Discovering a Reliable Heat-Shock Factor-1 Inhibitor to Treat Human Cancers: Potential Opportunity for Phytochemists.

Heat-shock factor-1 (HSF-1) is an important transcription factor that regulates pathogenesis of many human diseases through its extensive transcriptional regulation. Especially, it shows pleiotropic effects in human cancer, and hence it has recently received increased attention of cancer researchers. After myriad investigations on HSF-1, the field has advanced to the phase where there is consensus that finding a potent and selective pharmacological inhibitor for this transcription factor will be a major break-through in the treatment of various human cancers. Presently, all reported inhibitors have their limitations, made evident at different stages of clinical trials. This brief account summarizes the advances with tested natural products as HSF-1 inhibitors and highlights the necessity of phytochemistry in this endeavor of discovering a potent pharmacological HSF-1 inhibitor.

Heat shock factor-1 knockout enhances cholesterol 7α-hydroxylase (CYP7A1) and multidrug transporter (MDR1) gene expressions to attenuate atherosclerosis.

AIMS: Stress response, in terms of activation of stress factors, is known to cause obesity and coronary heart disease such as atherosclerosis in human. However, the underlying mechanism(s) of these pathways are not known. Here, we investigated the effect of heat shock factor-1 (HSF-1) on atherosclerosis. METHODS AND RESULTS: HSF-1 and low-density lipoprotein receptor (LDLr) double knockout (HSF-1(-/-)/LDLr(-/-)) and LDLr knockout (LDLr(-/-)) mice were fed with atherogenic western diet (WD) for 12 weeks. WD-induced weight gain and atherosclerotic lesion in aortic arch and carotid regions were reduced in HSF-1(-/-)/LDLr(-/-) mice, compared with LDLr(-/-) mice. Also, repression of PPAR-γ2 and AMPKα expression in adipose tissue, low hepatic steatosis, and lessened plasma adiponectins and lipoproteins were observed. In HSF-1(-/-)/LDLr(-/-) liver, higher cholesterol 7α-hydroxylase (CYP7A1) and multidrug transporter [MDR1/P-glycoprotein (P-gp)] gene expressions were observed, consistent with higher bile acid transport and larger hepatic bile ducts. Luciferase reporter gene assays with wild-type CYP7A1 and MDR1 promoters showed lesser luminescence than with mutant promoters (HSF-1 binding site deleted), indicating that HSF-1 binding is repressive of CYP7A1 and MDR1 gene expressions. CONCLUSION: HSF-1 ablation not only eliminates heat shock response, but it also transcriptionally up-regulates CYP7A1 and MDR1/P-gp axis in WD-diet fed HSF-1(-/-)/LDLr(-/-) mice to reduce atherosclerosis.

Sulfite Oxidase Activity of Cytochrome c: Role of Hydrogen Peroxide.

In humans, sulfite is generated endogenously by the metabolism of sulfur containing amino acids such as methionine and cysteine. Sulfite is also formed from exposure to sulfur dioxide, one of the major environmental pollutants. Sulfite is used as an antioxidant and preservative in dried fruits, vegetables, and beverages such as wine. Sulfite is also used as a stabilizer in many drugs. Sulfite toxicity has been associated with allergic reactions characterized by sulfite sensitivity, asthma, and anaphylactic shock. Sulfite is also toxic to neurons and cardiovascular cells. Recent studies suggest that the cytotoxicity of sulfite is mediated by free radicals; however, molecular mechanisms involved in sulfite toxicity are not fully understood. Cytochrome c (cyt c) is known to participate in mitochondrial respiration and has antioxidant and peroxidase activities. Studies were performed to understand the related mechanism of oxidation of sulfite and radical generation by ferric cytochrome c (Fe3+ cyt c) in the absence and presence of H2O2. Electron paramagnetic resonance (EPR) spin trapping studies using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) were performed with sulfite, Fe3+ cyt c, and H2O2. An EPR spectrum corresponding to the sulfite radical adducts of DMPO (DMPO-SO3-) was obtained. The amount of DMPO-SO3- formed from the oxidation of sulfite by the Fe3+ cyt c increased with sulfite concentration. In addition, the amount of DMPO-SO3- formed by the peroxidase activity of Fe3+ cyt c also increased with sulfite and H2O2 concentration. From these results, we propose a mechanism in which the Fe3+ cyt c and its peroxidase activity oxidizes sulfite to sulfite radical. Our results suggest that Fe3+ cyt c could have a novel role in the deleterious effects of sulfite in biological systems due to increased production of sulfite radical. It also shows that the increased production of sulfite radical may be responsible for neurotoxicity and some of the injuries which occur to humans born with molybdenum cofactor and sulfite oxidase deficiencies.

Oxidative Stress in Dilated Cardiomyopathy Caused by MYBPC3 Mutation.

Cardiomyopathies can result from mutations in genes encoding sarcomere proteins including MYBPC3, which encodes cardiac myosin binding protein-C (cMyBP-C). However, whether oxidative stress is augmented due to contractile dysfunction and cardiomyocyte damage in MYBPC3-mutated cardiomyopathies has not been elucidated. To determine whether oxidative stress markers were elevated in MYBPC3-mutated cardiomyopathies, a previously characterized 3-month-old mouse model of dilated cardiomyopathy (DCM) expressing a homozygous MYBPC3 mutation (cMyBP-C((t/t))) was used, compared to wild-type (WT) mice. Echocardiography confirmed decreased percentage of fractional shortening in DCM versus WT hearts. Histopathological analysis indicated a significant increase in myocardial disarray and fibrosis while the second harmonic generation imaging revealed disorganized sarcomeric structure and myocyte damage in DCM hearts when compared to WT hearts. Intriguingly, DCM mouse heart homogenates had decreased glutathione (GSH/GSSG) ratio and increased protein carbonyl and lipid malondialdehyde content compared to WT heart homogenates, consistent with elevated oxidative stress. Importantly, a similar result was observed in human cardiomyopathy heart homogenate samples. These results were further supported by reduced signals for mitochondrial semiquinone radicals and Fe-S clusters in DCM mouse hearts measured using electron paramagnetic resonance spectroscopy. In conclusion, we demonstrate elevated oxidative stress in MYPBC3-mutated DCM mice, which may exacerbate the development of heart failure.

Developing an irreversible inhibitor of human DDAH-1, an enzyme upregulated in melanoma.

Inhibitors of the human enzyme dimethylarginine dimethylaminohydrolase-1 (DDAH-1) can raise endogenous levels of asymmetric dimethylarginine (ADMA) and lead to a subsequent inhibition of nitric oxide synthesis. In this study, N(5) -(1-imino-2-chloroethyl)-L-ornithine (Cl-NIO) is shown to be a potent time- and concentration-dependent inhibitor of purified human DDAH-1 (KI =1.3±0.6 µM; kinact =0.34±0.07 min(-1) ), with >500-fold selectivity against two arginine-handling enzymes in the same pathway. An activity probe is used to measure the "in cell" IC50 value (6.6±0.2 µM) for Cl-NIO inhibition of DDAH-1 artificially expressed within cultured HEK293T cells. A screen of diverse melanoma cell lines reveals that a striking 50/64 (78 %) of melanoma lines tested showed increased levels of DDAH-1 relative to normal melanocyte control lines. Treatment of the melanoma A375 cell line with Cl-NIO shows a subsequent decrease in cellular nitric oxide production. Cl-NIO is a promising tool for the study of methylarginine-mediated nitric oxide control and a potential therapeutic lead compound for other indications with elevated nitric oxide production, such as septic shock and idiopathic pulmonary fibrosis.

Reversal of SIN-1-induced eNOS dysfunction by the spin trap, DMPO, in bovine aortic endothelial cells via eNOS phosphorylation.

BACKGROUND AND PURPOSE: Nitric oxide (NO) derived from eNOS is mostly responsible for the maintenance of vascular homeostasis and its decreased bioavailability is characteristic of reactive oxygen species (ROS)-induced endothelial dysfunction (ED). Because 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), a commonly used spin trap, can control intracellular nitroso-redox balance by scavenging ROS and donating NO, it was employed as a cardioprotective agent against ED but the mechanism of its protection is still not clear. This study elucidated the mechanism of protection by DMPO against SIN-1-induced oxidative injury to bovine aortic endothelial cells (BAEC). EXPERIMENTAL APPROACH: BAEC were treated with SIN-1, as a source of peroxynitrite anion (ONOO⁻), and then incubated with DMPO. Cytotoxicity following SIN-1 alone and cytoprotection by adding DMPO was assessed by MTT assay. Levels of ROS and NO generation from HEK293 cells transfected with wild-type and mutant eNOS cDNAs, tetrahydrobiopterin bioavailability, eNOS activity, eNOS and Akt kinase phosphorylation were measured. KEY RESULTS: Post-treatment of cells with DMPO attenuated SIN-1-mediated cytotoxicity and ROS generation, restoration of NO levels via increased in eNOS activity and phospho-eNOS levels. Treatment with DMPO alone significantly increased NO levels and induced phosphorylation of eNOS Ser¹¹79 via Akt kinase. Transfection studies with wild-type and mutant human eNOS confirmed the dual role of eNOS as a producer of superoxide anion (O2⁻) with SIN-1 treatment, and a producer of NO in the presence of DMPO. CONCLUSION AND IMPLICATIONS: Post-treatment with DMPO of oxidatively challenged cells reversed eNOS dysfunction and could have pharmacological implications in the treatment of cardiovascular diseases.

Differential effects of the peroxynitrite donor, SIN-1, on atrial and ventricular myocyte electrophysiology.

Oxidative stress has been implicated in the pathogenesis of heart failure and atrial fibrillation and can result in increased peroxynitrite production in the myocardium. Atrial and ventricular canine cardiac myocytes were superfused with 3-morpholinosydnonimine-N-ethylcarbamide (SIN-1), a peroxynitrite donor, to evaluate the acute electrophysiologic effects of peroxynitrite. Perforated whole-cell patch clamp techniques were used to record action potentials. SIN-1 (200 µM) increased the action potential duration (APD) in atrial and ventricular myocytes; however, in the atria, APD prolongation was rate independent, whereas in the ventricle APD, prolongation was rate dependent. In addition to prolongation of the action potential, beat-to-beat variability of repolarization was significantly increased in ventricular but not in atrial myocytes. We examined the contribution of intracellular calcium cycling to the effects of SIN-1 by treating myocytes with the SERCA blocker, thapsigargin (5-10 µM). Inhibition of calcium cycling prevented APD prolongation in the atrial and ventricular myocytes, and prevented the SIN-1-induced increase in ventricular beat-to-beat APD variability. Collectively, these data demonstrate that peroxynitrite affects atrial and ventricular electrophysiology differentially. A detailed understanding of oxidative modulation of electrophysiology in specific chambers is critical to optimize therapeutic approaches for cardiac diseases.

Diminazene attenuates pulmonary hypertension and improves angiogenic progenitor cell functions in experimental models.

RATIONALE: Studies have demonstrated that angiotensin-converting enzyme 2 (ACE2) plays a protective role against lung diseases, including pulmonary hypertension (PH). Recently, an antitrypanosomal drug, diminazene aceturate (DIZE), was shown to exert an "off-target" effect of enhancing the enzymatic activity of ACE2 in vitro. OBJECTIVES: To evaluate the pharmacological actions of DIZE in experimental models of PH. METHODS: PH was induced in male Sprague Dawley rats by monocrotaline, hypoxia, or bleomycin challenge. Subsets of animals were simultaneously treated with DIZE. In a separate set of experiments, DIZE was administered after 3 weeks of PH induction to determine whether the drug could reverse PH. MEASUREMENTS AND MAIN RESULTS: DIZE treatment significantly prevented the development of PH in all of the animal models studied. The protective effects were associated with an increase in the vasoprotective axis of the lung renin-angiotensin system, decreased inflammatory cytokines, improved pulmonary vasoreactivity, and enhanced cardiac function. These beneficial effects were abolished by C-16, an ACE2 inhibitor. Initiation of DIZE treatment after the induction of PH arrested disease progression. Endothelial dysfunction represents a hallmark of PH pathophysiology, and growing evidence suggests that bone marrow-derived angiogenic progenitor cells contribute to endothelial homeostasis. We observed that angiogenic progenitor cells derived from the bone marrow of monocrotaline-challenged rats were dysfunctional and were repaired by DIZE treatment. Likewise, angiogenic progenitor cells isolated from patients with PH exhibited diminished migratory capacity toward the key chemoattractant stromal-derived factor 1α, which was corrected by in vitro DIZE treatment. CONCLUSIONS: Our results identify a therapeutic potential of DIZE in PH therapy.

Hyperoxia and transforming growth factor ß1 signaling in the post-ischemic mouse heart.

AIMS: Following ischemic injury, myocardial healing and remodeling occur with characteristic myofibroblast trans-differentiation and scar formation. The current study tests the hypothesis that hyperoxia and nitric oxide (NO) regulate TGF-ß1 signaling in the post-ischemic myocardium. MAIN METHODS: C57BL/6 wild-type (WT), endothelial and inducible nitric oxide synthase knockout (eNOS(-/-) and iNOS(-/-)) mice were subjected to 30-min left anterior descending coronary artery occlusion followed by reperfusion. Myocardial tissue oxygenation was monitored with electron paramagnetic resonance oximetry. Protein expressions of TGF-ß1, receptor-activated small mothers against decapentaplegic homolog (Smad), p21 and α-smooth muscle actin (α-SMA) were measured with enzyme-linked immunosorbent assay (ELISA), Western immunoblotting, and immunohistochemical staining. KEY FINDINGS: There was a hyperoxic state in the post-ischemic myocardial tissue. Protein expressions of total and active TGF-ß1, p-Smad2/3 over t-Smad2/3 ratio, p21, and α-SMA were significantly increased in WT mice compared to Sham control. Knockout of eNOS or iNOS further increased protein expression of these signals. The expression of α-SMA was more abundant in the infarct of eNOS(-/-) and iNOS(-/-) mice than WT mice. A protein band indicating nitration of TGF-ß type-II receptor (TGFßRII) was observed from WT heart. Carbogen (95% O2 plus 5% CO2) treatment increased the ratio of p-Smad2/t-Smad2, which was inhibited by 10006329 EUK (EUK134) and sodium nitroprusside (SNP). In conclusion, hyperoxia up-regulated and NO/ONOO(-) inhibited cardiac TGF-ß1 signaling and myofibroblast trans-differentiation. SIGNIFICANCE: These findings may provide new insights in myocardial infarct healing and repair.

Age and exercise training alter signaling through reactive oxygen species in the endothelium of skeletal muscle arterioles.

Exercise training ameliorates age-related impairments in endothelium-dependent vasodilation in skeletal muscle arterioles. Additionally, exercise training is associated with increased superoxide production. The purpose of this study was to determine the role of superoxide and superoxide-derived reactive oxygen species (ROS) signaling in mediating endothelium-dependent vasodilation of soleus muscle resistance arterioles from young and old, sedentary and exercise-trained rats. Young (3 mo) and old (22 mo) male rats were either exercise trained or remained sedentary for 10 wk. To determine the impact of ROS signaling on endothelium-dependent vasodilation, responses to acetylcholine were studied under control conditions and during the scavenging of superoxide and/or hydrogen peroxide. To determine the impact of NADPH oxidase-derived ROS, endothelium-dependent vasodilation was determined following NADPH oxidase inhibition. Reactivity to superoxide and hydrogen peroxide was also determined. Tempol, a scavenger of superoxide, and inhibitors of NADPH oxidase reduced endothelium-dependent vasodilation in all groups. Similarly, treatment with catalase and simultaneous treatment with tempol and catalase reduced endothelium-dependent vasodilation in all groups. Decomposition of peroxynitrite also reduced endothelium-dependent vasodilation. Aging had no effect on arteriolar protein content of SOD-1, catalase, or glutathione peroxidase-1; however, exercise training increased protein content of SOD-1 in young and old rats, catalase in young rats, and glutathione peroxidase-1 in old rats. These data indicate that ROS signaling is necessary for endothelium-dependent vasodilation in soleus muscle arterioles, and that exercise training-induced enhancement of endothelial function occurs, in part, through an increase in ROS signaling.

Nitric oxide synthases and atrial fibrillation.

Oxidative stress has been implicated in the pathogenesis of atrial fibrillation. There are multiple systems in the myocardium which contribute to redox homeostasis, and loss of homeostasis can result in oxidative stress. Potential sources of oxidants include nitric oxide synthases (NOS), which normally produce nitric oxide in the heart. Two NOS isoforms (1 and 3) are normally expressed in the heart. During pathologies such as heart failure, there is induction of NOS 2 in multiple cell types in the myocardium. In certain conditions, the NOS enzymes may become uncoupled, shifting from production of nitric oxide to superoxide anion, a potent free radical and oxidant. Multiple lines of evidence suggest a role for NOS in the pathogenesis of atrial fibrillation. Therapeutic approaches to reduce atrial fibrillation by modulation of NOS activity may be beneficial, although further investigation of this strategy is needed.

Hydrogen sulfide increases nitric oxide production from endothelial cells by an akt-dependent mechanism.

Hydrogen sulfide (H(2)S) and nitric oxide (NO) are both gasotransmitters that can elicit synergistic vasodilatory responses in the in the cardiovascular system, but the mechanisms behind this synergy are unclear. In the current study we investigated the molecular mechanisms through which H(2)S regulates endothelial NO production. Initial studies were performed to establish the temporal and dose-dependent effects of H(2)S on NO generation using EPR spin trapping techniques. H(2)S stimulated a twofold increase in NO production from endothelial nitric oxide synthase (eNOS), which was maximal 30 min after exposure to 25-150 µM H(2)S. Following 30 min H(2)S exposure, eNOS phosphorylation at Ser 1177 was significantly increased compared to control, consistent with eNOS activation. Pharmacological inhibition of Akt, the kinase responsible for Ser 1177 phosphorylation, attenuated the stimulatory effect of H(2)S on NO production. Taken together, these data demonstrate that H(2)S up-regulates NO production from eNOS through an Akt-dependent mechanism. These results implicate H(2)S in the regulation of NO production in endothelial cells, and suggest that deficiencies in H(2)S signaling can directly impact processes regulated by NO.

Effects of high adherence to mediterranean or low-fat diets in medicated secondary prevention patients.

Although the Mediterranean diet (MD) and the low-fat Therapeutic Lifestyle Changes Diet (TLCD) promote equivalent increases in event-free survival in secondary coronary prevention, possible mechanisms of such complete dietary patterns in these patients, usually medicated, are unclear. The aim of this study was to investigate the effects of the MD versus the TLCD in markers of endothelial function, oxidative stress, and inflammation after acute coronary syndromes. Comparison was made between 3 months of the MD (n = 21; rich in whole grains, vegetables, fruits, nuts, and olive oil, plus red wine) and the TLCD (n = 19; plus phytosterols 2 g/day) in a highly homogenous population of stable patients who experienced coronary events in the previous 2 years (aged 45 to 65 years, all men) allocated to each diet under a strategy designed to optimize adherence, documented as >90%. Baseline demographics, body mass index and clinical data, and use of statins and other drugs were similar between groups. The MD and TLCD promoted similar decreases in body mass index and blood pressure (p ≤0.001) and particularly in plasma asymmetric dimethylarginine levels (p = 0.02) and l-arginine/asymmetric dimethylarginine ratios (p = 0.01). The 2 diets did not further enhance flow-mediated brachial artery dilation compared to baseline (4.4 ± 4.0%). Compared to the TLCD, the MD promoted decreases in blood leukocyte count (p = 0.025) and increases in high-density lipoprotein levels (p = 0.053) and baseline brachial artery diameter. Compared to the MD, the TLCD decreased low-density lipoprotein and oxidized low-density lipoprotein plasma levels, although the ratio of oxidized to total low-density lipoprotein remained unaltered. Glucose, high-sensitivity C-reactive protein, triglycerides, myeloperoxidase, intercellular adhesion molecular, vascular cell adhesion molecule, and glutathione serum and plasma levels remained unchanged with either diet. In conclusion, medicated secondary prevention patients show evident although small responses to the MD and the TLCD, with improved markers of redox homeostasis and metabolic effects potentially related to atheroprotection.

Cell death induced by the Jak2 inhibitor, G6, correlates with cleavage of vimentin filaments.

Hyperkinetic Jak2 tyrosine kinase signaling has been implicated in several human diseases including leukemia, lymphoma, myeloma, and the myeloproliferative neoplasms. Using structure-based virtual screening, we previously identified a novel Jak2 inhibitor named G6. We showed that G6 specifically inhibits Jak2 kinase activity and suppresses Jak2-mediated cellular proliferation. To elucidate the molecular and biochemical mechanisms by which G6 inhibits Jak2-mediated cellular proliferation, we treated Jak2-V617F expressing human erythroleukemia (HEL) cells for 12 h with either vehicle control or 25 µM of the drug and compared protein expression profiles using two-dimensional gel electrophoresis. One differentially expressed protein identified by electrospray mass spectroscopy was the intermediate filament protein, vimentin. It was present in DMSO treated cells but absent in G6 treated cells. HEL cells treated with G6 showed both time- and dose-dependent cleavage of vimentin as well as a marked reorganization of vimentin intermediate filaments within intact cells. In a mouse model of Jak2-V617F mediated human erythroleukemia, G6 also decreased the levels of vimentin protein, in vivo. The G6-induced cleavage of vimentin was found to be Jak2-dependent and calpain-mediated. Furthermore, we found that intracellular calcium mobilization is essential and sufficient for the cleavage of vimentin. Finally, we show that the cleavage of vimentin intermediate filaments, per se, is sufficient to reduce HEL cell viability. Collectively, these results suggest that G6-induced inhibition of Jak2-mediated pathogenic cell growth is concomitant with the disruption of intracellular vimentin filaments. As such, this work describes a novel pathway for the targeting of Jak2-mediated pathological cell growth.

Suppression of eNOS-derived superoxide by caveolin-1: a biopterin-dependent mechanism.

In the vasculature, nitric oxide (NO) is generated by endothelial NO synthase (eNOS) in a calcium/calmodulin-dependent reaction. In the absence of the requisite eNOS cofactor tetrahydrobiopterin (BH(4)), NADPH oxidation is uncoupled from NO generation, leading to the production of superoxide. Although this phenomenon is apparent with purified enzyme, cellular studies suggest that formation of the BH(4) oxidation product, dihydrobiopterin, is the molecular trigger for eNOS uncoupling rather than BH(4) depletion alone. In the current study, we investigated the effects of both BH(4) depletion and oxidation on eNOS-derived superoxide production in endothelial cells in an attempt to elucidate the molecular mechanisms regulating eNOS oxidase activity. Results demonstrated that pharmacological depletion of endothelial BH(4) does not result in eNOS oxidase activity, whereas BH(4) oxidation gave rise to significant eNOS-oxidase activity. These findings suggest that the endothelium possesses regulatory mechanisms, which prevent eNOS oxidase activity from pterin-free eNOS. Using a combination of gene silencing and pharmacological approaches, we demonstrate that eNOS-caveolin-1 association is increased under conditions of reduced pterin bioavailability and that this sequestration serves to suppress eNOS uncoupling. Using small interfering RNA approaches, we demonstrate that caveolin-1 gene silencing increases eNOS oxidase activity to 85% of that observed under conditions of BH(4) oxidation. Moreover, when caveolin-1 silencing was combined with a pharmacological inhibitor of AKT, BH(4) depletion increased eNOS-derived superoxide to 165% of that observed with BH(4) oxidation. This study identifies a critical role of caveolin-1 in the regulation of eNOS uncoupling and provides new insight into the mechanisms through which disease-associated changes in caveolin-1 expression may contribute to endothelial dysfunction.

Tetrahydrobiopterin depletion and NOS2 uncoupling contribute to heart failure-induced alterations in atrial electrophysiology.

AIMS: Heart failure is a common antecedent to atrial fibrillation; both heart failure and atrial fibrillation are associated with increased myocardial oxidative stress. Chronic canine heart failure reduces atrial action potential duration and atrial refractoriness. We hypothesized that inducible nitric oxide synthase 2 (NOS2) contributes to atrial oxidative stress and electrophysiologic alterations. METHODS AND RESULTS: A 16-week canine tachypacing model of heart failure was used (n= 21). At 10 weeks, dogs were randomized to either placebo (n = 12) or active treatment (n = 9) with NOS cofactor, tetrahydrobiopterin (BH(4), 50 mg), and NOS substrate (L-arginine, 3 g) twice daily for 6 weeks. A group of matched controls (n = 7) was used for comparison. Heart failure increased atrial NOS2 and reduced atrial BH(4), while L-arginine was unchanged. Treatment reduced inducible atrial fibrillation and normalized the heart failure-induced shortening of the left atrial myocyte action potential duration. Treatment increased atrial [BH(4)] while [L-arginine] was unchanged. Treatment did not improve left ventricular function or dimensions. Heart failure-induced reductions in atrial [BH(4)] resulted in NOS uncoupling, as measured by NO and superoxide anion (O(2)(·-)) production, while BH(4) and L-arginine treatment normalized NO and O(2)(·-). Heart failure resulted in left atrial oxidative stress, which was attenuated by BH(4) and L-arginine treatment. CONCLUSION: Chronic non-ischaemic heart failure results in atrial oxidative stress and electrophysiologic abnormalities by depletion of BH(4) and uncoupling of NOS2. Modulation of NOS2 activity by repletion of BH(4) may be a safe and effective approach to reduce the frequency of atrial arrhythmias during heart failure.

Vascular smooth muscle Jak2 mediates angiotensin II-induced hypertension via increased levels of reactive oxygen species.

AIMS: Angiotensin II (Ang II) type AT(1) receptors expressed on vascular smooth muscle cells (VSMCs) couple to the Jak2 signalling pathway. However, the importance of this tissue-specific coupling is poorly understood. The purpose of this investigation was to determine the importance of VSMC-derived Jak2 in angiotensin II-mediated hypertension. METHODS AND RESULTS: The Cre-loxP system was used to conditionally eliminate Jak2 tyrosine kinase expression within the smooth muscle cells of mice. Following chronic Ang II infusion, the resulting increase in mean arterial pressure (MAP) was significantly attenuated in the Jak2 null mice when compared with littermate controls. The VSMC Jak2 null mice were also protected from the Ang II-induced vascular remodelling. Aortic rings from the VSMC Jak2 null mice exhibited reduced Ang II-induced contraction and enhanced endothelial-dependent relaxation via increased nitric oxide (NO) bioavailability. When compared with controls, the VSMC Jak2 nulls also had lower levels of hydrogen peroxide, Rho kinase activity, and intracellular Ca(2+) in response to Ang II. CONCLUSIONS: The data indicate that VSMC Jak2 expression is involved in the pathogenesis of Ang II-dependent hypertension due to the increased presence of reactive oxygen species (ROS). As such, VSMC-derived Jak2 tyrosine kinase modulates overall vascular tone via multiple, non-redundant mechanisms.

Dimethylarginine dimethylaminohydrolase overexpression ameliorates atherosclerosis in apolipoprotein E-deficient mice by lowering asymmetric dimethylarginine.

Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase, is increasingly recognized as a novel biomarker in cardiovascular disease. To date, it remains unclear whether elevated ADMA levels are merely associated with cardiovascular risk or whether this molecule is of functional relevance in the pathogenesis of atherosclerotic vascular disease. To clarify this issue, we crossed dimethylarginine dimethylaminohydrolase (DDAH) transgenic mice that overexpress the human isoform 1 of the ADMA degrading enzyme DDAH into ApoE-deficient mice to generate ApoE(-/-)/hDDAH1(+/-) mice. In these mice, as well as ApoE(-/-) wild-type littermates, atherosclerosis within the aorta as well as vascular function of aortic ring preparations was assessed. We report here that overexpression of hDDAH1 reduces plaque formation in ApoE(-/-) mice by lowering ADMA. The extent of atherosclerosis closely correlated with plasma ADMA levels in male but not female mice fed either a standard rodent chow or an atherogenic diet. Functional analysis of aortic ring preparations revealed improved endothelial function in mice overexpressing hDDAH1. Our findings provide proof-of-principle that ADMA plays a causal role as a culprit molecule in atherosclerosis and support recent evidence indicating a functional relevance of DDAH enzymes in genetic mouse models. Together, these results demonstrate that pharmacological interventions targeting the ADMA/DDAH pathway may represent a novel approach in the prevention and management of cardiovascular diseases.

Hypertensive effects of central angiotensin II infusion and restraint stress are reduced with age.

OBJECTIVE: We investigated the effect of age on cardiovascular responses mediated by central angiotensin II (AngII) after intracerebroventricular infusion of AngII, and during restraint stress. METHODS: Blood pressure (BP) and heart rate (HR) of young (5-month-old) and old (27-month-old) male Fischer-344 x Brown-Norway rats were measured using radiotelemetry. AngII was infused intracerebroventricularly using osmotic minipumps (10 ng/0.5 microl/h for 11 days). BP and HR responses to stress were evaluated by placing animals in restrainers for 20 min before and after intracerebroventricular infusion of the AngII-type-1 receptor inhibitor losartan (15 microg/microl per h for 3 days). RESULTS: Resting BP was significantly elevated and HR was significantly lower in old rats compared with young. AngII-induced BP increase was markedly reduced in old rats, but HR responses were similar. Diurnal variation of both BP and HR was lower in old animals, and AngII reduced the amplitude of BP variation in young rats, but not in old. Restraint stress-induced BP and HR elevations were reduced with age. BP responses were diminished by central losartan infusion in both young and old, but this effect was more significant in young rats. In addition, expression of CuZn-superoxide dismutase and catalase declined significantly with age in the hypothalamus, whereas baseline oxidative stress increased. In contrast, AngII-induced increase in hypothalamic oxidative stress decreased with age. CONCLUSION: This study demonstrates that the role of central AngII diminishes with age in the regulation of BP both during baseline conditions and during stress, whereas the involvement of AngII in the regulation of HR remains unaffected.