Mitochondrial CypD Acetylation Promotes Endothelial Dysfunction and Hypertension.

BACKGROUND: Nearly half of adults have hypertension, a major risk factor for cardiovascular disease. Mitochondrial hyperacetylation is linked to hypertension, but the role of acetylation of specific proteins is not clear. We hypothesized that acetylation of mitochondrial CypD (cyclophilin D) at K166 contributes to endothelial dysfunction and hypertension. METHODS: To test this hypothesis, we studied CypD acetylation in patients with essential hypertension, defined a pathogenic role of CypD acetylation in deacetylation mimetic CypD-K166R mutant mice and endothelial-specific GCN5L1 (general control of amino acid synthesis 5 like 1)-deficient mice using an Ang II (angiotensin II) model of hypertension. RESULTS: Arterioles from hypertensive patients had 280% higher CypD acetylation coupled with reduced Sirt3 (sirtuin 3) and increased GCN5L1 levels. GCN5L1 regulates mitochondrial protein acetylation and promotes CypD acetylation, which is counteracted by mitochondrial deacetylase Sirt3. In human aortic endothelial cells, GCN5L1 depletion prevents superoxide overproduction. Deacetylation mimetic CypD-K166R mice were protected from vascular oxidative stress, endothelial dysfunction, and Ang II-induced hypertension. Ang II-induced hypertension increased mitochondrial GCN5L1 and reduced Sirt3 levels resulting in a 250% increase in GCN5L1/Sirt3 ratio promoting CypD acetylation. Treatment with mitochondria-targeted scavenger of cytotoxic isolevuglandins (mito2HOBA) normalized GCN5L1/Sirt3 ratio, reduced CypD acetylation, and attenuated hypertension. The role of mitochondrial acetyltransferase GCN5L1 in the endothelial function was tested in endothelial-specific GCN5L1 knockout mice. Depletion of endothelial GCN5L1 prevented Ang II-induced mitochondrial oxidative stress, reduced the maladaptive switch of vascular metabolism to glycolysis, prevented inactivation of endothelial nitric oxide, preserved endothelial-dependent relaxation, and attenuated hypertension. CONCLUSIONS: These data support the pathogenic role of CypD acetylation in endothelial dysfunction and hypertension. We suggest that targeting cytotoxic mitochondrial isolevuglandins and GCN5L1 reduces CypD acetylation, which may be beneficial in cardiovascular disease.

Immunoproteasomal Processing of IsoLG-Adducted Proteins Is Essential for Hypertension.

BACKGROUND: Hypertension is characterized by CD8+ (cluster differentiation 8) T cell activation and infiltration into peripheral tissues. CD8+ T cell activation requires proteasomal processing of antigenic proteins. It has become clear that isoLG (isolevuglandin)-adduced peptides are antigenic in hypertension; however, IsoLGs inhibit the constitutive proteasome. We hypothesized that immunoproteasomal processing of isoLG-adducts is essential for CD8+ T cell activation and inflammation in hypertension. METHODS: IsoLG adduct processing was studied in murine dendritic cells (DCs), endothelial cells (ECs), and B8 fibroblasts. The role of the proteasome and the immunoproteasome in Ang II (angiotensin II)-induced hypertension was studied in C57BL/6 mice treated with bortezomib or the immunoproteasome inhibitor PR-957 and by studying mice lacking 3 critical immunoproteasome subunits (triple knockout mouse). We also examined hypertension in mice lacking the critical immunoproteasome subunit LMP7 (large multifunctional peptidase 7) specifically in either DCs or ECs. RESULTS: We found that oxidant stress increases the presence of isoLG adducts within MHC-I (class I major histocompatibility complex), and immunoproteasome overexpression augments this. Pharmacological or genetic inhibition of the immunoproteasome attenuated hypertension and tissue inflammation. Conditional deletion of LMP7 in either DCs or ECs attenuated hypertension and vascular inflammation. Finally, we defined the role of the innate immune receptors STING (stimulator of interferon genes) and TLR7/8 (toll-like receptor 7/8) as drivers of LMP7 expression in ECs. CONCLUSIONS: These studies define a previously unknown role of the immunoproteasome in DCs and ECs in CD8+ T cell activation. The immunoproteasome in DCs and ECs is critical for isoLG-adduct presentation to CD8+ T cells, and in the endothelium, this guides homing and infiltration of T cells to specific tissues.

Repetitive sulfur dioxide exposure in mice models post-deployment respiratory syndrome.

Soldiers deployed to Iraq and Afghanistan have a higher prevalence of respiratory symptoms than nondeployed military personnel and some have been shown to have a constellation of findings on lung biopsy termed post-deployment respiratory syndrome (PDRS). Since many of the subjects in this cohort reported exposure to sulfur dioxide (SO2), we developed a model of repetitive exposure to SO2 in mice that phenocopies many aspects of PDRS, including adaptive immune activation, airway wall remodeling, and pulmonary vascular (PV) disease. Although abnormalities in small airways were not sufficient to alter lung mechanics, PV remodeling resulted in the development of pulmonary hypertension and reduced exercise tolerance in SO2-exposed mice. SO2 exposure led to increased formation of isolevuglandins (isoLGs) adducts and superoxide dismutase 2 (SOD2) acetylation in endothelial cells, which were attenuated by treatment with the isoLG scavenger 2-hydroxybenzylamine acetate (2-HOBA). In addition, 2-HOBA treatment or Siruin-3 overexpression in a transgenic mouse model prevented vascular remodeling following SO2 exposure. In summary, our results indicate that repetitive SO2 exposure recapitulates many aspects of PDRS and that oxidative stress appears to mediate PV remodeling in this model. Together, these findings provide new insights regarding the critical mechanisms underlying PDRS.NEW & NOTEWORTHY We developed a mice model of "post-deployment respiratory syndrome" (PDRS), a condition in Veterans with unexplained exertional dyspnea. Our model successfully recapitulates many of the pathological and physiological features of the syndrome, revealing involvement of the ROS-isoLGs-Sirt3-SOD2 pathway in pulmonary vasculature pathology. Our study provides additional knowledge about effects and long-term consequences of sulfur dioxide exposure on the respiratory system, serving as a valuable tool for future PDRS research.

Deacetylation Mimetic Mutation of Mitochondrial Superoxide Dismutase Attenuates Angiotensin II-Induced Hypertension by Protecting Against Oxidative Stress.

Almost one-half of adults have hypertension, and blood pressure is poorly controlled in a third of patients despite use of multiple drugs, likely due to mechanisms that are not affected by current treatments. Hypertension is linked to oxidative stress; however, common antioxidants are ineffective. Hypertension is associated with inactivation of key mitochondrial antioxidant, superoxide dismutase 2 (SOD2), due to hyperacetylation but the role of specific SOD2 lysine residues has not been defined. We proposed that inactivation of key intrinsic antioxidant, SOD2, is linked to Lysine 68 acetylation, and mutation of K68 to Arginine mimics SOD2 deacetylation, inhibits vascular oxidative stress and attenuates hypertension. To test this hypothesis, we have developed a new deacetylation mimic SOD2-K68R mice. We performed in vivo studies in SOD2-K68R mice using angiotensin II (AngII) model of vascular dysfunction and hypertension. AngII infusion in wildtype mice induced vascular inflammation and oxidative stress, and increased blood pressure to 160 mm Hg. SOD2-K68R mutation completely prevented increase in mitochondrial superoxide, abrogated vascular oxidative stress, preserved endothelial nitric oxide production, protected vasorelaxation and attenuated the AngII-induced hypertension. AngII and cytokines contribute to vascular oxidative stress and hypertension. Treatment of wildtype aortas with AngII and cytokines in organoid culture increased mitochondrial superoxide by 2-fold which was completely prevented in aortas isolated from SOD2-K68R mice. These data support an important role of SOD2-K68 acetylation in vascular oxidative stress and pathogenesis of hypertension. We conclude that strategies to reduce SOD2 acetylation may have therapeutic potential in the treatment of vascular dysfunction and hypertension.

Critical Role of Mitochondrial Fatty Acid Metabolism in Normal Cell Function and Pathological Conditions.

There is a "popular" belief that a fat-free diet is beneficial, supported by the scientific dogma indicating that high levels of fatty acids promote many pathological metabolic, cardiovascular, and neurodegenerative conditions. This dogma pressured scientists not to recognize the essential role of fatty acids in cellular metabolism and focus on the detrimental effects of fatty acids. In this work, we critically review several decades of studies and recent publications supporting the critical role of mitochondrial fatty acid metabolism in cellular homeostasis and many pathological conditions. Fatty acids are the primary fuel source and essential cell membrane building blocks from the origin of life. The essential cell membranes phospholipids were evolutionarily preserved from the earlier bacteria in human subjects. In the past century, the discovery of fatty acid metabolism was superseded by the epidemic growth of metabolic conditions and cardiovascular diseases. The association of fatty acids and pathological conditions is not due to their "harmful" effects but rather the result of impaired fatty acid metabolism and abnormal lifestyle. Mitochondrial dysfunction is linked to impaired metabolism and drives multiple pathological conditions. Despite metabolic flexibility, the loss of mitochondrial fatty acid oxidation cannot be fully compensated for by other sources of mitochondrial substrates, such as carbohydrates and amino acids, resulting in a pathogenic accumulation of long-chain fatty acids and a deficiency of medium-chain fatty acids. Despite popular belief, mitochondrial fatty acid oxidation is essential not only for energy-demanding organs such as the heart, skeletal muscle, and kidneys but also for metabolically "inactive" organs such as endothelial and epithelial cells. Recent studies indicate that the accumulation of long-chain fatty acids in specific organs and tissues support the impaired fatty acid oxidation in cell- and tissue-specific fashion. This work, therefore, provides a basis to challenge these established dogmas and articulate the need for a paradigm shift from the "pathogenic" role of fatty acids to the critical role of fatty acid oxidation. This is important to define the causative role of impaired mitochondrial fatty acid oxidation in specific pathological conditions and develop novel therapeutic approaches targeting mitochondrial fatty acid metabolism.

Pathological mechanisms of cigarette smoking, dietary, and sedentary lifestyle risks in vascular dysfunction: mitochondria as a common target of risk factors.

In the past century, the lifespan of the human population has dramatically increased to the 80 s, but it is hindered by a limited health span to the 60 s due to an epidemic increase in the cardiovascular disease which is a main cause of morbidity and mortality. We cannot underestimate the progress in understanding the major cardiovascular risk factors which include cigarette smoking, dietary, and sedentary lifestyle risks. Despite their clinical significance, these modifiable risk factors are still the major contributors to cardiovascular disease. It is, therefore, important to understand the specific molecular mechanisms behind their pathological effects to develop new therapies to improve the treatment of cardiovascular disease. In recent years, our group and others have made a progress in understanding how these risk factors can promote endothelial dysfunction, smooth muscle dysregulation, vascular inflammation, hypertension, lung, and heart diseases. These factors, despite differences in their nature, lead to stereotypical alterations in vascular metabolism and function. Interestingly, cigarette smoking has a tremendous impact on a very distant site from the initial epithelial exposure, namely circulation and vascular cells mediated by a variety of stable cigarette smoke components which promote vascular oxidative stress and alter vascular metabolism and function. Similarly, dietary and sedentary lifestyle risks facilitate vascular cell metabolic reprogramming promoting vascular oxidative stress and dysfunction. Mitochondria are critical in cellular metabolism, and in this work, we discuss a new concept that mitochondria are a common pathobiological target for these risk factors, and mitochondria-targeted treatments may have a therapeutic effect in the patients with cardiovascular disease.

Mitochondrial deacetylase Sirt3 in vascular dysfunction and hypertension.

PURPOSE OF REVIEW: Hypertension is a multifactorial disorder involving perturbations of the vasculature, the kidney, and the central nervous system. Hypertension represents a major risk factor for stroke, myocardial infarction, and heart failure. Despite treatment with multiple drugs, 37% of hypertensive patients remain hypertensive, likely due to the mechanisms contributing to blood pressure elevation that are not affected by current treatments. This review focuses on recently described novel role of mitochondrial deacetylase Sirt3 in vascular dysfunction and hypertension. RECENT FINDINGS: In the past several years, we have shown that the mitochondria are dysfunctional in hypertension; however, the role of mitochondria in the pathogenesis of hypertension remains elusive. We recently showed that patients with essential hypertension have decreased levels of the mitochondrial deacetylase Sirt3 leading to hyperacetylation of mitochondrial proteins. There is likely a causative role. Indeed, genetic deletion of Sirt3 in mice promotes vascular dysfunction and hypertension. Sirt3 depletion promotes endothelial dysfunction, increases smooth muscle cell hypertrophy, instigates vascular inflammation, and induces age-dependent hypertension. SUMMARY: Sirt3 is critical for vascular cell homeostasis, however, multiple risk factors impair Sirt3 leading to mitochondrial dysfunction and vascular dysregulation which contribute to hypertension and end-organ injury. Targeting Sirt3 may represent novel therapeutic approach to improve treatment of vascular dysfunction and reduce hypertension.

Mitochondrial Deacetylase Sirt3 Reduces Vascular Dysfunction and Hypertension While Sirt3 Depletion in Essential Hypertension Is Linked to Vascular Inflammation and Oxidative Stress.

RATIONALE: Hypertension represents a major risk factor for stroke, myocardial infarction, and heart failure and affects 30% of the adult population. Mitochondrial dysfunction contributes to hypertension, but specific mechanisms are unclear. The mitochondrial deacetylase Sirt3 (Sirtuin 3) is critical in the regulation of metabolic and antioxidant functions which are associated with hypertension, and cardiovascular disease risk factors diminish Sirt3 level. OBJECTIVE: We hypothesized that reduced Sirt3 expression contributes to vascular dysfunction in hypertension, but increased Sirt3 protects vascular function and decreases hypertension. METHODS AND RESULTS: To test the therapeutic potential of targeting Sirt3 expression, we developed new transgenic mice with global Sirt3OX (Sirt3 overexpression), which protects from endothelial dysfunction, vascular oxidative stress, and hypertrophy and attenuates Ang II (angiotensin II) and deoxycorticosterone acetate-salt induced hypertension. Global Sirt3 depletion in Sirt3-/- mice results in oxidative stress due to hyperacetylation of mitochondrial superoxide dismutase (SOD2), increases HIF1α (hypoxia-inducible factor-1), reduces endothelial cadherin, stimulates vascular hypertrophy, increases vascular permeability and vascular inflammation (p65, caspase 1, VCAM [vascular cell adhesion molecule-1], ICAM [intercellular adhesion molecule-1], and MCP1 [monocyte chemoattractant protein 1]), increases inflammatory cell infiltration in the kidney, reduces telomerase expression, and accelerates vascular senescence and age-dependent hypertension; conversely, increased Sirt3 expression in Sirt3OX mice prevents these deleterious effects. The clinical relevance of Sirt3 depletion was confirmed in arterioles from human mediastinal fat in patients with essential hypertension showing a 40% decrease in vascular Sirt3, coupled with Sirt3-dependent 3-fold increases in SOD2 acetylation, NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) activity, VCAM, ICAM, and MCP1 levels in hypertensive subjects compared with normotensive subjects. CONCLUSIONS: We suggest that Sirt3 depletion in hypertension promotes endothelial dysfunction, vascular hypertrophy, vascular inflammation, and end-organ damage. Our data support a therapeutic potential of targeting Sirt3 expression in vascular dysfunction and hypertension.

Long-Chain and Medium-Chain Fatty Acids in Energy Metabolism of Murine Kidney Mitochondria.

Scientists have long established that fatty acids are the primary substrates for kidney mitochondria. However, to date we still do not know how long-chain and middle-chain fatty acids are oxidized at the mitochondrial level. Our previous research has shown that mitochondria from the heart, brain, and kidney oxidize palmitoylcarnitine at a high rate only in the presence of succinate, glutamate, or pyruvate. In this paper, we report properties of the isolated kidney mitochondria and how malate and succinate affect the oxidation of C16 and C8 acylcarnitines. The isolated kidney mitochondria contain very few endogenous substrates and require malate to oxidize pyruvate, glutamate, and C16 or C8 acylcarnitines. We discovered that with 10 µM of C16 or C8 acylcarnitines, low concentrations of malate (0.2 mM) or succinate (0.5 mM) enhance the States 4 and 3 respiratory rates several times. The highest respiration rates were observed with C16 or C8 acylcarnitines and 5 mM succinate mixtures. Results show that kidney mitochondria, unlike the heart and brain mitochondria, lack the intrinsic inhibition of succinate dehydrogenase. Additionally, results show that the oxidation of fatty acid by the small respirasome's supercomplex generates a high level of CoQH2, and this makes SDH in the presence of succinate reverse the flow of electrons from CoQH2 to reduce fumarate to succinate. Finally, we report evidence that succinate dehydrogenase is a key mitochondrial enzyme that allows fast oxidation of fatty acids and turns the TCA cycle function from the catabolic to the anabolic and anaplerotic metabolic pathways.

Oxidant-resistant LRRC8A/C anion channels support superoxide production by NADPH oxidase 1.

KEY POINTS: LRRC8A-containing anion channels associate with NADPH oxidase 1 (Nox1) and regulate superoxide production and tumour necrosis factor-α (TNFα) signalling. Here we show that LRRC8C and 8D also co-immunoprecipitate with Nox1 in vascular smooth muscle cells. LRRC8C knockdown inhibited TNFα-induced O2•- production, receptor endocytosis, nuclear factor-κB (NF-κB) activation and proliferation while LRRC8D knockdown enhanced NF-κB activation. Significant changes in LRRC8 isoform expression in human atherosclerosis and psoriasis suggest compensation for increased inflammation. The oxidant chloramine-T (ChlorT, 1 mM) weakly (∼25%) inhibited LRRC8C currents but potently (∼80%) inhibited LRRC8D currents. Substitution of the extracellular loop (EL1, EL2) domains of 8D into 8C conferred significantly stronger (69%) ChlorT-dependent inhibition. ChlorT exposure impaired subsequent current block by DCPIB, which occurs through interaction with EL1, further implicating external oxidation sites. LRRC8A/C channels most effectively sustain Nox1 activity at the plasma membrane. This may result from their ability to remain active in an oxidized microenvironment. ABSTRACT: Tumour necrosis factor-α (TNFα) activates NADPH oxidase 1 (Nox1) in vascular smooth muscle cells (VSMCs), producing superoxide (O2•- ) required for subsequent signalling. LRRC8 family proteins A-E comprise volume-regulated anion channels (VRACs). The required subunit LRRC8A physically associates with Nox1, and VRAC activity is required for Nox activity and the inflammatory response to TNFα. VRAC currents are modulated by oxidants, suggesting that channel oxidant sensitivity and proximity to Nox1 may play a physiologically relevant role. In VSMCs, LRRC8C knockdown (siRNA) recapitulated the effects of siLRRC8A, inhibiting TNFα-induced extracellular and endosomal O2•- production, receptor endocytosis, nuclear factor-κB (NF-κB) activation and proliferation. In contrast, siLRRC8D potentiated NF-κB activation. Nox1 co-immunoprecipitated with 8C and 8D, and colocalized with 8D at the plasma membrane and in vesicles. We compared VRAC currents mediated by homomeric and heteromeric LRRC8C and LRRC8D channels expressed in HEK293 cells. The oxidant chloramine T (ChlorT, 1 mM) weakly inhibited 8C, but potently inhibited 8D currents. ChlorT exposure also impaired subsequent current block by the VRAC blocker DCPIB, implicating external sites of oxidation. Substitution of the 8D extracellular loop domains (EL1, EL2) into 8C conferred significantly stronger ChlorT-mediated inhibition of 8C currents. Our results suggest that LRRC8A/C channel activity can be effectively maintained in the oxidized microenvironment expected to result from Nox1 activation at the plasma membrane. Increased ratios of 8D:8C expression may potentially depress inflammatory responses to TNFα. LRRC8A/C channel downregulation represents a novel strategy to reduce TNFα-induced inflammation.

Combined l-citrulline and tetrahydrobiopterin therapy improves NO signaling and ameliorates chronic hypoxia-induced pulmonary hypertension in newborn pigs.

Newborn pigs with chronic hypoxia-induced pulmonary hypertension (PH) have evidence of endothelial nitric oxide synthase (eNOS) uncoupling. In this model, we showed that therapies that promote eNOS coupling, either tetrahydrobiopterin (BH4), a NOS cofactor, or l-citrulline, a NO-l-arginine precursor, inhibit PH. We wanted to determine whether cotreatment with l-citrulline and a BH4 compound, sapropterin dihydrochloride, improves NO signaling and chronic hypoxia-induced PH more markedly than either alone. Normoxic (control) and hypoxic piglets were studied. Some hypoxic piglets received sole treatment with l-citrulline or BH4, or were cotreated with l-citrulline and BH4, from day 3 through day 10 of hypoxia. Catheters were placed for hemodynamic measurements, and pulmonary arteries were dissected to assess eNOS dimer-to-monomer ratios and NO production. In untreated hypoxic piglets, pulmonary vascular resistance (PVR) was higher and NO production and eNOS dimer-to-monomer ratios were lower than in normoxic piglets. Compared with the untreated hypoxic group, PVR was lower in hypoxic piglets cotreated with l-citrulline and BH4 and in those treated with l-citrulline alone but not for those treated solely with BH4. NO production and eNOS dimer-to-monomer ratios were greater for all three treated hypoxic groups compared with the untreated group. Notably, greater improvements in PVR, eNOS dimer-to-monomer ratios, and NO production were found in hypoxic piglets cotreated with l-citrulline and BH4 than in piglets treated with either alone. Cotreatment with l-citrulline and BH4 more effectively improves NO signaling and inhibits chronic hypoxia-induced PH than either treatment alone. Combination therapies may offer enhanced therapeutic capacity for challenging clinical conditions, such as chronic neonatal PH.

Poldip2 knockdown inhibits vascular smooth muscle proliferation and neointima formation by regulating the expression of PCNA and p21.

Polymerase delta-interacting protein 2 (Poldip2) is a multi-functional protein with numerous roles in the vasculature, including the regulation of cell apoptosis and migration, as well as extracellular matrix deposition; however, its role in VSMC proliferation and neointimal formation is unknown. In this study, we investigated the role of Poldip2 in intraluminal wire-injury induced neointima formation and proliferation of vascular smooth muscle cells in vitro and in vivo. Poldip2 expression was observed in the intima and media of human atherosclerotic arteries, where it colocalized with proliferating cell nuclear antigen (PCNA). Wire injury of femoral arteries of Poldip2+/+ mice induced robust neointimal formation after 2 weeks, which was impaired in Poldip2+/‒ mice. PCNA expression was significantly reduced and expression of the cell cycle inhibitor p21 was significantly increased in wire-injured arteries of Poldip2+/‒ animals compared to wild-type controls. No difference was observed in apoptosis. Downregulation of Poldip2 in rat aortic smooth muscle cells significantly reduced serum-induced proliferation and PCNA expression, but upregulated p21 expression. Downregulation of p21 using siRNA reversed the inhibition of proliferation induced by knockdown of Poldip2. These results indicate that Poldip2 plays a critical role in the proliferation of VSMCs.

Lipoprotein modulation of proteinuric renal injury.

High-density lipoprotein (HDL) and its main protein, apolipoprotein AI (apoAI), have established benefits in various cells, but whether these cytoprotective effects of HDL pertain to renal cells is unclear. We investigated the in vitro consequences of exposing damaged podocytes to normal apoAI, HDL, and apoAI mimetic (L-4F), and the in vivo effects of L-4F on kidney and atherosclerotic injury in a podocyte-specific injury model of proteinuria. In vitro, primary mouse podocytes were injured by puromycin aminonucleoside (PAN). Cellular viability, migration, production of reactive oxygen species (ROS), apoptosis, and the underlying signaling pathway were assessed. In vivo, we used a proteinuric model, Nphs1-hCD25 transgenic (NEP25+) mice, which express human CD25 on podocytes. Podocyte injury was induced by using immunotoxin (LMB2) and generated a proteinuric atherosclerosis model, NEP25+:apoE-/- mice, was generated by mating apoE-deficient (apoE-/-) mice with NEP25+ mice. Animals received L-4F or control vehicle. Renal function, podocyte injury, and atherosclerosis were assessed. PAN reduced podocyte viability, migration, and increased ROS production, all significantly lessened by apoAI, HDL, and L-4F. L-4F attenuated podocyte apoptosis and diminished PAN-induced inactivation of Janus family protein kinase-2/signal transducers and activators of transcription 3. In NEP25+ mice, L-4F significantly lessened overall proteinuria, and preserved podocyte expression of synaptopodin and cell density. Proteinuric NEP25+:apoE-/- mice had more atherosclerosis than non-proteinuric apoE-/- mice, and these lesions were significantly decreased by L-4F. Normal human apoAI, HDL, and apoAI mimetic protect against podocyte damage. ApoAI mimetic provides in vivo beneficial effects on podocytes that culminate in reduced albuminuria and atherosclerosis. The results suggest supplemental apoAI/apoAI mimetic may be a novel candidate to lessen podocyte damage and its complications.

Apoptosis signal-regulating kinase 1 activation by Nox1-derived oxidants is required for TNFα receptor endocytosis.

Tumor necrosis factor-α (TNFα) is a proinflammatory cytokine that is closely linked to the development of cardiovascular disease. TNFα activates NADPH oxidase 1 (Nox1) and reactive oxygen species (ROS), including superoxide (O2·-), production extracellularly is required for subsequent signaling in vascular smooth muscle cells (VSMCs). Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein kinase kinase kinase that is activated by oxidation of associated thioredoxin. The role of ASK1 in Nox1-mediated signaling by TNFα is poorly defined. We hypothesized that ASK1 is required for TNFα receptor endocytosis and subsequent inflammatory TNFα signaling. We employed a knockdown strategy to explore the role of ASK1 in TNFα signaling in VSMCs. siRNA targeting ASK1 had no effect on TNFα-induced extracellular O2·- production. However, siASK1 inhibited receptor endocytosis as well as phosphorylation of two endocytosis-related proteins, dynamin1 and caveolin1. Intracellular O2·- production was subsequently reduced, as were other inflammatory signaling steps including NF-κB activation, IL-6 production, inducible nitric oxide synthase and VCAM expression, and VSMC proliferation. Prolonged exposure to TNFα (24 h) increased tumor necrosis factor receptor (TNFR) subtype 1 and 2 expression, and these effects were also attenuated by siASK1. ASK1 coimmunoprecipitated with both Nox1 and the leucine rich repeat containing 8A anion channel, two essential components of the TNFR1 signaling complex. Activation of ASK1 by autophosphorylation at Thr845 occurs following thioredoxin dissociation, and this requires the presence of Nox1. Thus, Nox1 is part of the multiprotein ASK1 signaling complex. In response to TNFα, ASK1 is activated by Nox1-derived oxidants, and this plays a critical role in translating these ROS into a physiologic response in VSMCs. NEW & NOTEWORTHY Apoptosis signal-regulating kinase 1 (ASK1) drives dynamin1 and caveolin1 phosphorylation and TNFα receptor endocytosis. ASK1 modulates TNFα-induced NF-κB activation, survival, and proliferation. ASK1 and NADPH oxidase 1 (Nox1) physically associate in a multiprotein signaling complex. Nox1 is required for TNFα-induced ASK1 activation.

Reactive oxygen species modulate Na+-coupled neutral amino acid transporter 1 expression in piglet pulmonary arterial endothelial cells.

We have previously shown that Na+-coupled neutral amino acid transporter 1 (SNAT1) modulates nitric oxide (NO) production in pulmonary arterial endothelial cells (PAECs) from newborn piglets. Specifically, the ability to increase NO production in response to the l-arginine-NO precursor l-citrulline is dependent on SNAT1 expression. Elucidating factors that regulate SNAT1 expression in PAECs could provide new insights and therapeutic targets relevant to NO production. Our major goals were to determine if reactive oxygen species (ROS) modulate SNAT1 expression in PAECs from newborn piglets and to evaluate the role of NADPH oxidase 1 (NOX1) and uncoupled endothelial NO synthase, enzymatic sources of ROS, in hypoxia-induced increases in SNAT1 expression. Treatment with either H2O2 or xanthine plus xanthine oxidase increased SNAT1 expression in PAECs from newborn piglets cultured under normoxic conditions. Hypoxia-induced increases in SNAT1 expression were inhibited by treatments with the ROS-removing agents catalase and superoxide dismutase, NOX1 siRNA, and the NO synthase inhibitor NG-nitro-l-arginine methyl ester. Both tetrahydropbiopterin (BH4) and l-citrulline, two therapies that decrease ROS by recoupling endothelial NO synthase, reduced the hypoxia-induced increase in SNAT1 expression. BH4 and l-citrulline treatment improved NO production in hypoxic PAECs despite a reduction in SNAT1 expression. In conclusion, SNAT1 expression is modulated by ROS in PAECs from newborn piglets. However, ROS-mediated decreases in SNAT1 expression per se do not implicate a reduction in NO production. Although SNAT1 may be critical to l-citrulline-induced increases in NO production, therapies designed to alter SNAT1 expression may not lead to a concordant change in NO production. NEW & NOTEWORTHY Na+-coupled neutral amino acid transporter 1 (SNAT1) modulates nitric oxide (NO) production in piglet pulmonary arterial endothelial cells. Factors that regulate SNAT1 expression in pulmonary arterial endothelial cells are unclear. Here, we show that ROS-reducing strategies inhibit hypoxia-induced increases in SNAT1 expression. l-Citrulline and tetrahydropbiopterin decrease SNAT1 expression but increase NO production. Although SNAT1 is modulated by ROS, changes in SNAT1 expression may not cause a concordant change in NO production.

Tobacco smoking induces cardiovascular mitochondrial oxidative stress, promotes endothelial dysfunction, and enhances hypertension.

Tobacco smoking is a major risk factor for cardiovascular disease and hypertension. It is associated with the oxidative stress and induces metabolic reprogramming, altering mitochondrial function. We hypothesized that cigarette smoke induces cardiovascular mitochondrial oxidative stress, which contributes to endothelial dysfunction and hypertension. To test this hypothesis, we studied whether the scavenging of mitochondrial H2O2 in transgenic mice expressing mitochondria-targeted catalase (mCAT) attenuates the development of cigarette smoke/angiotensin II-induced mitochondrial oxidative stress and hypertension compared with wild-type mice. Two weeks of exposure of wild-type mice with cigarette smoke increased systolic blood pressure by 17 mmHg, which was similar to the effect of a subpresssor dose of angiotensin II (0.2 mg·kg-1·day-1), leading to a moderate increase to the prehypertensive level. Cigarette smoke exposure and a low dose of angiotensin II cooperatively induced severe hypertension in wild-type mice, but the scavenging of mitochondrial H2O2 in mCAT mice completely prevented the development of hypertension. Cigarette smoke and angiotensin II cooperatively induced oxidation of cardiolipin (a specific biomarker of mitochondrial oxidative stress) in wild-type mice, which was abolished in mCAT mice. Cigarette smoke and angiotensin II impaired endothelium-dependent relaxation and induced superoxide overproduction, which was diminished in mCAT mice. To mimic the tobacco smoke exposure, we used cigarette smoke condensate, which induced mitochondrial superoxide overproduction and reduced endothelial nitric oxide (a hallmark of endothelial dysfunction in hypertension). Western blot experiments indicated that tobacco smoke and angiotensin II reduce the mitochondrial deacetylase sirtuin-3 level and cause hyperacetylation of a key mitochondrial antioxidant, SOD2, which promotes mitochondrial oxidative stress. NEW & NOTEWORTHY This work demonstrates tobacco smoking-induced mitochondrial oxidative stress, which contributes to endothelial dysfunction and development of hypertension. We suggest that the targeting of mitochondrial oxidative stress can be beneficial for treatment of pathological conditions associated with tobacco smoking, such as endothelial dysfunction, hypertension, and cardiovascular diseases.

Sirt3 Impairment and SOD2 Hyperacetylation in Vascular Oxidative Stress and Hypertension.

RATIONALE: Clinical studies have shown that Sirt3 (Sirtuin 3) expression declines by 40% by 65 years of age paralleling the increased incidence of hypertension and metabolic conditions further inactivate Sirt3 because of increased NADH (nicotinamide adenine dinucleotide, reduced form) and acetyl-CoA levels. Sirt3 impairment reduces the activity of a key mitochondrial antioxidant enzyme, superoxide dismutase 2 (SOD2) because of hyperacetylation. OBJECTIVE: In this study, we examined whether the loss of Sirt3 activity increases vascular oxidative stress because of SOD2 hyperacetylation and promotes endothelial dysfunction and hypertension. METHODS AND RESULTS: Hypertension was markedly increased in Sirt3-knockout (Sirt3-/-) and SOD2-depleted (SOD2+/-) mice in response to low dose of angiotensin II (0.3 mg/kg per day) compared with wild-type C57Bl/6J mice. Sirt3 depletion increased SOD2 acetylation, elevated mitochondrial O2· -, and diminished endothelial nitric oxide. Angiotensin II-induced hypertension was associated with Sirt3 S-glutathionylation, acetylation of vascular SOD2, and reduced SOD2 activity. Scavenging of mitochondrial H2O2 in mCAT mice expressing mitochondria-targeted catalase prevented Sirt3 and SOD2 impairment and attenuated hypertension. Treatment of mice after onset of hypertension with a mitochondria-targeted H2O2 scavenger, mitochondria-targeted hydrogen peroxide scavenger ebselen, reduced Sirt3 S-glutathionylation, diminished SOD2 acetylation, and reduced blood pressure in wild-type but not in Sirt3-/- mice, whereas an SOD2 mimetic, (2-[2,2,6,6-tetramethylpiperidin-1-oxyl-4-ylamino]-2-oxoethyl) triphenylphosphonium (mitoTEMPO), reduced blood pressure and improved vasorelaxation both in Sirt3-/- and wild-type mice. SOD2 acetylation had an inverse correlation with SOD2 activity and a direct correlation with the severity of hypertension. Analysis of human subjects with essential hypertension showed 2.6-fold increase in SOD2 acetylation and 1.4-fold decrease in Sirt3 levels, whereas SOD2 expression was not affected. CONCLUSIONS: Our data suggest that diminished Sirt3 expression and redox inactivation of Sirt3 lead to SOD2 inactivation and contributes to the pathogenesis of hypertension.

NOX4 (NADPH Oxidase 4) and Poldip2 (Polymerase δ-Interacting Protein 2) Induce Filamentous Actin Oxidation and Promote Its Interaction With Vinculin During Integrin-Mediated Cell Adhesion.

Objective- Actin cytoskeleton assembly and organization, as a result of focal adhesion (FA) formation during cell adhesion, are dependent on reactive oxygen species and the cellular redox environment. Poldip2 (polymerase δ-interacting protein 2), a novel regulator of NOX4 (NADPH oxidase 4), plays a significant role in reactive oxygen species production and cytoskeletal remodeling. Thus, we hypothesized that endogenous reactive oxygen species derived from Poldip2/NOX4 contribute to redox regulation of actin and cytoskeleton assembly during integrin-mediated cell adhesion. Approach and Results- Using vascular smooth muscle cells, we verified that hydrogen peroxide (H2O2) levels increase during integrin-mediated cell attachment as a result of activation of NOX4. Filamentous actin (F-actin) was oxidized by sulfenylation during cell attachment, with a peak at 3 hours (0.80±0.04 versus 0.08±0.13 arbitrary units at time zero), which was enhanced by overexpression of Poldip2. Depletion of Poldip2 or NOX4 using siRNA, or scavenging of endogenous H2O2 with catalase, inhibited F-actin oxidation by 78±26%, 99±1%, and 98±1%, respectively. To determine the consequence of F-actin oxidation, we examined the binding of F-actin to vinculin, a protein involved in FA complexes that regulates FA maturation. Vinculin binding during cell adhesion as well as migration capacity were inhibited after transfection with actin containing 2 oxidation-resistant point mutations (C272A and C374A). Silencing of Poldip2 or NOX4 also impaired actin-vinculin interaction, which disturbed maturation of FAs and inhibited cell migration. Conclusions- These results suggest that integrin engagement during cell attachment activates Poldip2/Nox4 to oxidize actin, which modulates FA assembly.

Tetrahydrobiopterin oral therapy recouples eNOS and ameliorates chronic hypoxia-induced pulmonary hypertension in newborn pigs.

We previously showed that newborn piglets who develop pulmonary hypertension during exposure to chronic hypoxia have diminished pulmonary vascular nitric oxide (NO) production and evidence of endothelial NO synthase (eNOS) uncoupling (Fike CD, Dikalova A, Kaplowitz MR, Cunningham G, Summar M, Aschner JL. Am J Respir Cell Mol Biol 53: 255-264, 2015). Tetrahydrobiopterin (BH4) is a cofactor that promotes eNOS coupling. Current clinical strategies typically invoke initiating treatment after the diagnosis of pulmonary hypertension, rather than prophylactically. The major purpose of this study was to determine whether starting treatment with an oral BH4 compound, sapropterin dihydrochloride (sapropterin), after the onset of pulmonary hypertension would recouple eNOS in the pulmonary vasculature and ameliorate disease progression in chronically hypoxic piglets. Normoxic (control) and hypoxic piglets were studied. Some hypoxic piglets received oral sapropterin starting on day 3 of hypoxia and continued throughout an additional 7 days of hypoxic exposure. Catheters were placed for hemodynamic measurements, and pulmonary arteries were dissected to assess eNOS dimer-to-monomer ratios (a measure of eNOS coupling), NO production, and superoxide (O2·-) generation. Although higher than in normoxic controls, pulmonary vascular resistance was lower in sapropterin-treated hypoxic piglets than in untreated hypoxic piglets. Consistent with eNOS recoupling, eNOS dimer-to-monomer ratios and NO production were greater and O2·- generation was less in pulmonary arteries from sapropterin-treated than untreated hypoxic animals. When started after disease onset, oral sapropterin treatment inhibits chronic hypoxia-induced pulmonary hypertension at least in part by recoupling eNOS in the pulmonary vasculature of newborn piglets. Rescue treatment with sapropterin may be an effective strategy to inhibit further development of pulmonary hypertension in newborn infants suffering from chronic cardiopulmonary conditions associated with episodes of prolonged hypoxia.