Adolescent Stress-Induced Ventral Hippocampus Redox Dysregulation Underlies Behavioral Deficits and Excitatory/Inhibitory Imbalance Related to Schizophrenia.

BACKGROUND AND HYPOTHESIS: Redox dysregulation has been proposed as a convergent point of childhood trauma and the emergence of psychiatric disorders, such as schizophrenia (SCZ). A critical region particularly vulnerable to environmental insults during adolescence is the ventral hippocampus (vHip). However, the impact of severe stress on vHip redox states and their functional consequences, including behavioral and electrophysiological changes related to SCZ, are not entirely understood. STUDY DESIGN: After exposing adolescent animals to physical stress (postnatal day, PND31-40), we explored social and cognitive behaviors (PND47-49), the basal activity of pyramidal glutamate neurons, the number of parvalbumin (PV) interneurons, and the transcriptomic signature of the vHip (PND51). We also evaluated the impact of stress on the redox system, including mitochondrial respiratory function, reactive oxygen species (ROS) production, and glutathione (GSH) levels in the vHip and serum. STUDY RESULTS: Adolescent-stressed animals exhibited loss of sociability, cognitive impairment, and vHip excitatory/inhibitory (E/I) imbalance. Genome-wide transcriptional profiling unveiled the impact of stress on redox system- and synaptic-related genes. Stress impacted mitochondrial respiratory function and changes in ROS levels in the vHip. GSH and glutathione disulfide (GSSG) levels were elevated in the serum of stressed animals, while GSSG was also increased in the vHip and negatively correlated with sociability. Additionally, PV interneuron deficits in the vHip caused by adolescent stress were associated with oxidative stress. CONCLUSIONS: Our results highlight the negative impact of adolescent stress on vHip redox regulation and mitochondrial function, which are partially associated with E/I imbalance and behavioral abnormalities related to SCZ.

Reduced acetylation of TFAM promotes bioenergetic dysfunction in the failing heart.

General control of amino acid synthesis 5-like 1 (GCN5L1) was previously identified as a key regulator of protein lysine acetylation in mitochondria. Subsequent studies demonstrated that GCN5L1 regulates the acetylation status and activity of mitochondrial fuel substrate metabolism enzymes. However, the role of GCN5L1 in response to chronic hemodynamic stress is largely unknown. Here, we show that cardiomyocyte-specific GCN5L1 knockout mice (cGCN5L1 KO) display exacerbated heart failure progression following transaortic constriction (TAC). Mitochondrial DNA and protein levels were decreased in cGCN5L1 KO hearts after TAC, and isolated neonatal cardiomyocytes with reduced GCN5L1 expression had lower bioenergetic output in response to hypertrophic stress. Loss of GCN5L1 expression led to a decrease in the acetylation status of mitochondrial transcription factor A (TFAM) after TAC in vivo, which was linked to a reduction in mtDNA levels in vitro. Together, these data suggest that GCN5L1 may protect from hemodynamic stress by maintaining mitochondrial bioenergetic output.

Nitrate and nitrite-based therapy to attenuate cardiovascular remodelling in arterial hypertension.

Hypertension is a highly prevalent disease marked by vascular and cardiac maladaptive remodelling induced mainly by renin-angiotensin system activation followed by oxidative stress. Here, we briefly describe these damages and review the current evidence supporting a potential role for nitrate and nitrite as antihypertensive molecules that act via nitric oxide (NO) formation-dependent and NO formation-independent mechanisms and how nitrate/nitrite inhibits cardiovascular remodelling in hypertension. The renin-angiotensin system activation and oxidative stress converge to activate proteases involved in cardiovascular remodelling in hypertension. Besides these proteases, several investigations have demonstrated that reduced endogenous NO bioavailability is a central pathological event in hypertension. In this regard, nitrate/nitrite, long considered inert products of NO, is now known as physiological molecules able to reduce blood pressure in hypertensive patients and in different experimental models of hypertension. These effects are associated with the formation of NO and other NO-related molecules, which could induce S-nitrosylation of target proteins. However, it remains unclear whether S-nitrosylation is an essential mechanism for the anti-remodelling effects of nitrate/nitrite in hypertension. Moreover, nitrate/nitrite produces antioxidant effects associated with the inhibition of signalling pathways involved in cardiovascular remodelling. Together, these findings may help to establish nitrate and nitrite as effective therapies in hypertension-induced cardiovascular remodelling.

IMPACT OF DIABETIC NEUROPATHY ON QUALITY OF LIFE AND POSTURAL BALANCE IN BRAZILIAN OLDER ADULTS.

OBJECTIVE: To analyze the impact of the Peripheral Diabetic Neuropathy (PDN) on the postural and functional balance and quality of life of Brazilian older adults. METHODS: A cross-sectional study. Sixty older men and women (60-79 years) were divided into three groups: control, DM without and with PDN. The following parameters were evaluated: anthropometry; quality of life; postural balance (BESTest); functional balance in force plate (NeuroCom Balance). RESULTS: PDN group presented significant differences compared with the other groups, with the worst performance in quality of life than DM2 without PDN in: sensory functioning (p = 0.030); past and future (p = 0.036); death and dying (p = 0.035). Postural balance deficit in the total score (p = 0.025) and biomedical constraints section (p = 0.043) of the BESTest, compared with DM2 without PDN (p = 0.007). In the functional balance (Neurocom), PDN group presented a worse performance in the time spent on the left side (p = 0.030) than the control group. During step up over test, the control group performed the task faster than the group with PDN (p = 0.004). CONCLUSION: This study showed that neuropaths presented worse physical performance and postural balance deficits, sensorial limitations, affecting the daily tasks and, as a consequence, decreasing the quality of life in Brazilian older adults. Level of Evidence II, Cross-sectional observational study.

OBJETIVO: Analisar a influência da neuropatia diabética periférica (NDP) no equilíbrio postural, atividades funcionais e na qualidade de vida em idosos. MÉTODOS: Estudo transversal. Avaliamos 60 homens e mulheres idosos (60-79 anos) divididos em três grupos: controle, DM sem e com NDP. Foram avaliados: antropometria; qualidade de vida; equilíbrio postural (BESTest); atividades funcionais pelo equilíbrio funcional na placa de força (NeuroCom Balance). RESULTADOS: Grupo NDP apresentou diferenças comparado a outros grupos, pior desempenho na qualidade de vida que o DM2 sem NDP em: funcionamento sensorial (p = 0,030); passado e futuro (p = 0,036); morte e morrer (p = 0,035). Déficit de equilíbrio postural no escore total (p = 0,025) e seção de restrições biomédicas (p = 0,043) do BESTest comparado ao DM2 sem NDP (p = 0,007). No equilíbrio funcional (Neurocom), o grupo NDP apresentou pior desempenho no tempo gasto no lado esquerdo (p = 0,030) comparado ao grupo controle. Durante a etapa de teste, o grupo controle executou a tarefa mais rapidamente que o grupo NDP (p = 0,004). CONCLUSÃO: Neuropatas apresentaram pior desempenho físico e déficits no equilíbrio postural, limitações sensoriais, afetando as tarefas diárias da doença e, consequentemente, diminuição da qualidade de vida em idosos brasileiros. Nível de Evidência II, Estudo observacional transversal.

Impact of diabetic neuropathy on quality of life and postural balance in brazilian older adults / O impacto da neuropatia diabética na qualidade de vida e equilíbrio postural em idosos brasileiros

ABSTRACT Objective: To analyze the impact of the Peripheral Diabetic Neuropathy (PDN) on the postural and functional balance and quality of life of Brazilian older adults. Methods: A cross-sectional study. Sixty older men and women (60-79 years) were divided into three groups: control, DM without and with PDN. The following parameters were evaluated: anthropometry; quality of life; postural balance (BESTest); functional balance in force plate (NeuroCom Balance). Results: PDN group presented significant differences compared with the other groups, with the worst performance in quality of life than DM2 without PDN in: sensory functioning (p = 0.030); past and future (p = 0.036); death and dying (p = 0.035). Postural balance deficit in the total score (p = 0.025) and biomedical constraints section (p = 0.043) of the BESTest, compared with DM2 without PDN (p = 0.007). In the functional balance (Neurocom), PDN group presented a worse performance in the time spent on the left side (p = 0.030) than the control group. During step up over test, the control group performed the task faster than the group with PDN (p = 0.004). Conclusion: This study showed that neuropaths presented worse physical performance and postural balance deficits, sensorial limitations, affecting the daily tasks and, as a consequence, decreasing the quality of life in Brazilian older adults. Level of Evidence II, Cross-sectional observational study.

RESUMO Objetivo: Analisar a influência da neuropatia diabética periférica (NDP) no equilíbrio postural, atividades funcionais e na qualidade de vida em idosos. Métodos: Estudo transversal. Avaliamos 60 homens e mulheres idosos (60-79 anos) divididos em três grupos: controle, DM sem e com NDP. Foram avaliados: antropometria; qualidade de vida; equilíbrio postural (BESTest); atividades funcionais pelo equilíbrio funcional na placa de força (NeuroCom Balance). Resultados: Grupo NDP apresentou diferenças comparado a outros grupos, pior desempenho na qualidade de vida que o DM2 sem NDP em: funcionamento sensorial (p = 0,030); passado e futuro (p = 0,036); morte e morrer (p = 0,035). Déficit de equilíbrio postural no escore total (p = 0,025) e seção de restrições biomédicas (p = 0,043) do BESTest comparado ao DM2 sem NDP (p = 0,007). No equilíbrio funcional (Neurocom), o grupo NDP apresentou pior desempenho no tempo gasto no lado esquerdo (p = 0,030) comparado ao grupo controle. Durante a etapa de teste, o grupo controle executou a tarefa mais rapidamente que o grupo NDP (p = 0,004). Conclusão: Neuropatas apresentaram pior desempenho físico e déficits no equilíbrio postural, limitações sensoriais, afetando as tarefas diárias da doença e, consequentemente, diminuição da qualidade de vida em idosos brasileiros. Nível de Evidência II, Estudo observacional transversal.

Myoglobin promotes nitrite-dependent mitochondrial S-nitrosation to mediate cytoprotection after hypoxia/reoxygenation.

It is well established that myoglobin supports mitochondrial respiration through the storage and transport of oxygen as well as through the scavenging of nitric oxide. However, during ischemia/reperfusion (I/R), myoglobin and mitochondria both propagate myocardial injury through the production of oxidants. Nitrite, an endogenous signaling molecule and dietary constituent, mediates potent cardioprotection after I/R and this effect relies on its interaction with both myoglobin and mitochondria. While independent mechanistic studies have demonstrated that nitrite-mediated cardioprotection requires the presence of myoglobin and the post-translational S-nitrosation of critical cysteine residues on mitochondrial complex I, it is unclear whether myoglobin directly catalyzes the S-nitrosation of complex I or whether mitochondrial-dependent nitrite reductase activity contributes to S-nitrosation. Herein, using purified myoglobin and isolated mitochondria, we characterize and directly compare the nitrite reductase activities of mitochondria and myoglobin and assess their contribution to mitochondrial S-nitrosation. We demonstrate that myoglobin is a significantly more efficient nitrite reductase than isolated mitochondria. Further, deoxygenated myoglobin catalyzes the nitrite-dependent S-nitrosation of mitochondrial proteins. This reaction is enhanced in the presence of oxidized (Fe3+) myoglobin and not significantly affected by inhibitors of mitochondrial respiration. Using a Chinese Hamster Ovary cell model stably transfected with human myoglobin, we show that both myoglobin and mitochondrial complex I expression are required for nitrite-dependent attenuation of cell death after anoxia/reoxygenation. These data expand the understanding of myoglobin's role both as a nitrite reductase to a mediator of S-nitrosation and as a regulator of mitochondrial function, and have implications for nitrite-mediated cardioprotection after I/R.

A neuroglobin-based high-affinity ligand trap reverses carbon monoxide-induced mitochondrial poisoning.

Carbon monoxide (CO) remains the most common cause of human poisoning. The consequences of CO poisoning include cardiac dysfunction, brain injury, and death. CO causes toxicity by binding to hemoglobin and by inhibiting mitochondrial cytochrome c oxidase (CcO), thereby decreasing oxygen delivery and inhibiting oxidative phosphorylation. We have recently developed a CO antidote based on human neuroglobin (Ngb-H64Q-CCC). This molecule enhances clearance of CO from red blood cells in vitro and in vivo Herein, we tested whether Ngb-H64Q-CCC can also scavenge CO from CcO and attenuate CO-induced inhibition of mitochondrial respiration. Heart tissue from mice exposed to 3% CO exhibited a 42 ± 19% reduction in tissue respiration rate and a 33 ± 38% reduction in CcO activity compared with unexposed mice. Intravenous infusion of Ngb-H64Q-CCC restored respiration rates to that of control mice correlating with higher electron transport chain CcO activity in Ngb-H64Q-CCC-treated compared with PBS-treated, CO-poisoned mice. Further, using a Clark-type oxygen electrode, we measured isolated rat liver mitochondrial respiration in the presence and absence of saturating solutions of CO (160 µm) and nitric oxide (100 µm). Both CO and NO inhibited respiration, and treatment with Ngb-H64Q-CCC (100 and 50 µm, respectively) significantly reversed this inhibition. These results suggest that Ngb-H64Q-CCC mitigates CO toxicity by scavenging CO from carboxyhemoglobin, improving systemic oxygen delivery and reversing the inhibitory effects of CO on mitochondria. We conclude that Ngb-H64Q-CCC or other CO scavengers demonstrate potential as antidotes that reverse the clinical and molecular effects of CO poisoning.

Loss of GCN5L1 in cardiac cells limits mitochondrial respiratory capacity under hyperglycemic conditions.

The mitochondrial acetyltransferase-related protein GCN5L1 controls the activity of fuel substrate metabolism enzymes in several tissues. While previous studies have demonstrated that GCN5L1 regulates fatty acid oxidation in the prediabetic heart, our understanding of its role in overt diabetes is not fully developed. In this study, we examined how hyperglycemic conditions regulate GCN5L1 expression in cardiac tissues, and modeled the subsequent effect in cardiac cells in vitro. We show that GCN5L1 abundance is significantly reduced under diabetic conditions in vivo, which correlated with reduced acetylation of known GCN5L1 fuel metabolism substrate enzymes. Treatment of cardiac cells with high glucose reduced Gcn5l1 expression in vitro, while expression of the counteracting deacetylase enzyme, Sirt3, was unchanged. Finally, we show that genetic depletion of GCN5L1 in H9c2 cells leads to reduced mitochondrial oxidative capacity under high glucose conditions. These data suggest that GCN5L1 expression is highly responsive to changes in cellular glucose levels, and that loss of GCN5L1 activity under hyperglycemic conditions impairs cardiac energy metabolism.

Electrophilic nitro-oleic acid reverses obesity-induced hepatic steatosis.

Non-alcoholic fatty liver disease (NAFLD) is linked to obesity and insulin resistance and is the most prevalent chronic liver disease. During the development of obesity and NAFLD, mitochondria adapt to the increased lipid load in hepatocytes by increasing the rate of fatty acid oxidation. In concert with this, reactive species (RS) generation is increased, damaging hepatocytes and inducing inflammation. Hepatic mitochondrial dysfunction is central to the pathogenesis of NAFLD via undefined mechanisms. There are no FDA approved treatments for NAFLD other than weight loss and management of glucose tolerance. Electrophilic nitro-oleic acid (NO2-OA) displays anti-inflammatory and antioxidant signaling actions, thus mitochondrial dysfunction, RS production and inflammatory responses to NO2-OA and the insulin sensitizer rosiglitazone were evaluated in a murine model of insulin resistance and NAFLD. Mice on HFD for 20 wk displayed increased adiposity, insulin resistance and hepatic lipid accumulation (steatosis) compared to mice on normal chow (NC). The HFD mice had mitochondrial dysfunction characterized by lower hepatic mitochondrial complex I, IV and V activity compared to mice on NC. Treatment with NO2-OA or rosiglitazone for the last 42 days (out of 20 wk) abrogated HFD-mediated decreases in hepatic mitochondrial complex I, IV and V activity. Notably, NO2-OA treatment normalized hepatic triglyceride levels and significantly reversed hepatic steatosis. Despite the improved glucose tolerance observed upon rosiglitazone treatment, liver weight and hepatic triglycerides were significantly increased over vehicle-treated HFD mice. These observations support that the pleiotropic signaling actions of electrophilic fatty acids limit the complex hepatic and systemic pathogenic responses instigated by obesity, without the adverse effects of thiazolidinedione drugs such as rosiglitazone.

Nox1/Ref-1-mediated activation of CREB promotes Gremlin1-driven endothelial cell proliferation and migration.

Pulmonary arterial hypertension (PAH) is a complex degenerative disorder marked by aberrant vascular remodeling associated with hyperproliferation and migration of endothelial cells (ECs). Previous reports implicated bone morphogenetic protein antagonist Gremlin 1 in this process; however, little is known of the molecular mechanisms involved. The current study was designed to test whether redox signaling initiated by NADPH oxidase 1 (Nox1) could promote transcription factor CREB activation by redox factor 1 (Ref-1), transactivation of Gremlin1 transcription, EC migration, and proliferation. Human pulmonary arterial EC (HPAECs) exposed in vitro to hypoxia to recapitulate PAH signaling displayed induced Nox1 expression, reactive oxygen species (ROS) production, PKA activity, CREB phosphorylation, and CREB:CRE motif binding. These responses were abrogated by selective Nox1 inhibitor NoxA1ds and/or siRNA Nox1. Nox1-activated CREB migrated to the nucleus and bound to Ref-1 leading to CREB:CRE binding and Gremlin1 transcription. CHiP assay and CREB gene-silencing illustrated that CREB is pivotal for hypoxia-induced Gremlin1, which, in turn, stimulates EC proliferation and migration. In vivo, participation of Nox1, CREB, and Gremlin1, as well as CREB:CRE binding was corroborated in a rat PAH model. Activation of a previously unidentified Nox1-PKA-CREB/Ref-1 signaling pathway in pulmonary endothelial cells leads to Gremlin1 transactivation, proliferation and migration. These findings reveal a new signaling pathway by which Nox1 via induction of CREB and Gremlin1 signaling contributes to vascular remodeling and provide preclinical indication of its significance in PAH.

Nitrite treatment downregulates vascular MMP-2 activity and inhibits vascular remodeling in hypertension independently of its antihypertensive effects.

Hypertension is associated with cardiovascular remodeling. Given that impaired redox state activates matrix metalloproteinase (MMP)- 2 and promotes vascular remodeling, we hypothesized that nitrite treatment at a non-antihypertensive dose exerts antioxidant effects and attenuates both MMP-2 activation and vascular remodeling of hypertension. We examined the effects of oral sodium nitrite at antihypertensive (15 mg/kg) or non-antihypertensive (1 mg/kg) daily dose in hypertensive rats (two kidney, one clip; 2K1C model). Sham-operated and 2K1C hypertensive rats received vehicle or nitrite by gavage for four weeks. Systolic blood pressure decreased only in hypertensive rats treated with nitrite 15 mg/Kg/day. Both low and high nitrite doses decreased 2K1C-induced vascular remodeling assessed by measuring aortic cross-sectional area, media/lumen ratio, and number of vascular smooth muscle cells/aortic length. Both low and high nitrite doses decreased 2K1C-induced vascular oxidative stress assessed in situ with the fluorescent dye DHE and with the lucigenin chemiluminescence assay. Vascular MMP-2 expression and activity were assessed by gel zymography, Western blot, and in situ zymography increased with hypertension. While MMP-2 levels did not change in response to both doses of nitrite, both doses completely prevented hypertension-induced increases in vascular MMP activity. Moreover, incubation of aortas from hypertensive rats with nitrite at 1-20 µmol/L reduced gelatinolytic activity by 20-30%. This effect was fully inhibited by the xanthine oxidase (XOR) inhibitor febuxostat, suggesting XOR-mediated generation of nitric oxide (NO) from nitrite as a mechanism explaining the responses to nitrite. In vitro incubation of aortic extracts with nitrite 20 µmol/L did not affect MMP-2 activity. These results show that nitrite reverses the vascular structural alterations of hypertension, independently of anti-hypertensive effects. This response is mediated, at least in part, by XOR and is attributable to antioxidant effects of nitrite blunting vascular MMP-2 activation. Our findings suggest nitrite therapy to reverse structural alterations of hypertension.

Adropin regulates pyruvate dehydrogenase in cardiac cells via a novel GPCR-MAPK-PDK4 signaling pathway.

Mitochondria supply ~90% of the ATP required for contractile function in cardiac cells. While adult cardiomyocytes preferentially utilize fatty acids as a fuel source for oxidative phosphorylation, cardiac mitochondria can switch to other substrates when required. This change is driven in part by a combination of extracellular and intracellular signal transduction pathways that alter mitochondrial gene expression and enzymatic activity. The mechanisms by which extracellular metabolic information is conveyed to cardiac mitochondria are not currently well defined. Recent work has shown that adropin - a liver-secreted peptide hormone - can induce changes in mitochondrial fuel substrate utilization in skeletal muscle, leading to increased glucose use. In this study, we examined whether adropin could regulate mitochondrial glucose utilization pathways in cardiac cells. We show that stimulation of cultured cardiac cells with adropin leads to decreased expression of the pyruvate dehydrogenase (PDH) negative regulator PDK4, which reduces inhibitory PDH phosphorylation. The downregulation of PDK4 expression by adropin is lost when GPR19 - a putative adropin receptor - is genetically depleted in H9c2 cells. Loss of GRP19 expression alone increased PDK4 expression, leading to a reduction in mitochondrial respiration. Finally, we show that adropin-mediated GPR19 signaling relies on the p44/42 MAPK pathway, and that pharmacological disruption of this pathway blocks the effects of adropin on PDK4 in cardiac cells. These findings suggest that adropin may be a key regulator of fuel substrate utilization in the heart, and implicates an orphan G-protein coupled receptor in a novel signaling pathway controlling mitochondrial fuel metabolism.

Nitrite exerts antioxidant effects, inhibits the mTOR pathway and reverses hypertension-induced cardiac hypertrophy.

Cardiac hypertrophy is a common consequence of chronic hypertension and leads to heart failure and premature death. The anion nitrite is now considered as a bioactive molecule able to exert beneficial cardiovascular effects. Previous results showed that nitrite attenuates hypertension-induced increases in reactive oxygen species (ROS) production in the vasculature. Whether antioxidant effects induced by nitrite block critical signaling pathways involved in cardiac hypertrophy induced by hypertension has not been determined yet. The Akt/mTOR signaling pathway is responsible to activate protein synthesis during cardiac remodeling and is activated by increased ROS production, which is commonly found in hypertension. Here, we investigated the effects of nitrite treatment on cardiac remodeling and activation of this hypertrophic signaling pathway in 2 kidney-1 clip (2K1C) hypertension. Sham and 2K1C rats were treated with oral nitrite at 1 or 15 mg/kg for four weeks. Nitrite treatment (15 mg/kg) reduced systolic blood pressure and decreased ROS production in the heart tissue from hypertensive rats. This nitrite dose also blunted hypertension-induced activation of mTOR pathway and cardiac hypertrophy. While the lower nitrite dose (1 mg/kg) did not affect blood pressure, it exerted antioxidant effects and tended to attenuate mTOR pathway activation and cardiac hypertrophy induced by hypertension. Our findings provide strong evidence that nitrite treatment decreases cardiac remodeling induced by hypertension as a result of its antioxidant effects and downregulation of mTOR signaling pathway. This study may help to establish nitrite as an effective therapy in hypertension-induced cardiac hypertrophic remodeling.

Direct renin inhibition is not enough to prevent reactive oxygen species generation and vascular dysfunction in renovascular hypertension.

Renin-angiotensin system activation promotes oxidative stress and endothelial dysfunction. However, no previous study has examined the effects of the renin inhibitor aliskiren, either alone or combined with angiotensin II type 1 antagonists on alterations induced by two-kidney, one-clip (2K1C) hypertension. We compared the vascular effects of aliskiren (50mg/kg/day), losartan (10mg/kg/day), or both by gavage for 4 weeks in 2K1C and control rats. Treatment with losartan, aliskiren, or both exerted similar antihypertensive effects. Aliskiren lowered plasma Ang I concentrations in sham rats and in hypertensive rats treated with aliskiren or with both drugs. Aliskiren alone or combined with losartan decreased plasma angiotensin II concentrations measured by high performance liquid chromatography, whereas losartan alone had no effects. In contrast, losartan alone or combined with aliskiren abolished hypertension-induced increases in aortic angiotensin II concentrations, whereas aliskiren alone exerted no such effects. While hypertension enhanced aortic oxidative stress assessed by dihydroethidium fluorescence and by lucigenin chemiluminescence, losartan alone or combined with aliskiren, but not aliskiren alone, abolished this alteration. Hypertension impaired aortic relaxation induced by acetylcholine, and losartan alone or combined with aliskiren, but not aliskiren alone, reversed this alteration. Losartan alone or combined with aliskiren, but not aliskiren alone, increased plasma nitrite concentrations in 2K1C rats. These findings show that antihypertensive effects of aliskiren do not prevent hypertension-induced vascular oxidative stress and endothelial dysfunction. These findings contrast those found with losartan and suggest that renin inhibition is not enough to prevent hypertension-induced impaired redox biology and vascular dysfunction.

Acetylation of mitochondrial proteins by GCN5L1 promotes enhanced fatty acid oxidation in the heart.

Lysine acetylation is a reversible posttranslational modification and is particularly important in the regulation of mitochondrial metabolic enzymes. Acetylation uses acetyl-CoA derived from fuel metabolism as a cofactor, thereby linking nutrition to metabolic activity. In the present study, we investigated how mitochondrial acetylation status in the heart is controlled by food intake and how these changes affect mitochondrial metabolism. We found that there was a significant increase in cardiac mitochondrial protein acetylation in mice fed a long-term high-fat diet and that this change correlated with an increase in the abundance of the mitochondrial acetyltransferase-related protein GCN5L1. We showed that the acetylation status of several mitochondrial fatty acid oxidation enzymes (long-chain acyl-CoA dehydrogenase, short-chain acyl-CoA dehydrogenase, and hydroxyacyl-CoA dehydrogenase) and a pyruvate oxidation enzyme (pyruvate dehydrogenase) was significantly upregulated in high-fat diet-fed mice and that the increase in long-chain and short-chain acyl-CoA dehydrogenase acetylation correlated with increased enzymatic activity. Finally, we demonstrated that the acetylation of mitochondrial fatty acid oxidation proteins was decreased after GCN5L1 knockdown and that the reduced acetylation led to diminished fatty acid oxidation in cultured H9C2 cells. These data indicate that lysine acetylation promotes fatty acid oxidation in the heart and that this modification is regulated in part by the activity of GCN5L1.NEW & NOTEWORTHY Recent research has shown that acetylation of mitochondrial fatty acid oxidation enzymes has greatly contrasting effects on their activity in different tissues. Here, we provide new evidence that acetylation of cardiac mitochondrial fatty acid oxidation enzymes by GCN5L1 significantly upregulates their activity in diet-induced obese mice.

Omeprazole impairs vascular redox biology and causes xanthine oxidoreductase-mediated endothelial dysfunction.

Proton pump inhibitors (PPIs) are widely used drugs that may increase the cardiovascular risk by mechanisms not entirely known. While PPIs increase asymmetric dimethylarginine (ADMA) levels and inhibit nitric oxide production, it is unknown whether impaired vascular redox biology resulting of increased xanthine oxidoreductase (XOR) activity mediates PPIs-induced endothelial dysfunction (ED). We examined whether increased XOR activity impairs vascular redox biology and causes ED in rats treated with omeprazole. We also examined whether omeprazole aggravates the ED found in hypertension. Treatment with omeprazole reduced endothelium-dependent aortic responses to acetylcholine without causing hypertension. However, omeprazole did not aggravate two-kidney, one-clip (2K1C) hypertension, nor hypertension-induced ED. Omeprazole and 2K1C increased vascular oxidative stress as assessed with dihydroethidium (DHE), which reacts with superoxide, and by the lucigenin chemiluminescence assay. The selective XOR inhibitor febuxostat blunted both effects induced by omeprazole. Treatment with omeprazole increased plasma ADMA concentrations, XOR activity and systemic markers of oxidative stress. Incubation of aortic rings with ADMA increased XOR activity, DHE fluorescence and lucigenin chemiluminescence signals, and febuxostat blunted these effects. Providing functional evidence that omeprazole causes ED by XOR-mediated mechanisms, we found that febuxostat blunted the ED caused by omeprazole treatment. This study shows that treatment with omeprazole impairs the vascular redox biology by XOR-mediated mechanisms leading to ED. While omeprazole did not further impair hypertension-induced ED, further studies in less severe animal models are warranted. Our findings may have major relevance, particularly to patients with cardiovascular diseases taking PPIs.

Tempol improves xanthine oxidoreductase-mediated vascular responses to nitrite in experimental renovascular hypertension.

Upregulation of xanthine oxidoreductase (XOR) increases vascular reactive oxygen species (ROS) levels and contributes to nitroso-redox imbalance. However, XOR can generate nitric oxide (NO) from nitrite, and increased superoxide could inactivate NO formed from nitrite. This study tested the hypothesis that XOR contributes to the cardiovascular effects of nitrite in renovascular hypertension, and that treatment with the antioxidant tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl) improves XOR-mediated effects of nitrite. Blood pressure was assessed weekly in two-kidney one-clip (2K1C) and control rats. After six weeks of hypertension, the relaxing responses to nitrite were assessed in aortic rings in the presence of the XOR inhibitor oxypurinol (or vehicle), either in the absence or in the presence of tempol. Moreover, in vivo hypotensive responses to nitrite were also examined in the presence of oxypurinol (or vehicle) and tempol (or vehicle). Aortic XOR activity and expression were evaluated by fluorescence and Western blot, respectively. Vascular ROS production was assessed by the dihydroethidium assay. 2K1C hypertensive rats showed increased aortic XOR activity and vascular ROS production compared with control rats. Oxypurinol shifted the nitrite concentration-response curve to the right in aortic rings from 2K1C rats (but not in controls). Oxypurinol also attenuated the hypotensive responses to nitrite in 2K1C rats (but not in controls). These functional findings agree with increased aortic and plasma XOR activity found in 2K1C rats. Tempol treatment enhanced oxypurinol-induced shift of the nitrite concentration-response curve to the right. However, antioxidant treatment did not affect XOR-mediated hypotensive effects of nitrite. Our results show that XOR is important to the cardiovascular responses to nitrite in 2K1C hypertension, and XOR inhibitors commonly used by patients may cancel this effect. This finding suggests that nitrite treatment may not be effective in patients being treated with XOR inhibitors. Moreover, while tempol may improve the vascular responses to nitrite, antihypertensive responses are not affected.

Sodium nitrite attenuates MMP-9 production by endothelial cells and may explain similar effects of atorvastatin.

Imbalanced matrix metalloproteinase (MMP) activity promotes cardiovascular alterations that are attenuated by statins. These drugs exert pleiotropic effects independent of cholesterol concentrations, including upregulation of nitric oxide (NO) formation and MMP downregulation. However, statins also increase tissue concentrations of nitrites, which activate new signaling pathways independent of NO. We examined whether atorvastatin attenuates MMP-9 production by human umbilical vein endothelial cells (HUVEC) stimulated with phorbol 12-myristate 13-acetate (PMA) by mechanisms possibly involving increased nitrite, and whether this effect results of NO formation. We also examined whether such an effect is improved by sildenafil, an inhibitor of phosphodiesterase-5 which potentiates NO-induced increases in cyclic GMP. MMP activity and nitrite concentrations were measured by gelatin zymography and ozone-based reductive chemiluminescence, respectively, in the conditioned medium of HUVECs incubated for 24 h with these drugs. Phospho-NFκB p65 concentrations were measured in cell lysate to assess NFκB activation. Atorvastatin attenuated PMA-induced MMP-9 gelatinolytic activity by mechanisms not involving NO, although it increased nitrite concentrations, whereas sildenafil had no effects. Combining both drugs showed no improved responses compared to atorvastatin alone. While sodium nitrite attenuated MMP-9 production by HUVECs, adding hemoglobin (NO scavenger) did not affect the responses to nitrite. Neither atorvastatin nor nitrite inhibited PMA-induced increases in phospho-NFκB p65 concentrations. These findings show that sodium nitrite attenuates MMP-9 production by endothelial cells and may explain similar effects exerted by atorvastatin. With both drugs, the inhibitory effects on MMP-9 production are not dependent on NO formation or on inhibition of NFκB activation. Our findings may help to elucidate important new nitrite-mediated mechanisms by which statins affect imbalanced MMP activity in a variety of cardiovascular disease.

Atorvastatin and sildenafil decrease vascular TGF-ß levels and MMP-2 activity and ameliorate arterial remodeling in a model of renovascular hypertension.

Imbalanced matrix metalloproteinase (MMP)-2 activity and transforming growth factor expression (TGF-ß) are involved in vascular remodeling of hypertension. Atorvastatin and sildenafil exert antioxidant and pleiotropic effects that may result in cardiovascular protection. We hypothesized that atorvastatin and sildenafil alone or in association exert antiproliferative effects by down-regulating MMP-2 and TGF-ß, thus reducing the vascular hypertrophy induced by two kidney, one clip (2K1C) hypertension. Sham and 2K1C rats were treated with oral atorvastatin 50 mg/kg, sildenafil 45 mg/kg, or both, daily for 8 weeks. Blood pressure was monitored weekly. Morphologic changes in the aortas were studied. TGF-ß levels were determined by immunofluorescence. MMP-2 activity and expression were determined by in situ zymography, gel zymography, Western blotting, and immunofluorescence. The effects of both drugs on proliferative responses of aortic smooth muscle cells to PDGF and on on MMP-2 activity in vitro were determined. Atorvastatin, sildenafil, or both drugs exerted antiproliferative effects in vitro. All treatments attenuated 2K1C-induced hypertension and prevented the increases in the aortic cross-sectional area and media/lumen ratio in 2K1C rats. Aortas from 2K1C rats showed higher collagen deposition, TGF-ß levels and MMP-2 activity and expression when compared with Sham-operated animals. Treatment with atorvastatin and/or sildenafil was associated with attenuation of 2K1C hypertension-induced increases in these pro-fibrotic factors. However, these drugs had no in vitro effects on hr-MMP-2 activity. Atorvastatin and sildenafil was associated with decreased vascular TGF-ß levels and MMP-2 activity in renovascular hypertensive rats, thus ameliorating the vascular remodeling. These novel pleiotropic effects of both drugs may translate into protective effects in patients.