Search

Background Ischemia/reperfusion (I/R) injury causes overproduction of reactive oxygen species, which are the major culprits of oxidative stress that leads to inflammation, apoptosis, myocardial damage, and dysfunction. Bilirubin acts as a potent endogenous antioxidant that is capable of scavenging various reactive oxygen species. We have previously generated bilirubin nanoparticles (BRNPs) consisting of polyethylene glycol–conjugated bilirubin. In this study, we examined the therapeutic effects of BRNPs on myocardial I/R injury in mice. Methods and Results In vivo imaging using fluorophore encapsulated BRNPs showed BRNPs preferentially targeted to the site of I/R injury in the heart. Cardiac I/R surgery was performed by first ligating the left anterior descending coronary artery. After 45 minutes, reperfusion was achieved by releasing the ligation. BRNPs were administered intraperitoneally at 5 minutes before and 24 hours after reperfusion. Mice that received BRNPs showed significant improvements in their cardiac output, assessed by echocardiogram and pressure volume loop measurements, compared with the ones that received vehicle treatment. BRNPs treatment also significantly reduced the myocardial infarct size in mice that underwent cardiac I/R, compared with the vehicle‐treatment group. In addition, BRNPs effectively suppressed reactive oxygen species and proinflammatory factor levels, as well as the amount of cardiac apoptosis. Conclusions Taken together, BRNPs could exert their therapeutic effects on cardiac I/R injury through attenuation of oxidative stress, apoptosis, and inflammation, providing a novel therapeutic modality for myocardial I/R injury.

Background Aldehyde dehydrogenase‐2 (ALDH2), a mitochondrial enzyme, detoxifies reactive aldehydes such as 4‐hydroxy‐2‐nonenal (4HNE). A highly prevalent E487K mutation in ALDH2 (ALDH2*2) in East Asian people with intrinsic low ALDH2 activity is implicated in diabetic complications. 4HNE‐induced cardiomyocyte dysfunction was studied in diabetic cardiac damage; however, coronary endothelial cell (CEC) injury in myocardial ischemia‐reperfusion injury (IRI) in diabetic mice has not been studied. Therefore, we hypothesize that the lack of ALDH2 activity exacerbates 4HNE‐induced CEC dysfunction which leads to cardiac damage in ALDH2*2 mutant diabetic mice subjected to myocardial IRI. Methods and Results Three weeks after diabetes mellitus (DM) induction, hearts were subjected to IRI either in vivo via left anterior descending artery occlusion and release or ex vivo IRI by using the Langendorff system. The cardiac performance was assessed by conscious echocardiography in mice or by inserting a balloon catheter in the left ventricle in the ex vivo model. Just 3 weeks of DM led to an increase in cardiac 4HNE protein adducts and, cardiac dysfunction, and a decrease in the number of CECs along with reduced myocardial ALDH2 activity in ALDH2*2 mutant diabetic mice compared with their wild‐type counterparts. Systemic pretreatment with Alda‐1 (10 mg/kg per day), an activator of both ALDH2 and ALDH2*2, led to a reduction in myocardial infarct size and dysfunction, and coronary perfusion pressure upon cardiac IRI by increasing CEC population and coronary arteriole opening. Conclusions Low ALDH2 activity exacerbates 4HNE‐mediated CEC injury and thereby cardiac dysfunction in diabetic mouse hearts subjected to IRI, which can be reversed by ALDH2 activation.

Background Left ventricular diastolic dysfunction, an early stage in the pathogenesis of heart failure with preserved ejection fraction, is exacerbated by joint exposure to hypertension and obesity; however, the molecular mechanisms involved remain uncertain. The mitochondrial UCP3 (uncoupling protein 3) is downregulated in the heart with obesity. Here, we used a rat model of UCP3 haploinsufficiency (ucp3 +/‐ ) to test the hypothesis that decreased UCP3 promotes left ventricular diastolic dysfunction during hypertension. Methods and Results Ucp3 +/‐ rats and ucp3 +/+ littermates fed a high‐salt diet (HS; 2% NaCl) and treated with angiotensin II (190 ng/kg per min for 28 days) experienced a similar rise in blood pressure (158±4 versus 155±7 mm Hg). However, UCP3 insufficiency worsened diastolic dysfunction according to echocardiographic assessment of left ventricular filling pressures (E/e’; 18.8±1.0 versus 14.9±0.6; P P P +/‐ rats. Histological and molecular analyses further revealed that UCP3 insufficiency accelerated left ventricular concentric remodeling, detrimental interstitial matrix remodeling, and fetal gene reprogramming during hypertension. Moreover, UCP3 insufficiency increased oxidative stress and led to greater impairment of protein kinase G signaling. Conclusions Our findings identified UCP3 insufficiency as a cause for increased incidence of left ventricular diastolic dysfunction during hypertension. The results add further support to the use of antioxidants targeting mitochondrial reactive oxygen species as an adjuvant therapy for preventing heart failure with preserved ejection fraction in individuals with obesity.

Background The loss of endothelial integrity increases the risk of intracerebral hemorrhage during ischemic stroke. Adjunct therapeutic targets for reperfusion in ischemic stroke are in need to prevent blood‐brain barrier disruption. Recently, we have shown that endothelial permeability is mediated by lysophosphatidic acid (LPA), but the role of autotaxin, which produces LPA, remains unclear in stroke. We investigate whether autotaxin/LPA axis regulates blood‐brain barrier integrity after cerebral ischemia. Methods and Results Ischemic stroke was induced in mice by middle cerebral artery occlusion for 90 minutes, followed by 24‐hour reperfusion. The therapeutic efficacy of autotaxin/LPA receptor blockade was evaluated using triphenyl tetrazolium chloride staining, Evans blue permeability, infrared imaging, mass spectrometry, and XF24 analyzer to evaluate blood‐brain barrier integrity, autotaxin activity, and mitochondrial bioenergetics. In our mouse model of ischemic stroke, the mRNA levels of autotaxin were elevated 1.7‐fold following the cerebral ischemia and reperfusion (I/R) group compared with the sham. The enzymatic activity of autotaxin was augmented by 4‐fold in the I/R group compared with the sham. Plasma and brain tissues in I/R group showed elevated LPA levels. The I/R group also demonstrated mitochondrial dysfunction, as evidenced by decreased ( P Conclusions Autotaxin‐LPA signaling blockade attenuates blood‐brain barrier disruption and mitochondrial function following I/R, suggesting targeting this axis could be a new therapeutic approach toward treating ischemic stroke.

Background Age‐related heart diseases are significant contributors to increased morbidity and mortality. Emerging evidence indicates that mitochondria within cardiomyocytes contribute to age‐related increased reactive oxygen species (ROS) generation that plays an essential role in aging‐associated cardiac diseases. Methods and Results The present study investigated differences between ROS production in cardiomyocytes isolated from adult (6 months) and aged (24 months) Fischer 344 rats, and in cardiac tissue of adult (18–65 years) and elderly (>65 years) patients with preserved cardiac function. Superoxide dismutase inhibitable ferricytochrome c reduction assay (1.32±0.63 versus 0.76±0.31 nMol/mg per minute; P =0.001) superoxide and H 2 O 2 production, measured as dichlorofluorescein diacetate fluorescence (1646±428 versus 699±329, P =0.04), were significantly higher in the aged versus adult cardiomyocytes. Similarity in age‐related alteration between rats and humans was identified in mitochondrial‐electron transport chain‐complex‐I‐associated increased oxidative‐stress by MitoSOX fluorescence (53.66±18.58 versus 22.81±12.60; P =0.03) and in 4‐HNE adduct levels (187.54±54.8 versus 47.83±16.7 ng/mg protein, P =0.0063), indicative of increased peroxidation in the elderly. These differences correlated with changes in functional enrichment of genes regulating ROS homeostasis pathways in aged human and rat hearts. Functional merged collective network and pathway enrichment analysis revealed common genes prioritized in human and rat aging‐associated networks that underlay enriched functional terms of mitochondrial complex I and common pathways in the aging human and rat heart. Conclusions Aging sensitizes mitochondrial and extramitochondrial mechanisms of ROS buildup within the heart. Network analysis of the transcriptome highlights the critical elements involved with aging‐related ROS homeostasis pathways common in rat and human hearts as targets.

Background Dietary Mg intake is associated with a decreased risk of developing heart failure, whereas low circulating Mg level is associated with increased cardiovascular mortality. We investigated whether Mg deficiency alone could cause cardiomyopathy. Methods and Results C57BL/6J mice were fed with a low Mg (low‐Mg, 15–30 mg/kg Mg) or a normal Mg (nl‐Mg, 600 mg/kg Mg) diet for 6 weeks. To test reversibility, half of the low‐Mg mice were fed then with nl‐Mg diet for another 6 weeks. Low‐Mg diet significantly decreased mouse serum Mg (0.38±0.03 versus 1.14±0.03 mmol/L for nl‐Mg; P P =0.011). Cellular ATP was decreased significantly in low‐Mg hearts. The changes were accompanied by mitochondrial dysfunction with mitochondrial reactive oxygen species overproduction and membrane depolarization. cMyBPC (cardiac myosin‐binding protein C) was S ‐glutathionylated in low‐Mg mouse hearts. All these changes were normalized with Mg repletion. In vivo (2‐(2,2,6,6‐tetramethylpiperidin‐1‐oxyl‐4‐ylamino)‐2‐oxoethyl)triphenylphosphonium chloride treatment during low‐Mg diet improved cardiac relaxation, increased ATP levels, and reduced S ‐glutathionylated cMyBPC. Conclusions Mg deficiency caused a reversible diastolic cardiomyopathy associated with mitochondrial dysfunction and oxidative modification of cMyBPC. In deficiency states, Mg supplementation may represent a novel treatment for diastolic heart failure.

Background Cardiac arrest (CA) can induce oxidative stress after resuscitation, which causes cellular and organ damage. We hypothesized that post‐resuscitation normoxic therapy would protect organs against oxidative stress and improve oxygen metabolism and survival. We tested the oxygen‐sensitive reactive oxygen species from mitochondria to determine the association with hyperoxia‐induced oxidative stress. Methods and Results Sprague–Dawley rats were subjected to 10‐minute asphyxia‐induced CA with a fraction of inspired O 2 of 0.3 or 1.0 (normoxia versus hyperoxia, respectively) after resuscitation. The survival rate at 48 hours was higher in the normoxia group than in the hyperoxia group (77% versus 28%, P P P P Conclusions Post‐resuscitation normoxic therapy attenuated the oxidative stress in multiple organs and improved post‐CA organ injury, oxygen metabolism, and survival. Additionally, post‐CA hyperoxia increased the mitochondrial reactive oxygen species and activated the antioxidation system.

Background In complex congenital heart disease patients such as those with tetralogy of Fallot, the right ventricle (RV) is subject to pressure overload, leading to RV hypertrophy and eventually RV failure. The mechanisms that promote the transition from stable RV hypertrophy to RV failure are unknown. We evaluated the role of mitochondrial bioenergetics in the development of RV failure. Methods and Results We created a murine model of RV pressure overload by pulmonary artery banding and compared with sham‐operated controls. Gene expression by RNA‐sequencing, oxidative stress, mitochondrial respiration, dynamics, and structure were assessed in pressure overload‐induced RV failure. RV failure was characterized by decreased expression of electron transport chain genes and mitochondrial antioxidant genes (aldehyde dehydrogenase 2 and superoxide dismutase 2) and increased expression of oxidant stress markers (heme oxygenase, 4‐hydroxynonenal). The activities of all electron transport chain complexes decreased with RV hypertrophy and further with RV failure (oxidative phosphorylation: sham 552.3±43.07 versus RV hypertrophy 334.3±30.65 versus RV failure 165.4±36.72 pmol/(s×mL), P Conclusions Pressure overload‐induced RV failure is characterized by decreased transcription and activity of electron transport chain complexes and increased oxidative stress which are associated with decreased energy generation. An improved understanding of the complex processes of energy generation could aid in developing novel therapies to mitigate mitochondrial dysfunction and delay the onset of RV failure.

Background Impaired endothelial function is thought to contribute to the increased cardiovascular risk associated with above‐normal blood pressure (BP). However, the association between endothelial function and BP classified by 2017 American College of Cardiology/American Heart Association guidelines is unknown. Our objective was to determine if endothelial function decreases in midlife/older adults across the 2017 American College of Cardiology/American Heart Association guidelines BP classifications and identify associated mechanisms of action. Methods and Results A retrospective analysis of endothelial function (brachial artery flow‐mediated dilation) from 988 midlife/older adults (aged 50+ years) stratified by BP status (normal BP; elevated BP; stage 1 hypertension; stage 2 hypertension) was performed. Endothelium‐independent dilation (sublingual nitroglycerin), reactive oxygen species–mediated suppression of endothelial function (∆brachial artery flow‐mediated dilation with vitamin C infusion), and endothelial cell and plasma markers of oxidative stress and inflammation were assessed in subgroups. Compared with normal BP (n=411), brachial artery flow‐mediated dilation was 12% ( P =0.04), 15% ( P P P =0.14). Vitamin C infusion increased brachial artery flow‐mediated dilation in those with above‐normal BP ( P ≤0.02) but not normal BP ( P =0.11). Endothelial cell p47 phox ( P P =0.01) were higher in individuals with above‐normal BP. Conclusions Vascular endothelial function is progressively impaired with increasing BP in otherwise healthy adults classified by 2017 American College of Cardiology/American Heart Association guidelines. Impaired endothelial function with above‐normal BP is mediated by excessive reactive oxygen species signaling associated with increased endothelial expression of nicotinamide adenine dinucleotide phosphate oxidase and circulating interleukin‐6.

Background Patients with peripheral artery disease (PAD) undergo frequent episodes of ischemia-reperfusion in lower extremity muscles that may negatively affect mitochondrial health and are associated with impaired mobility. We hypothesized that skeletal muscle from PAD patients will show high mitochondrial DNA heteroplasmy, especially in regions more susceptible to oxidative damage, such as the displacement loop, and that the degree of heteroplasmy will be correlated with the severity of ischemia and mobility impairment. Methods and Results Mitochondrial mutations and deletions and their relative abundance were identified by targeted mitochondrial DNA sequencing in biopsy specimens of gastrocnemius muscle from 33 PAD (ankle brachial index 0.9) subjects aged >= 60 years. The probability of heteroplasmy per DNA base was significantly higher for PAD subjects than non-PAD within each region. In adjusted models, PAD was associated with higher heteroplasmy than non-PAD (P=0.003), but the association was limited to microheteroplasmy, that is heteroplasmy found in 1% to 5% of all mitochondrial genomes (P=0.004). Heteroplasmy in the displacement loop and coding regions were significantly higher for PAD than non-PAD subjects after adjustment for age, sex, race, and diabetes mellitus (P=0.037 and 0.004, respectively). Low mitochondrial damage, defined by both low mitochondrial DNA copy number and low microheteroplasmy, was associated with better walking performance. Conclusions People with PAD have higher low frequency heteroplasmy in gastrocnemius muscle compared with people without PAD. Among people with PAD, those who had evidence of least mitochondrial damage, had better walking performance than those with more mitochondrial damage. Registration URL: http://www.clinicaltrials.gov. Unique identifier: NCT02246660., This work was supported by the National Institute on Aging (R21-AG047510), by the National Heart, Lung, and Blood Institute (R01-HL107510, R01HL109244, R01-HL088589, R01HL122846), the Office of Dietary Supplements, and the Nathan Shock Center for Aging.

Background Oxidative stress and high salt intake could be independent or intertwined risk factors in the origin of hypertension. Kidneys are the major organ to regulate sodium homeostasis and blood pressure and the renal dopamine system plays a pivotal role in sodium regulation during sodium replete conditions. Oxidative stress has been implicated in renal dopamine dysfunction and development of hypertension, especially in salt‐sensitive animal models. Here we show the nexus between high salt intake and oxidative stress causing renal tubular dopamine oxidation, which leads to mitochondrial and lysosomal dysfunction and subsequently causes renal inflammation and hypertension. Methods and Results Male Sprague Dawley rats were divided into the following groups, vehicle (V)—tap water, high salt ( HS )—1% NaCl, L‐buthionine‐sulfoximine ( BSO ), a prooxidant, and HS plus BSO without and with antioxidant resveratrol (R) for 6 weeks. Oxidative stress was significantly higher in BSO and HS + BSO –treated rat compared with vehicle; however, blood pressure was markedly higher in the HS + BSO group whereas an increase in blood pressure in the BSO group was modest. HS + BSO –treated rats had significant renal dopamine oxidation, lysosomal and mitochondrial dysfunction, and increased renal inflammation; however, HS alone had no impact on organelle function or inflammation. Resveratrol prevented oxidative stress, dopamine oxidation, organelle dysfunction, inflammation, and hypertension in BSO and HS + BSO rats. Conclusions These data suggest that dopamine oxidation, especially during increased sodium intake and oxidative milieu, leads to lysosomal and mitochondrial dysfunction and renal inflammation with subsequent increase in blood pressure. Resveratrol, while preventing oxidative stress, protects renal function and mitigates hypertension.

Background Translocation of miR‐181c into cardiac mitochondria downregulates the mitochondrial gene, mt‐ COX 1. miR‐181c/d −/− hearts experience less oxidative stress during ischemia/reperfusion (I/R) and are protected against I/R injury. Additionally, miR‐181c overexpression can increase mitochondrial matrix Ca 2+ ([Ca 2+ ] m ), but the mechanism by which miR‐181c regulates [Ca 2+ ] m is unknown. Methods and Results By RNA sequencing and analysis, here we show that hearts from miR‐181c/d −/− mice overexpress nuclear‐encoded Ca 2+ regulatory and metabolic pathway genes, suggesting that alterations in miR‐181c and mt‐ COX 1 perturb mitochondria‐to‐nucleus retrograde signaling and [Ca 2+ ] m regulation. Quantitative polymerase chain reaction validation of transcription factors that are known to initiate retrograde signaling revealed significantly higher Sp1 (specificity protein) expression in the miR‐181c/d −/− hearts. Furthermore, an association of Sp1 with the promoter region of MICU 1 was confirmed by chromatin immunoprecipitation‐quantitative polymerase chain reaction and higher expression of MICU 1 was found in the miR‐181c/d −/− hearts. Conversely, downregulation of Sp1 by small interfering RNA decreased MICU 1 expression in neonatal mouse ventricular myocytes. Changes in PDH activity provided evidence for a change in [Ca 2+ ] m via the miR‐181c/ MICU 1 axis. Moreover, this mechanism was implicated in the pathology of I/R injury. When MICU 1 was knocked down in the miR‐181c/d −/− heart by lentiviral expression of a short‐hairpin RNA against MICU 1, cardioprotective effects against I/R injury were abrogated. Furthermore, using an in vitro I/R model in miR‐181c/d −/− neonatal mouse ventricular myocytes, we confirmed the contribution of both Sp1 and MICU 1 in ischemic injury. Conclusions miR‐181c regulates mt‐ COX 1, which in turn regulates MICU 1 expression through the Sp1‐mediated mitochondria‐to‐nucleus retrograde pathway. Loss of miR‐181c can protect the heart from I/R injury by modulating [Ca 2+ ] m through the upregulation of MICU 1.

Background Uric acid (UA) is a plasmatic antioxidant that has possible effects on blood pressure. The effects of UA on endothelial function are unclear. We hypothesize that endothelial function is not impaired unless significant UA depletion is achieved through selective xanthine oxidase inhibition with febuxostat and recombinant uricase (rasburicase). Methods and Results Microvascular hyperemia, induced by iontophoresis of acetylcholine and sodium nitroprusside, and heating-induced local hyperemia after iontophoresis of saline and a specific nitric oxide synthase inhibitor were assessed by laser Doppler imaging. Blood pressure and renin-angiotensin system markers were measured, and arterial stiffness was assessed. CRP (C-reactive protein), allantoin, chlorotyrosine/tyrosine ratio, homocitrulline/lysine ratio, myeloperoxidase activity, malondialdehyde, and interleukin-8 were used to characterize inflammation and oxidative stress. Seventeen young healthy men were enrolled in a randomized, double-blind, placebo-controlled, 3-way crossover study. The 3 compared conditions were placebo, febuxostat alone, and febuxostat together with rasburicase. The allantoin (μmol/L)/UA (μmol/L) ratio differed between sessions (P, info:eu-repo/semantics/published

Background Reduced oxygen delivery in congenital heart disease causes delayed brain maturation and white matter abnormalities in utero. No treatment currently exists. Tetrahydrobiopterin ( BH 4) is a cofactor for neuronal nitric oxide synthase. BH 4 availability is reduced upon NOS activation, such as during hypoxic conditions, and leads to toxin production. We hypothesize that BH 4 levels are depleted in the hypoxic brain and that BH4 replacement therapy mitigates the toxic effects of hypoxia on white matter. Methods and Results Transgenic mice were used to visualize oligodendrocytes. Hypoxia was introduced during a period of white matter development equivalent to the human third trimester. BH 4 was administered during hypoxia. BH 4 levels were depleted in the hypoxic brain by direct quantification (n=7–12). The proliferation (n=3–6), apoptosis (n=3–6), and developmental stage (n=5–8) of oligodendrocytes were determined immunohistologically. Total oligodendrocytes increased after hypoxia, consistent with hypoxia‐induced proliferation seen previously; however, mature oligodendrocytes were less prevalent in hypoxia, and there was accumulation of immature oligodendrocytes. BH 4 treatment improved the mature oligodendrocyte number such that it did not differ from normoxia, and accumulation of immature oligodendrocytes was not observed. These results persisted beyond the initial period of hypoxia (n=3–4). Apoptosis increased with hypoxia but decreased with BH 4 treatment to normoxic levels. White matter myelin levels decreased following hypoxia by western blot. BH 4 treatment normalized myelination (n=6–10). Hypoxia worsened sensory‐motor coordination on balance beam tasks, and BH 4 therapy normalized performance (n=5–9). Conclusions Suboptimal BH 4 levels influence hypoxic white matter abnormalities. Repurposing BH 4 for use during fetal brain development may limit white matter dysmaturation in congenital heart disease.

Background Patients resuscitated from cardiac arrest ( CA ) have highly variable neurological, circulatory, and systemic ischemia‐reperfusion injuries. After the initial hypoxic‐ischemic insult, a cascade of immune and inflammatory responses develops and is often fatal. The role of the immune response in pathophysiological characteristics and recovery is not well understood. We studied immune cell activity and its association with outcomes in a cohort of CA survivors. Methods and Results After informed consent, we collected blood samples at intervals over a week after resuscitation from CA . We examined the expression of CD 39 and CD 73 (alias 5′‐nucleotidase), production of tumor necrosis factor‐α, generation of reactive oxygen species, and secretion of vascular endothelial growth factor by circulating myeloid and lymphoid cells, in comparison to cells obtained from control subjects before coronary artery bypass grafting surgery. The number of circulating total and CD 73‐expressing lymphocytes correlated with survival after CA . Incubation of immune cells, obtained from post‐ CA subjects, with AMP , a substrate for CD 73, resulted in inhibition of tumor necrosis factor‐α production and generation of reactive oxygen species. This effect was blocked by adenosine 5′‐(α, β‐methylene) diphosphate, a specific inhibitor of CD 73 and ZM 241385, an A2 adenosine receptor antagonist. We also found that AMP ‐dependent activation of CD 73 induces production of vascular endothelial growth factor. Conclusions CD 73‐expressing lymphocytes mediate cellular protection from inflammation after CA through inhibition of proinflammatory activation of myeloid cells and promotion of vascular endothelial growth factor secretion. The contribution of CD 73 lymphocytes in the regulation of acute inflammation and tissue injury after CA warrants further study.

Background The mechanistic target of rapamycin complex 1 ( mTORC 1) is an important intracellular energy sensor that regulates gene expression and protein synthesis through its downstream signaling components, the S6‐kinase and the ribosomal S6 protein. Recently, signaling arising from mTORC 1 has been implicated in regulation of the cardiovascular system with implications for disease. Here, we examined the contribution of mTORC 1 signaling to the regulation of vascular function. Methods and Results Activation of mTORC 1 pathway in aortic rings with leucine or an adenoviral vector expressing a constitutively active S6‐kinase reduces endothelial‐dependent vasorelaxation in an mTORC 1‐dependent manner without affecting smooth muscle relaxation responses. Moreover, activation of mTORC 1 signaling in endothelial cells increases reactive oxygen species ( ROS ) generation and ROS gene expression resulting in a pro‐oxidant gene environment. Blockade of ROS signaling with Tempol restores endothelial function in vascular rings with increased mTORC 1 activity indicating a crucial interaction between mTORC 1 and ROS signaling. We then tested the role of nuclear factor‐κB transcriptional complex in connecting mTORC 1 and ROS signaling in endothelial cells. Blockade of inhibitor of nuclear factor κ‐B kinase subunit β activity with BMS ‐345541 prevented the increased ROS generation associated with increased mTORC 1 activity in endothelial cells but did not improve vascular endothelial function in aortic rings with increased mTORC 1 and ROS signaling. Conclusions These results implicate mTORC 1 as a critical molecular signaling hub in the vascular endothelium in mediating vascular endothelial function through modulation of ROS signaling.

Background Kallistatin exerts beneficial effects on organ injury by inhibiting oxidative stress and inflammation. However, the role of kallistatin in atherosclerosis is largely unknown. Here, we investigated the role and mechanisms of kallistatin in patients with coronary artery disease ( CAD ), atherosclerotic plaques of apoE −/− mice, and endothelial activation. Methods and Results Plasma kallistatin levels were analyzed in 453 patients at different stages of CAD . Kallistatin levels were significantly lower in patients with CAD and negatively associated with CAD severity and oxidative stress. Human kallistatin cDNA in an adenoviral vector was injected intravenously into apoE −/− mice after partial carotid ligation, with or without nitric oxide synthase inhibitor (N ω ‐nitro‐L‐arginine methyl ester) or sirtuin 1 inhibitor (nicotinamide). Kallistatin gene delivery significantly reduced macrophage deposition, oxidative stress, and plaque volume in the carotid artery, compared with control adenoviral injection. Kallistatin administration increased endothelial nitrous oxide synthase, sirtuin 1, interleukin‐10, superoxide dismutase 2, and catalase expression in carotid plaques. The beneficial effects of kallistatin in mice were mitigated by N ω ‐nitro‐L‐arginine methyl ester or nicotinamide. Furthermore, human kallistatin protein suppressed tumor necrosis factor‐α–induced NADPH oxidase activity and increased endothelial nitrous oxide synthase and sirtuin 1 expression in cultured human endothelial cells. These effects were also abolished by N ω ‐nitro‐L‐arginine methyl ester or nicotinamide. Conclusions This was the first study to demonstrate that reduced plasma kallistatin levels in patients are associated with CAD severity and oxidative stress. Kallistatin treatment prevents carotid atherosclerotic plaque formation in mice by stimulating the sirtuin 1/endothelial nitrous oxide synthase pathway. These findings indicate the potential protective effects of kallistatin on atherosclerosis in human subjects and mouse models.

Background Aortic valve sclerosis (AVSc), the early asymptomatic presentation of calcific aortic valve (AV) disease, affects 25% to 30% of patients aged >65 years. In vitro and ex vivo experiments with antioxidant strategies and antagonists of osteogenic differentiation revealed that AVSc is reversible. In this study, we characterized the underlying changes in the extracellular matrix architecture and valve interstitial cell activation in AVSc and tested in vitro and in vivo the activity of a clinically approved SOD (superoxide dismutase) mimic and redox‐active drug MnTnBuOE‐2‐PyP5+ (BMX‐001). Methods and Results After receiving informed consent, samples from patients with AVSc, AV stenosis, and controls were collected. Uniaxial mechanical stimulation and in vitro studies on human valve interstitial cells were performed. An angiotensin II chronic infusion model was used to impose AV thickening and remodeling. We characterized extracellular matrix structures by small‐angle light scattering, scanning electron microscopy, histology, and mass spectrometry. Diseased human valves showed altered collagen fiber alignment and ultrastructural changes in AVSc, accumulation of oxidized cross‐linking products in AV stenosis, and reversible expression of extracellular matrix regulators ex vivo. We demonstrated that MnTnBuOE‐2‐PyP5+ inhibits human valve interstitial cell activation and extracellular matrix remodeling in a murine model (C57BL/6J) of AVSc by electron microscopy and histology. Conclusions AVSc is associated with architectural remodeling despite marginal effects on the mechanical properties in both human and mice. MnTnBuOE‐2‐PyP5+ controls AV thickening in a murine model of AVSc. Because this compound has been approved recently for clinical use, this work could shift the focus for the treatment of calcific AV disease, moving from AV stenosis to an earlier presentation (AVSc) that could be more responsive to medical therapies.

Background Neurological deficits in hypoxic‐ischemic encephalopathy, even with therapeutic hypothermia, are partially attributed to white matter injury. We theorized that proteasome insufficiency contributes to white matter injury. Methods and Results Neonatal piglets received hypoxia‐ischemia ( HI ) or sham procedure with normothermia, hypothermia, or hypothermia+rewarming. Some received a proteasome activator drug (oleuropein) or white matter–targeted, virus‐mediated proteasome knockdown. We measured myelin oligodendrocyte glycoprotein, proteasome subunit 20S (P20S), proteasome activity, and carbonylated and ubiquitinated protein levels in white matter and cerebral cortex. HI reduced myelin oligodendrocyte glycoprotein levels regardless of temperature, and myelin oligodendrocyte glycoprotein loss was associated with increased ubiquitinated and carbonylated protein levels. Ubiquitinated and carbonyl‐damaged proteins increased in white matter 29 hours after HI during hypothermia to exceed levels at 6 to 20 hours. In cortex, ubiquitinated proteins decreased. Ubiquitinated and carbonylated protein accumulation coincided with lower P20S levels in white matter; P20S levels also decreased in cerebral cortex. However, proteasome activity in white matter lagged behind that in cortex 29 hours after HI during hypothermia. Systemic oleuropein enhanced white matter P20S and protected the myelin, whereas proteasome knockdown exacerbated myelin oligodendrocyte glycoprotein loss and ubiquitinated protein accumulation after HI . At the cellular level, temperature and HI interactively affected macroglial P20S enrichment in subcortical white matter. Rewarming alone increased macroglial P20S immunoreactivity, but this increase was blocked by HI . Conclusions Oxidized and ubiquitinated proteins accumulate with HI ‐induced white matter injury. Proteasome insufficiency may drive this injury. Hypothermia did not prevent myelin damage, protect the proteasome, or preserve oxidized and ubiquitinated protein clearance after HI .

Background Oxidative stress–mediated Ca 2+ /calmodulin‐dependent protein kinase II (Ca MKII) phosphorylation of cardiac ion channels has emerged as a critical contributor to arrhythmogenesis in cardiac pathology. However, the link between mitochondrial‐derived reactive oxygen species (md ROS ) and increased Ca MKII activity in the context of cardiac arrhythmias has not been fully elucidated and is difficult to establish experimentally. Methods and Results We hypothesize that pathological md ROS can cause erratic action potentials through the oxidation‐dependent Ca MKII activation pathway. We further propose that Ca MKII ‐dependent phosphorylation of sarcolemmal slow Na + channels alone is sufficient to elicit early afterdepolarizations. To test the hypotheses, we expanded our well‐established guinea pig cardiomyocyte e xcitation‐ c ontraction coupling, m itochondrial e nergetics, and R OS ‐ i nduced‐ R OS ‐ r elease model by incorporating oxidative Ca MKII activation and Ca MKII ‐dependent Na + channel phosphorylation in silico. Simulations show that md ROS mediated‐Ca MKII activation elicits early afterdepolarizations by augmenting the late Na + currents, which can be suppressed by blocking L‐type Ca 2+ channels or Na + /Ca 2+ exchangers. Interestingly, we found that oxidative Ca MKII activation–induced early afterdepolarizations are sustained even after md ROS has returned to its physiological levels. Moreover, mitochondrial‐targeting antioxidant treatment can suppress the early afterdepolarizations, but only if given in an appropriate time window. Incorporating concurrent md ROS ‐induced ryanodine receptors activation further exacerbates the proarrhythmogenic effect of oxidative Ca MKII activation. Conclusions We conclude that oxidative Ca MKII activation–dependent Na channel phosphorylation is a critical pathway in mitochondria‐mediated cardiac arrhythmogenesis.

Background 8‐Oxo‐2′‐deoxyguanosine (8‐oxo‐2′‐ dG ) is a biomarker of oxidative DNA damage that is associated with cardiovascular disease and premature mortality in the general population. Although oxidative stress has a proven role in cardiovascular complications in diabetes mellitus, evidence for a relationship between plasma 8‐oxo‐2′‐ dG and major cardiovascular outcomes in diabetes mellitus is weak. Methods and Results A case‐cohort study was performed in 3766 participants with prevalent diabetes mellitus in the ADVANCE (Action in Diabetes and Vascular Disease: Preterax and Diamicron Modified Release Controlled Evaluation) trial ( ClinicalTrials.gov number NCT 00145925). The hazard ratios for mortality and major acute cardiovascular events were derived using Cox regression models. During a median of 5 years of follow‐up, 695 (18.4%) participants in this enriched cohort died (including 354 deaths from cardiovascular disease). Individuals with higher levels of 8‐oxo‐2′‐ dG were more likely to die. After adjusting for cardiovascular disease risk factors, the hazard ratio for a 1‐SD increase in plasma 8‐oxo‐2′‐ dG was 1.10 (95% confidence interval, 1.01–1.20; P =0.03). This was driven by an independent association between plasma 8‐oxo‐2′‐ dG and cardiovascular death (hazard ratio, 1.23; 95% confidence interval, 1.10–1.37 [ P dG and noncardiovascular disease death (of which cancer was the major single cause). 8‐Oxo‐2′‐ dG was also not significantly associated with either nonfatal myocardial infarction or nonfatal stroke. Conclusions In adults with type 2 diabetes mellitus, increased levels of 8‐oxo‐2′‐ dG are independently associated with all‐cause mortality and cardiovascular mortality in adults with longstanding type 2 diabetes mellitus who participated in the ADVANCE trial, consistent with the role of oxidative damage in the development and progression of cardiovascular decompensation in diabetes mellitus. Clinical Trial Registration URL : http://www.clinicaltrials.gov . Unique identifier: NCT 00145925.

Background There are several mechanisms, including inflammation, oxidative stress and abnormal calcium homeostasis, involved in the pathogenesis of atrial fibrillation. In diabetes mellitus ( DM ), increased oxidative stress may be attributable to higher xanthine oxidase activity. In this study, we examined the relationship between oxidative stress and atrial electrical and structural remodeling, and calcium handling abnormalities, and the potential beneficial effects of the xanthine oxidase inhibitor allopurinol upon these pathological changes. Methods and Results Ninety rabbits were randomly and equally divided into 3 groups: control, DM, and allopurinol‐treated DM group. Echocardiographic and hemodynamic assessments were performed in vivo. Serum and tissue markers of oxidative stress and atrial fibrosis, including the protein expression were examined. Atrial interstitial fibrosis was evaluated by Masson trichrome staining. I CaL was measured from isolated left atrial cardiomyocytes using voltage‐clamp techniques. Confocal microscopy was used to detect intracellular calcium transients. The Ca 2+ handling protein expression was analyzed by Western blotting. Mitochondrial‐related proteins were analyzed as markers of mitochondrial function. Compared with the control group, rabbits with DM showed left ventricular hypertrophy, increased atrial interstitial fibrosis, oxidative stress and fibrosis markers, I CaL and intracellular calcium transient, and atrial fibrillation inducibility. These abnormalities were alleviated by allopurinol treatment. Conclusions Allopurinol, via its antioxidant effects, reduces atrial mechanical, structural, ion channel remodeling and mitochondrial synthesis abnormalities induced by DM‐related increases in oxidative stress.

Background Subarachnoid hemorrhage is a serious clinical condition that impairs local cerebral blood flow perfusion and consequently initiates neuronal dysfunction. Pressure‐sensitive myogenic vasomotor regulation is an important mechanism involved in the regulation of cerebral blood flow. We hypothesized that extravascular hemolyzed blood enhances arteriolar myogenic constriction, which in vivo may contribute to the reduction of local cerebral blood flow after subarachnoid hemorrhage. Methods and Results Arterioles isolated from the middle cerebral artery ( MCA arterioles) of mice were cannulated in a perfusion chamber. Arteriolar diameters in response to step increases in intraluminal pressure (20–120 mm Hg) were measured in various experimental conditions. In response to increases in intraluminal pressure, all MCA arterioles exhibited myogenic vasoconstrictions. Compared with controls, the pressure‐induced constriction was significantly enhanced in arterioles (in vitro) exposed to extravascular hemolyzed blood or different concentrations of extracellular erythrocyte lysate (1%, 10%, and 20%) for different exposure durations (1–6 hours). The magnitude of enhancement was proportional to the lysate concentration and exposure duration. In in vivo experiments, 10 μL of autologous blood lysate were injected into the mouse subarachnoid space on the surface of the left MCA . Two hours later, MCA arterioles were isolated and left MCA arterioles displayed enhanced myogenic responses compared with the right MCA . The enhanced myogenic response was prevented by scavenge of superoxide in both in vitro and in vivo experiments. Conclusions Extravascular hemolyzed blood, perhaps by promoting vascular production of superoxide, augments myogenic constriction of cerebral arterioles, which plays a crucial role in the subarachnoid hemorrhage–induced cerebral ischemia.

Background Vascular complications and abnormal platelet function contribute to morbidity and mortality in diabetes mellitus. We hypothesized that the Rho‐related GTP ase protein, Rac1, can influence both endothelial and platelet function and might represent a potential novel therapeutic target in diabetes mellitus. Methods and Results We used both in vitro and ex vivo approaches to test the effects of pharmacological inhibition of Rac1 during hyperglycemic condition. We evaluated the effect of NSC 23766, a pharmacological inhibitor of Rac1, on vascular function in diabetic mice and platelet aggregation in diabetic subjects. We demonstrated that the administration of NSC 23766 protects from hyperglycemia‐induced endothelial dysfunction, restoring NO levels, and reduces oxidative stress generated by nicotinamide adenine dinucleotide phosphate oxidase. Mechanistically, we identified Rho‐associated coiled‐coil serine/threonine kinase‐1 as a downstream target of Rac1. Moreover, we reported that during hyperglycemic conditions, human platelets showed hyperactivation of Rac1 and impaired NO release, which were both partially restored after NSC 23766 treatment. Finally, we characterized the antiplatelet effect of NSC 23766 during hyperglycemic conditions, demonstrating the additional role of Rac1 inhibition in reducing platelet aggregation in diabetic patients treated with common antiplatelet drugs. Conclusions Our data suggest that the pharmacological inhibition of Rac1 could represent a novel therapeutic strategy to reduce endothelial dysfunction and platelet hyperaggregation in diabetes mellitus.

Background Germinal matrix hemorrhage ( GMH ) is a leading cause of mortality and lifelong morbidity in preterm infants. Posthemorrhagic hydrocephalus ( PHH ) is a common complication of GMH . A sodium‐coupled bicarbonate exchanger ( NCBE ) encoded by solute carrier family 4 member 10 gene is expressed on the choroid plexus basolateral membrane and may play a role in cerebrospinal fluid production and the development of PHH . Following GMH , iron degraded from hemoglobin has been linked to PHH . Choroid plexus epithelial cells also contain iron‐responsive element‐binding proteins ( IRP s), IRP 1, and IRP 2 that bind to mRNA iron‐responsive elements. The present study aims to resolve the following issues: (1) whether the expression of NCBE is regulated by IRP s; (2) whether NCBE regulates the formation of GMH ‐induced hydrocephalus; and (3) whether inhibition of NCBE reduces PHH development. Methods and Results GMH model was established in P7 rat pups by injecting bacterial collagenase into the right ganglionic eminence. Another group received iron trichloride injections instead of collagenase. Deferoxamine was administered intraperitoneally for 3 consecutive days after GMH /iron trichloride. Solute carrier family 4 member 10 small interfering RNA or scrambled small interfering RNA was administered by intracerebroventricular injection 24 hours before GMH and followed with an injection every 7 days over 21 days. NCBE expression increased while IRP 2 expression decreased after GMH /iron trichloride. Deferoxamine ameliorated both the GMH ‐induced and iron trichloride–induced decrease of IRP 2 and decreased NCBE expressions. Deferoxamine and solute carrier family 4 member 10 small interfering RNA improved cognitive and motor functions at 21 to 28 days post GMH and reduced cerebrospinal fluid production as well as the degree of hydrocephalus at 28 days after GMH . Conclusions Targeting iron‐induced overexpression of NCBE may be a translatable therapeutic strategy for the treatment of PHH following GMH .

Background The matrine‐type alkaloids are bioactive components extracted from Sophora flavescens , which is used in treatment of diabetes mellitus in traditional Chinese medicine. Advanced glycation end products mediate diabetic vascular complications. This study was aimed to investigate the protective effects and molecular mechanisms of matrine‐type alkaloids on advanced glycation end products–induced reactive oxygen species–mediated endothelial apoptosis. Methods and Results Rats aorta and cultured rat aortic endothelial cells were exposed to advanced glycation end products. Matrine‐type alkaloids, p38 mitogen‐activated protein kinase (MAPK) inhibitor, and small interference RNA s against p38 MAPK kinases MAPK kinase kinase ( MKK )3 and MKK 6 were administrated. Intracellular reactive oxygen species production, cell apoptosis, phosphorylation of MKK s/p38 MAPK, and expression levels of heme oxygenase/ NADPH quinone oxidoreductase were assessed. The nuclear factor erythroid 2‐related factor 2 nuclear translocation and the binding activity of nuclear factor erythroid 2‐related factor 2 with antioxidant response element were also evaluated. Matrine‐type alkaloids suppressed intracellular reactive oxygen species production and inhibited endothelial cell apoptosis in vivo and in vitro by recovering phosphorylation of MKK 3/6 and p38 MAPK , nuclear factor erythroid 2‐related factor 2 nuclear translocation, and antioxidant response element binding activity, as well as the expression levels of heme oxygenase/ NADPH quinone oxidoreductase. p38 MAPK inhibitor treatment impaired the effects of matrine‐type alkaloids in vivo and in vitro. MKK 3/6 silencing impaired the effects of matrine‐type alkaloids in vitro. Conclusions Matrine‐type alkaloids exert endothelial protective effects against advanced glycation end products induced reactive oxygen species–mediated apoptosis by targeting MKK 3/6 and enhancing their phosphorylation.

Background Pulmonary arterial remodeling, a main characteristic of hypoxic pulmonary hypertension, can gradually reverse once oxygen has been restored. Previous studies documented that apoptosis increased markedly during the reversal of remodeled pulmonary arteries, but the types of cells and mechanisms related to the apoptosis have remained elusive. This study aimed to determine whether pulmonary artery smooth muscle cell ( PASMC )‐specific apoptosis was involved in the reoxygenation‐induced reversal of hypoxic pulmonary arterial remodeling and elucidate the underlying mechanism. Methods and Results Hypoxic pulmonary hypertension was induced in adult male Sprague‐Dawley rats (n=6/group) by chronic hypobaric hypoxia. and the hypoxic pulmonary hypertension rats were then transferred to a normoxia condition. During reoxygenation, hypoxia‐induced pulmonary arterial remodeling gradually reversed. The reversal of remodeled pulmonary arteries was associated with increased H 2 O 2 and with changes in lung expression of cleaved caspase3/ PARP , Bax, and Bcl‐2, consistent with increased apoptosis. The PASMC apoptosis, in particular, increased remarkably during this reversal. In vitro, reoxygenation induced the apoptosis of cultured rat primary PASMC s accompanied by increased mitochondrial reactive oxygen species, mitochondrial dysfunction, and the release of cytochrome C from mitochondria to cytoplasm. Clearance of reactive oxygen species alleviated mitochondrial dysfunction as well as the release of cytochrome C and, finally, decreased PASMC apoptosis. Conclusions Reoxygenation‐induced apoptosis of PASMC s is implicated in the reversal of hypoxic pulmonary arterial remodeling, which may be attributed to the mitochondrial reactive oxygen species–mediated mitochondrial dysfunction.

Background Atherosclerosis progression during aging culminates in the development of vulnerable plaques, which may increase the risk of cardiovascular events. Increased generation and/or decreased scavenging of reactive oxygen species in the vascular wall are major contributors to atherogenesis. We previously showed that superoxide dismutase 2 deficiency increased vascular oxidative stress and reduced aortic compliance in aged wild‐type mice and that young Apoe −/− / Sod2 +/− had increased mitochondrial DNA damage and atherosclerosis versus young Apoe −/− mice. Here we investigated the effects of superoxide dismutase 2 deficiency on atherosclerosis progression and plaque morphology in middle‐aged Apoe −/− mice. Methods and Results Compared with Apoe −/− , middle‐aged Apoe −/− / Sod2 +/− mice had increased vascular wall reactive oxygen species ( P P Apoe −/− / Sod2 +/− mice had an increased necrotic core with higher inflammatory cell infiltration, a thinned fibrous cap with depleted smooth muscle content, and intraplaque hemorrhage. In addition, the plaque shoulder area had higher levels of calpain‐2, caspase‐3, and matrix metalloproteinase‐2 in intimal smooth muscle cells and depleted fibrous cap collagen. Targeting mitochondrial reactive oxygen species with Mito TEMPO attenuated features of atherosclerotic plaque vulnerability in middle‐aged Apoe −/− / Sod2 +/− mice by lowering expression of calpain‐2, caspase‐3, and matrix metalloproteinase‐2 and decreasing smooth muscle cell apoptosis and matrix degradation. Conclusions Enhanced mitochondrial oxidative stress under hyperlipidemic conditions in aging induces plaque instability, in part by increasing smooth muscle cell apoptosis, necrotic core expansion, and matrix degradation. Targeting mitochondrial reactive oxygen species or its effectors may be a viable therapeutic strategy to prevent aging‐associated and oxidative stress–related atherosclerosis complications.

Background Emerging evidence indicates that impaired angiogenesis may contribute to hypertension‐induced cardiac remodeling. The nicotinamide adenine dinucleotide–dependent deacetylase Sirtuin 3 ( SIRT 3) has the potential to modulate angiogenesis, but this has not been confirmed. As such, the aim of this study was to examine the relationship between SIRT 3‐mediated angiogenesis and cardiac remodeling. Methods and Results Our experiments were performed on SIRT 3 knockout and age‐matched wild‐type mice infused with angiotensin II (1400 ng/kg per minute) or saline for 14 days. After angiotensin II infusion, SIRT 3 knockout mice developed more severe microvascular rarefaction and functional hypoxia in cardiac tissues compared with wild‐type mice. These events were concomitant with mitochondrial dysfunction and enhanced collagen I and collagen III expression, leading to cardiac fibrosis. Silencing SIRT 3 facilitated angiotensin II –induced aberrant Pink/Parkin acetylation and impaired mitophagy, while excessive mitochondrial reactive oxygen species generation limited angiogenic capacity in primary mouse cardiac microvascular endothelial cells. Moreover, SIRT 3 overexpression in cardiac microvascular endothelial cells enhanced Pink/Parkin‐mediated mitophagy, attenuated mitochondrial reactive oxygen species generation, and restored vessel sprouting and tube formation. In parallel, endothelial cell–specific SIRT 3 transgenic mice showed decreased fibrosis, as well as improved cardiac function and microvascular network, compared with wild‐type mice with similar stimuli. Conclusions Collectively, these findings suggest that SIRT 3 could promote angiogenesis through attenuating mitochondrial dysfunction caused by defective mitophagy.

Background Mitochondrial Ca 2+ homeostasis is fundamental to the regulation of mitochondrial reactive oxygen species ( ROS ) generation and adenosine triphosphate production. Recently, transient receptor potential channel, canonical type 3 ( TRPC 3), has been shown to localize to the mitochondria and to play a role in maintaining mitochondrial calcium homeostasis. Inhibition of TRPC 3 attenuates vascular calcium influx in spontaneously hypertensive rats ( SHR s). However, it remains elusive whether mitochondrial TRPC 3 participates in hypertension by increasing mitochondrial calcium handling and ROS production. Methods and Results In this study we demonstrated increased TRPC 3 expression in purified mitochondria in the vasculature from SHR s, which facilitates enhanced mitochondrial calcium uptake and ROS generation compared with Wistar‐Kyoto rats. Furthermore, inhibition of TRPC 3 by its specific inhibitor, Pyr3, significantly decreased the vascular mitochondrial ROS production and H 2 O 2 synthesis and increased adenosine triphosphate content. Administration of telmisartan can improve these abnormalities. This beneficial effect was associated with improvement of the mitochondrial respiratory function through recovering the activity of pyruvate dehydrogenase in the vasculature of SHR s. In vivo, chronic administration of telmisartan suppressed TRPC 3‐mediated excessive mitochondrial ROS generation and vasoconstriction in the vasculature of SHR s. More importantly, TRPC 3 knockout mice exhibited significantly ameliorated hypertension through reduction of angiotensin II –induced mitochondrial ROS generation. Conclusions Together, we give experimental evidence for a potential mechanism by which enhanced TRPC 3 activity at the cytoplasmic and mitochondrial levels contributes to redox signaling and calcium dysregulation in the vasculature from SHR s. Angiotensin II or telmisartan can regulate [Ca 2+ ] mito , ROS production, and mitochondrial energy metabolism through targeting TRPC 3.

Background Sex‐related differences in cardiac function and iron metabolism exist in humans and experimental animals. Male patients and preclinical animal models are more susceptible to cardiomyopathies and heart failure. However, whether similar differences are seen in iron‐overload cardiomyopathy is poorly understood. Methods and Results Male and female wild‐type and hemojuvelin‐null mice were injected and fed with a high‐iron diet, respectively, to develop secondary iron overload and genetic hemochromatosis. Female mice were completely protected from iron‐overload cardiomyopathy, whereas iron overload resulted in marked diastolic dysfunction in male iron‐overloaded mice based on echocardiographic and invasive pressure‐volume analyses. Female mice demonstrated a marked suppression of iron‐mediated oxidative stress and a lack of myocardial fibrosis despite an equivalent degree of myocardial iron deposition. Ovariectomized female mice with iron overload exhibited essential pathophysiological features of iron‐overload cardiomyopathy showing distinct diastolic and systolic dysfunction, severe myocardial fibrosis, increased myocardial oxidative stress, and increased expression of cardiac disease markers. Ovariectomy prevented iron‐induced upregulation of ferritin, decreased myocardial SERCA2a levels, and increased NCX1 levels. 17β‐Estradiol therapy rescued the iron‐overload cardiomyopathy in male wild‐type mice. The responses in wild‐type and hemojuvelin‐null female mice were remarkably similar, highlighting a conserved mechanism of sex‐dependent protection from iron‐overload‐mediated cardiac injury. Conclusions Male and female mice respond differently to iron‐overload‐mediated effects on heart structure and function, and females are markedly protected from iron‐overload cardiomyopathy. Ovariectomy in female mice exacerbated iron‐induced myocardial injury and precipitated severe cardiac dysfunction during iron‐overload conditions, whereas 17β‐estradiol therapy was protective in male iron‐overloaded mice.

Background Bioavailability of nitric oxide ( NO ) and hydrogen sulfide (H 2 S) is reduced in heart failure ( HF ). Recent studies suggest cross‐talk between NO and H 2 S signaling. We previously reported that sodium nitrite (Na NO 2 ) ameliorates myocardial ischemia‐reperfusion injury and HF. Nuclear factor‐erythroid‐2‐related factor 2 (Nrf2) regulates the antioxidant proteins expression and is upregulated by H 2 S. We examined the Na NO 2 effects on endogenous H 2 S bioavailability and Nrf2 activation in mice subjected to ischemia‐induced chronic heart failure (CHF). Methods and Results Mice underwent 60 minutes of left coronary artery occlusion and 4 weeks of reperfusion. Na NO 2 (165 μg/kgic) or vehicle was administered at reperfusion and then in drinking water (100 mg/L) for 4 weeks. Left ventricular (LV), ejection fraction (EF), LV end diastolic ( LVEDD ) and systolic dimensions ( LVESD ) were determined at baseline and at 4 weeks of reperfusion. Myocardial tissue was analyzed for oxidative stress and respective gene/protein‐related assays. We found that Na NO 2 therapy preserved LVEF, LVEDD and LVSD at 4 weeks during ischemia‐induced HF. Myocardial malondialdehyde and protein carbonyl content were significantly reduced in Na NO 2 ‐treated mice as compared to vehicle, suggesting a reduction in oxidative stress. Na NO 2 therapy markedly increased expression of Cu,Zn‐superoxide dismutase, catalase, and glutathione peroxidase during 4 weeks of reperfusion. Furthermore, Na NO 2 upregulated the activity of Nrf2, as well as H 2 S‐producing enzymes, and ultimately increased H 2 S bioavailability in ischemia‐induced CHF in mice as compared with vehicle. Conclusions Our results demonstrate that Na NO 2 therapy significantly improves LV function via increasing H 2 S bioavailability, Nrf2 activation, and antioxidant defenses.

Background Zofenopril, a sulfhydrylated angiotensin‐converting enzyme inhibitor (ACEI), reduces mortality and morbidity in infarcted patients to a greater extent than do other ACEIs. Zofenopril is a unique ACE I that has been shown to increase hydrogen sulfide (H 2 S) bioavailability and nitric oxide ( NO ) levels via bradykinin‐dependent signaling. Both H 2 S and NO exert cytoprotective and antioxidant effects. We examined zofenopril effects on H 2 S and NO bioavailability and cardiac damage in murine and swine models of myocardial ischemia/reperfusion ( I /R) injury. Methods and Results Zofenopril (10 mg/kg PO ) was administered for 1, 8, and 24 hours to establish optimal dosing in mice. Myocardial and plasma H 2 S and NO levels were measured along with the levels of H 2 S and NO enzymes (cystathionine β‐synthase, cystathionine γ‐lyase, 3‐mercaptopyruvate sulfur transferase, and endothelial nitric oxide synthase). Mice received 8 hours of zofenopril or vehicle pretreatment followed by 45 minutes of ischemia and 24 hours of reperfusion. Pigs received placebo or zofenopril (30 mg/daily orally) 7 days before 75 minutes of ischemia and 48 hours of reperfusion. Zofenopril significantly augmented both plasma and myocardial H 2 S and NO levels in mice and plasma H 2 S (sulfane sulfur) in pigs. Cystathionine β‐synthase, cystathionine γ‐lyase, 3‐mercaptopyruvate sulfur transferase, and total endothelial nitric oxide synthase levels were unaltered, while phospho‐endothelial nitric oxide synthase 1177 was significantly increased in mice. Pretreatment with zofenopril significantly reduced myocardial infarct size and cardiac troponin I levels after I /R injury in both mice and swine. Zofenopril also significantly preserved ischemic zone endocardial blood flow at reperfusion in pigs after I/R. Conclusions Zofenopril‐mediated cardioprotection during I /R is associated with an increase in H 2 S and NO signaling.

Background Recent studies have failed to establish a causal relationship between high‐density lipoprotein cholesterol levels ( HDL ‐C) and cardiovascular disease ( CVD ), shifting focus to other HDL measures. We previously reported that smaller/denser HDL levels are protective against cerebrovascular disease. This study sought to determine which of small+medium HDL particle concentration ( HDL ‐P) or large HDL ‐P was more strongly associated with carotid intima‐media thickening ( cIMT ) in an ethnically diverse cohort. Methods and Results In cross‐sectional analyses of participants from the Multi Ethnic Study of Atherosclerosis ( MESA ), we evaluated the associations of nuclear magnetic resonance spectroscopy–measured small+medium versus large HDL ‐P with cIMT measured in the common and internal carotid arteries, through linear regression. After adjustment for CVD confounders, low‐density lipoprotein cholesterol ( LDL ‐C), HDL ‐C, and small+medium HDL ‐P remained significantly and inversely associated with common (coefficient=−1.46 μm; P =0.00037; n=6512) and internal cIMT (coefficient=−3.82 μm; P =0.0051; n=6418) after Bonferroni correction for 4 independent tests (threshold for significance=0.0125; α=0.05/4). Large HDL ‐P was significantly and inversely associated with both cIMT outcomes before HDL ‐C adjustment; however, after adjustment for HDL ‐C, the association of large HDL ‐P with both common (coefficient=1.55 μm; P =0.30; n=6512) and internal cIMT (coefficient=4.84 μm; P =0.33; n=6418) was attenuated. In a separate sample of 126 men, small/medium HDL ‐P was more strongly correlated with paraoxonase 1 activity ( r p =0.32; P =0.00023) as compared to both total HDL ‐P ( r p =0.27; P =0.0024) and large HDL ‐P ( r p =0.02; P =0.41) measures. Conclusions Small+medium HDL ‐P is significantly and inversely correlated with cIMT measurements. Correlation of small+medium HDL ‐P with cardioprotective paraoxonase 1 activity may reflect a functional aspect of HDL responsible for this finding.

Background Nitric oxide synthase uncoupling occurs under conditions of oxidative stress modifying the enzyme's function so it generates superoxide rather than nitric oxide. Nitric oxide synthase uncoupling occurs with chronic pressure overload, and both are ameliorated by exogenous tetrahydrobiopterin ( BH 4)—a cofactor required for normal nitric oxide synthase function—supporting a pathophysiological link. Genetically augmenting BH 4 synthesis in endothelial cells fails to replicate this benefit, indicating that other cell types dominate the effects of exogenous BH 4 administration. We tested whether the primary cellular target of BH 4 is the cardiomyocyte or whether other novel mechanisms are invoked. Methods and Results Mice with cardiomyocyte‐specific overexpression of GTP cyclohydrolase 1 ( mGCH 1) and wild‐type littermates underwent transverse aortic constriction. The mGCH 1 mice had markedly increased myocardial BH 4 and, unlike wild type, maintained nitric oxide synthase coupling after transverse aortic constriction; however, the transverse aortic constriction–induced abnormalities in cardiac morphology and function were similar in both groups. In contrast, exogenous BH 4 supplementation improved transverse aortic constricted hearts in both groups, suppressed multiple inflammatory cytokines, and attenuated infiltration of inflammatory macrophages into the heart early after transverse aortic constriction. Conclusions BH 4 protection against adverse remodeling in hypertrophic cardiac disease is not driven by its prevention of myocardial nitric oxide synthase uncoupling, as presumed previously. Instead, benefits from exogenous BH 4 are mediated by a protective effect coupled to suppression of inflammatory pathways and myocardial macrophage infiltration.

Background Persistent thromboxane ( TX ) generation while receiving aspirin therapy is associated with an increased risk of cardiovascular events. The Reduction in Graft Occlusion Rates ( RIGOR ) study found that aspirin‐insensitive TXA 2 generation, indicated by elevated urine 11‐dehydro‐ TXB 2 ( UTXB 2 ) 6 months after coronary artery bypass graft surgery, was a potent risk factor for vein graft thrombosis and originated predominantly from nonplatelet sources. Our goal was to identify risks factors for nonplatelet TXA 2 generation. Methods and Results Multivariable modeling was performed by using clinical and laboratory variables obtained from 260 RIGOR subjects with verified aspirin‐mediated inhibition of platelet TXA 2 generation. The strongest variable associated with UTXB 2 6 months after surgery, accounting for 47.2% of the modeled effect, was urine 8‐iso‐prostaglandin ( PG)F 2α , an arachidonic acid metabolite generated nonenzymatically by oxidative stress (standardized coefficient 0.442, P UTXB 2 ( P PGF 2α correlated with risk of vein graft occlusion (odds ratio 1.67, P =0.001) but was not independent of UTXB 2 . In vitro studies revealed that endothelial cells generate TXA 2 in response to oxidative stress and direct exposure to 8‐iso‐ PGF 2α . Conclusions Oxidative stress–induced formation of 8‐iso‐ PGF 2α is strongly associated with nonplatelet thromboxane formation and early vein graft thrombosis after coronary artery bypass graft surgery. The endothelium is potentially an important source of oxidative stress–induced thromboxane generation. These findings suggest therapies that reduce oxidative stress could be useful in reducing cardiovascular risks associated with aspirin‐insensitive thromboxane generation.

Background Perturbed balance between NO and O 2 •− . (ie, NO/redox imbalance) is central in the pathobiology of diabetes‐induced vascular dysfunction. We examined whether stimulation of β 3 adrenergic receptors (β 3 ARs), coupled to endothelial nitric oxide synthase (eNOS) activation, would re‐establish NO/redox balance, relieve oxidative inhibition of the membrane proteins eNOS and Na + ‐K + (NK) pump, and improve vascular function in a new animal model of hyperglycemia. Methods and Results We established hyperglycemia in male White New Zealand rabbits by infusion of S961, a competitive high‐affinity peptide inhibitor of the insulin receptor. Hyperglycemia impaired endothelium‐dependent vasorelaxation by “uncoupling” of eNOS via glutathionylation (eNOS‐GSS) that was dependent on NADPH oxidase activity. Accordingly, NO levels were lower while O 2 •− levels were higher in hyperglycemic rabbits. Infusion of the β 3 AR agonist CL316243 (CL) decreased eNOS‐GSS, reduced O 2 •− , restored NO levels, and improved endothelium‐dependent relaxation. CL decreased hyperglycemia‐induced NADPH oxidase activation as suggested by co‐immunoprecipitation experiments, and it increased eNOS co‐immunoprecipitation with glutaredoxin‐1, which may reflect promotion of eNOS de‐glutathionylation by CL. Moreover, CL reversed hyperglycemia‐induced glutathionylation of the β 1 NK pump subunit that causes NK pump inhibition, and improved K + ‐induced vasorelaxation that reflects enhancement in NK pump activity. Lastly, eNOS‐GSS was higher in vessels of diabetic patients and was reduced by CL, suggesting potential significance of the experimental findings in human diabetes. Conclusions β 3 AR activation restored NO/redox balance and improved endothelial function in hyperglycemia. β 3 AR agonists may confer protection against diabetes‐induced vascular dysfunction.

Background The homologous to the E6‐AP carboxyl terminus (HECT) –type ubiquitin E3 ligase ITCH is an enzyme that plays a pivotal role in posttranslational modification by ubiquitin proteasomal protein degradation. Thioredoxin‐interacting protein ( TXNIP ) is a negative regulator of the thioredoxin system and an endogenous reactive oxygen species scavenger. In the present study, we focused on the functional role of ubiquitin E3 ligase ITCH and its interaction with TXNIP to elucidate the mechanism of cardiotoxicity induced by reactive oxygen species , such as doxorubicin and hydrogen peroxide. Methods and Results Protein interaction between TXNIP and ITCH in cardiomyocyte was confirmed by immunoprecipitation assays. Overexpression of ITCH increased proteasomal TXNIP degradation and augmented thioredoxin activity, leading to inhibition of reactive oxygen species generation, p38 MAPK , p53, and subsequent intrinsic pathway cardiomyocyte apoptosis in reactive oxygen species –induced cardiotoxicity. Conversely, knockdown of ITCH using small interfering RNA inhibited TXNIP degradation and resulted in a subsequent increase in cardiomyocyte apoptosis. Next, we generated a transgenic mouse with cardiac‐specific overexpression of ITCH , called the ITCH ‐Tg mouse. The expression level of TXNIP in the myocardium in ITCH ‐Tg mice was significantly lower than WT littermates. In ITCH ‐Tg mice, cardiac dysfunction and remodeling were restored compared with WT littermates after doxorubicin injection and myocardial infarction surgery. Kaplan–Meier analysis revealed that ITCH ‐Tg mice had a higher survival rate than WT littermates after doxorubicin injection and myocardial infarction surgery. Conclusion We demonstrated, for the first time, that ITCH targets TXNIP for ubiquitin‐proteasome degradation in cardiomyocytes and ameliorates reactive oxygen species –induced cardiotoxicity through the thioredoxin system.

Background NOX ‐2, the catalytic subunit of NADPH oxidase, has a key role in the formation of reactive oxidant species and is implicated in impairing flow‐mediated dilation ( FMD ). Dark chocolate exerts artery dilatation via down‐regulating NOX 2‐mediated oxidative stress. The aim of this study was to investigate whether dark chocolate improves walking autonomy in peripheral artery disease ( PAD ) patients via an oxidative stress‐mediated mechanism. Methods and Results FMD , serum levels of isoprostanes, nitrite/nitrate (NOx) and sNOX 2‐dp, a marker of blood NOX 2 activity, maximal walking distance ( MWD ) and maximal walking time ( MWT ) were studied in 20 PAD patients (14 males and 6 females, mean age: 69±9 years) randomly allocated to 40 g of dark chocolate (>85% cocoa) or 40 g of milk chocolate (≤35% cocoa) in a single blind, cross‐over design. The above variables were assessed at baseline and 2 hours after chocolate ingestion. Dark chocolate intake significantly increased MWD (+11%; P MWT (+15%; P P P =0.01) and sNOX 2‐dp (−37%; P MWD was independently associated with Δ of MWT ( P P =0.018). In vitro study demonstrated that HUVEC incubated with a mixture of polyphenols significantly increased nitric oxide ( P P VCAM 1 ( P Conclusion In PAD patients dark but not milk chocolate acutely improves walking autonomy with a mechanism possibly related to an oxidative stress‐mediated mechanism involving NOX 2 regulation. Clinical Trial Registration URL : http://www.clinicaltrials.gov . Unique identifier: NCT 01947712.

Background A sedentary lifestyle is an independent risk factor for cardiovascular disease and exercise has been shown to ameliorate this risk. Inactivity is associated with a loss of muscle mass, which is also reversed with isometric exercise training. The relationship between muscle mass and vascular function is poorly defined. The aims of the current study were to determine whether increasing muscle mass by genetic deletion of myostatin, a negative regulator of muscle growth, can influence vascular function in mesenteric arteries from obese db/db mice. Methods and Results Myostatin expression was elevated in skeletal muscle of obese mice and associated with reduced muscle mass (30% to 50%). Myostatin deletion increased muscle mass in lean (40% to 60%) and obese (80% to 115%) mice through increased muscle fiber size ( P NO ) synthase inhibitor l ‐ NG ‐nitroarginine methyl ester ( l ‐ NAME) . Prostacyclin ( PGI 2 )‐ and endothelium‐derived hyperpolarizing factor ( EDHF)‐ mediated vasodilation were preserved in obese mice and unaffected by myostatin deletion. Reactive oxygen species) was elevated in the mesenteric endothelium of obese mice and down‐regulated by deletion of myostatin in obese mice. Impaired vasodilation in obese mice was improved by NADPH oxidase inhibitor ( GKT 136901). Treatment with sepiapterin, which increases levels of tetrahydrobiopterin, improved vasodilation in obese mice, an improvement blocked by l ‐ NAME . Conclusions Increasing muscle mass by genetic deletion of myostatin improves NO‐ , but not PGI 2 ‐ or EDHF‐ mediated vasodilation in obese mice; this vasodilation improvement is mediated by down‐regulation of superoxide.

Background Clinical studies suggest that hypercholesterolemia may cause ageing in hematopoietic stem cells ( HSC s) because ageing‐associated alterations were found in peripheral blood cells and their bone marrow residing precursors in patients with advanced atherosclerosis. We hypothesized that hypercholesterolemia induces oxidant stress in hematopoietic stems cells that accelerates their ageing. Methods and Results Here we show that HSC s from ApoE −/− mice, as well as HSC s from C57Bl/6 mice fed a high cholesterol diet ( HCD ) accumulated ox LDL and had greater ROS levels. In accordance, the expression pattern of the genes involved in ROS metabolism changed significantly in HSC s from ApoE −/− mice. Hypercholesterolemia caused a significant reduction in phenotypically defined long‐term HSC compartment, telomere length, and repopulation capacity of KTLS cells, indicating accelerated ageing in these cells. Gene array analysis suggested abnormal cell cycle status, and the key cell cycle regulators including p19 ARF , p27 Kip1 and p21 Waf1 were upregulated in KTLS cells from hypercholesterolemic mice. These effects were p38‐dependent and reversed in vivo by treatment of hypercholesterolemic mice with antioxidant N ‐acetylcysteine. The oxidant stress also caused aberrant expression of Notch1 that caused loss of quiescence and proliferation leading to the expansion of KTLS compartment in hypercholesterolemic mice. Conclusion Taken together, we provide evidence that hypercholesterolemia can cause oxidant stress that accelerates the ageing and impairs the reconstitution capacity of HSC s.

Background Ischemia–reperfusion (I/R) studies have implicated oxidant stress, the mitochondrial permeability transition pore ( mPTP ), and poly(ADP‐ribose) polymerase ( PARP ) as contributing factors in myocardial cell death. However, the interdependence of these factors in the intact, blood‐perfused heart is not known. We therefore wanted to determine whether oxidant stress, mPTP opening, and PARP activity contribute to the same death pathway after myocardial I/R. Methods and Results A murine left anterior descending coronary artery ( LAD ) occlusion (30 minutes) and release (1 to 4 hours) model was employed. Experimental groups included controls and antioxidant‐treated, mPTP ‐inhibited, or PARP ‐inhibited hearts. Antioxidant treatment prevented oxidative damage, mPTP opening, ATP depletion, and PARP activity, placing oxidant stress as the proximal death trigger. Genetic deletion of cyclophilin D (CypD −/− ) prevented loss of total NAD + and PARP activity, and mPTP ‐mediated loss of mitochondrial function. Control hearts showed progressive mitochondrial depolarization and loss of ATP from 1.5 to 4 hours of reperfusion, but not outer mitochondrial membrane rupture. Neither genetic deletion of PARP ‐1 nor its pharmacological inhibition prevented the initial mPTP ‐mediated depolarization or loss of ATP , but PARP ablation did allow mitochondrial recovery by 4 hours of reperfusion. Conclusions These results indicate that oxidant stress, the mPTP , and PARP activity contribute to a single death pathway after I/R in the heart. PARP activation undermines cell survival by preventing mitochondrial recovery after mPTP opening early in reperfusion. This suggests that PARP ‐mediated prolongation of mitochondrial depolarization contributes significantly to cell death via an energetic crisis rather than by mitochondrial outer membrane rupture.

Background This study tested the hypothesis that type 2 diabetes restricts multipotency of db/db mesenchymal stem cells ( MSC s), promotes their terminal differentiation into adipocytes rather than endothelial cells, thereby promotes adipocytic infiltration into ischemic muscles, and reduces their capacity to participate in postischemic neovascularization. Methods and Results To test this hypothesis, we transplanted MSC s from db/db or wild‐type ( WT ) mice into WT recipients after induction of hind limb ischemia. WT recipients of db/db MSC s demonstrated adipocyte infiltration of ischemic muscle and impaired neovascularization; WT recipients of WT MSC s showed no intramuscular adipocyte infiltration and had significantly enhanced neovascularization ( P MSC s that differentiated into an adipocyte phenotype was greater and into an endothelial cell was less in WT recipients transplanted with db/db MSC s than those transplanted with WT MSC s ( P db/db MSC s exhibited greater oxidant stress, greater adipocyte differentiation, and less endothelial differentiation than WT MSC s, and these differences were reversed by treatment with N ‐acetylcysteine or Nox4 siRNA ( P Nox4 expression, oxidant stress, and adipocyte differentiation in WT MSC s, and these insulin‐induced effects were reversed by Nox4 siRNA ( P db/db MSC oxidant stress by in vivo pretreatment with Nox4 siRNA before transplantation reversed their impaired capacity to augment postischemic neovascularization. Conclusions Type 2 diabetes–induced oxidant stress restricts the multipotency of MSC s and impairs their capacity to increase blood flow recovery after the induction of hind‐limb ischemia. Reversal of MSC oxidant stress might permit greater leverage of the therapeutic potential of MSC transplantation in the setting of diabetes.

Background Decreased serum arylesterase activity, catalyzed by the high-density lipoprotein–associated paraoxonase (PON)-1, is associated with increased oxidant stress and atherosclerosis risk. We sought to determine the prognostic value of serum PON-1 activity, as monitored by PON or arylesterase activities, in subjects with chronic kidney disease (CKD), particularly in relation to established cardiac biomarkers. Methods and Results Serum arylesterase and PON activities were measured in sequential subjects with CKD (n=630; estimated glomerular filtration rate [eGFR]