Res. Further study utilizing a prenatal restraint stress (PRS) animal model indicated that maternal supplementation of Res may activate AMPK in the hippocampi of both male and female offspring, and that PRS-induced mitochondrial loss in the offspring hippocampus was inhibited by Res maternal supplementation. In addition, Res activated Nrf2-mediated phase II enzymes and reduced PRS-induced oxidative damage in both male and female offspring. Moreover, PRS markedly decreased mRNA levels of various neuron markers, as well as resultant offspring cognitive function, based on spontaneous alternation performance and Morris water maze tests, the results of which were significantly improved by maternal Res supplementation. Our results provide evidence indicating that AMPK may modulate mitochondrial content and phase II enzymes in neuronal cells, a process which may play an essential role in preventing PRS-induced cognitive impairment. Through the coupling of mitochondrial biogenesis and the Nrf2 pathway, AMPK may modulate oxidative stress and be a promising target against neurological disorders.     

Involvement of Kv1.5 Protein in Oxidative Vascular Endothelial Cell Injury

Endothelial injury related to oxidative stress is a key event in cardiovascular diseases, such as hypertension and atherosclerosis. The activation of the redox-sensitive Kv1.5 potassium channel mediates mitochondrial reactive oxygen species (ROS)-induced apoptosis in vascular smooth muscle cells and some cancer cells. Kv1.5 channel is therefore taken as a new potential therapeutic target for pulmonary hypertension and cancers. Although Kv1.5 is abundantly expressed in vascular endothelium, there is little knowledge of its role in endothelial injury related to oxidative stress. We found that DPO-1, a specific inhibitor of Kv1.5, attenuated H₂O₂-evoked endothelial cell apoptosis in an in vivo rat carotid arterial model. In human umbilical vein endothelial cells (HUVECs) and human pulmonary arterial endothelial cells (HPAECs), angiotensin II and oxLDL time- or concentration-dependently enhanced Kv1.5 protein expression in parallel with the production of intracellular ROS and endothelial cell injury. Moreover, siRNA-mediated knockdown of Kv1.5 attenuated, whereas adenovirus-mediated Kv1.5 cDNA overexpression enhanced oxLDL–induced cellular damage, NADPH oxidase and mitochondria-derived ROS production and restored the decrease in protein expression of mitochondria uncoupling protein 2 (UCP2). Collectively, these data suggest that Kv1.5 may play an important role in oxidative vascular endothelial injury.