Arginase overexpression and NADPH oxidase stimulation underlie impaired vasodilation induced by advanced glycation end products.

BACKGROUND: Advanced glycation endproducts (AGEs) play a major role in the development of many vascular complications that are mediated by endothelial dysfunction. The present work aimed to investigate the mechanism by which AGEs impair vasodilation. METHODS: The effect of AGEs on vasodilation induced by acetylcholine or D NONOate was examined by incubating isolated rat aortae with different AGEs concentrations. ACh-induced nitric oxide generation was assessed using the fluorescent probe diaminofluorecein (DAF-FM). The effect of AGEs on expression of mRNA for arginase 2, NADPH oxidase and endothelial nitric oxide synthase (eNOS) were determined by real-time PCR. RESULTS: One-hour in vitro incubation of rat aortae with AGEs impaired endothelial-dependent vasodilation produced by ACh, while increasing D NONOate-induced vasodilation. Preincubation of aortae with l-ornithine, an arginase 2-inhibitor, prevented the impairment effect induced by AGEs on endothelial-dependent vasodilation. Superoxide scavenging by tempol or NADPH oxidase inhibition by apocynin also blocked the effect of AGEs. AGEs decreased ACh-induced NO production and this was inhibited by both l-ornithine and apocynin. Furthermore, AGEs exposure increased arginase mRNA expression but decreased mRNA expression for eNOS in isolated rat aortae. CONCLUSION: The present results indicate that AGEs impairs endothelial-dependent vasodilation, and this effect is mediated via arginase overexpression and NADPH oxidase stimulation.

Cardioprotection by 6-gingerol in diabetic rats.

The current study was conducted to evaluate the effect of 6-gingerol (6G) on cardiac complications in streptozotocin (STZ)-induced diabetic (DM) rats. STZ-induced DM rats (single 50 mg/kg i.p. injection, 15 days prior to drug treatment) or time-matched controls were treated with 6G (75 mg/day route orally). After a further 8 weeks, blood was collected for biochemical analysis and 8-isoprostenol was measured in urine. Cardiac hemodynamics and ECG was assessed. 6G significantly attenuated the increased level of blood glucose in diabetic rats and improved cardiac hemodynamics in including RR interval, max dP/dt, min dP/dt and Tau. In addition, 6G alleviated the elevated ST segment, T amplitude and R amplitude with no significant effect on disturbed levels of adiponectin, TGF-ß or 8-isoprostenol induced by diabetes. The results showed that treatment with 6G has an ameliorative effect on cardiac dysfunction induced by diabetes. Which may be not related to its potential antioxidant effect.

Xanthine oxidase inhibition alleviates the cardiac complications of insulin resistance: effect on low grade inflammation and the angiotensin system.

BACKGROUND: We have previously shown that hyperuricemia plays an important role in the vascular complications of insulin resistance (IR). Here we investigated the effect of xanthine oxidase (XO) inhibition on the cardiac complications of IR. METHODS: IR was induced in rats by a high fructose high fat diet for 12 weeks. Allopurinol, a standard XO inhibitor, was administered in the last 4 weeks before cardiac hemodynamics and electrocardiography, serum glucose, insulin, tumor necrosis factor alpha (TNFα), 8-isoprostane, uric acid, lactate dehydrogenase (LDH) and XO activity were measured. Expression of cardiac angiotensin II (AngII) and angiotensin receptor 1 (AT1) were assessed by immunofluorescence. RESULTS: IR animals had significant hyperuricemia which was inhibited by allopurinol administration. IR was associated with impaired ventricular relaxation (reflected by a decreased diastolic pressure increment and prolonged diastolic duration) and XO inhibition greatly attenuated impaired relaxation. IR was accompanied by cardiac ischemia (reflected by increased QTc and T peak trend intervals) while XO inhibition alleviated the ECG abnormalities. When subjected to isoproterenol-induced ischemia, IR hearts were less resistant (reflected by larger ST height depression and higher LDH level) while XO inhibition alleviated the accompanying ischemia. In addition, XO inhibition prevented the elevation of serum 8-isoprostane and TNFα, and blocked elevated AngII and AT1 receptor expression in the heart tissue of IR animals. However, XO inhibition did not affect the developed hyperinsulinemia or dyslipidemia. CONCLUSIONS: XO inhibition alleviates cardiac ischemia and impaired relaxation in IR through the inhibition of low grade inflammation and the angiotensin system.

Expression and agonist responsiveness of CXCR3 variants in human T lymphocytes.

The chemokine receptor CXCR3 and its ligands CXCL9, CXCL10 and CXCL11 are involved in variety of inflammatory disorders including multiple sclerosis, rheumatoid arthritis, psoriasis and sarcoidosis. Two alternatively spliced variants of the human CXCR3-A receptor have been described, termed CXCR3-B and CXCR3-alt. Human CXCR3-B binds CXCL9, CXCL10, CXCL11 as well as an additional ligand CXCL4. In contrast, CXCR3-alt only binds CXCL11. We report that CXCL4 induces intracellular calcium mobilization as well as Akt and p44/p42 extracellular signal-regulated kinase phosphorylation, in activated human T lymphocytes. These responses have similar concentration dependence and time-courses to those induced by established CXCR3 agonists. Moreover, phosphorylation of Akt and p44/p42 is inhibited by pertussis toxin, suggesting coupling to Gα(i) protein. Surprisingly, and in contrast with the other CXCR3 agonists, stimulation of T lymphocytes with CXCL4 failed to elicit migratory responses and did not lead to loss of surface CXCR3 expression. Taken together, our findings show that, although CXCL4 is coupled to downstream biochemical machinery, its role in T cells is probably distinct from that of CXCR3-A agonists.

Staphylococcus aureus products subvert the Burkholderia cenocepacia-induced inflammatory response in airway epithelial cells.

Introduction. Chronic pulmonary infection is associated with colonization with multiple micro-organisms but host-microbe and microbe-microbe interactions are poorly understood.Aim. This study aims to investigate the differences in host responses to mono- and co-infection with S. aureus and B. cenocepacia in human airway epithelial cells.Methodology. We assessed the effect of co-infection with B. cenocepacia and S. aureus on host signalling and inflammatory responses in the human airway epithelial cell line 16HBE, using ELISA and western blot analysis.Results. The results show that B. cenocepacia activates MAPK and NF-κB signalling pathways, subsequently eliciting robust interleukin (IL)-8 production. However, when airway epithelial cells were co-treated with live B. cenocepacia bacteria and S. aureus supernatants (conditioned medium), the pro-inflammatory response was attenuated. This anti-inflammatory effect was widely exhibited in the S. aureus isolates tested and was mediated via reduced MAPK and NF-κB signalling, but not via IL-1 receptor or tumour necrosis factor receptor modulation. The staphylococcal effectors were characterized as small, heat-stable, non-proteinaceous and not cell wall-related factors.Conclusion. This study demonstrates for the first time the host response in a S. aureus/B. cenocepacia co-infection model and provides insight into a staphylococcal immune evasion mechanism, as well as a therapeutic intervention for excessive inflammation.

Interleukin 13 increases contractility of murine tracheal smooth muscle by a phosphoinositide 3-kinase p110delta-dependent mechanism.

The Th2 cytokine interleukin (IL) 13 can elicit a number of responses consistent with a key role in the pathogenesis of asthma. We have used pharmacological and genetic approaches to demonstrate the role of signaling via the class I phosphoinositide 3-kinase p110delta isoform in IL-13-induced hyper-responsiveness of murine tracheal smooth muscle contractility in vitro. IL-13 treatment of tracheal tissue is associated with an early activation of phosphoinositide 3-kinase (PI3K), as assessed by phosphorylation of Akt. Tracheal smooth muscle contractility is enhanced by overnight incubation with IL-13, resulting in increased maximal contractions (E(max)) to carbachol (CCh) and KCl. Inhibition of PI3K by the non-isoform-selective inhibitors wortmannin or 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), or the selective inhibitor of the PI3K p110delta isoform 2-(6-aminopurin-9-ylmethyl)-5-methyl-3-O-tolyl-3H-quinazolin-4-one (IC87114), prevented IL-13-induced hyper-responsiveness. Consistent with a role for PI3K p110delta in IL-13-induced hyper-responsiveness, IL-13 was unable to induce hyper-responsiveness in tissues from mice expressing the catalytically inactive form of p110delta (p110delta(D910A)). These data indicate that IL-13 contributes to tracheal smooth muscle hyper-responsiveness via the PI3K p110delta isoform. In addition to previously reported effects on airway inflammation, inhibition of PI3K p110delta may be a useful target for the treatment of asthma by preventing IL-13-induced airway smooth muscle hyper-responsiveness.

Zingerone alleviates the delayed ventricular repolarization and AV conduction in diabetes: Effect on cardiac fibrosis and inflammation.

BACKGROUND: The study aims to analyse the action of zingerone in diabetes-related cardiac arrhythmias. METHODS: Diabetes was induced by streptozocin while treatment groups received 20 mg/kg zingerone daily. Following extra seven weeks, electrocardiography, extraction of blood, urine and heart for biochemical analysis, histopathology and immunofluorescence were undertaken. RESULTS: The suppression of QT and QTc prolongation in diabetic rats was indicative of prolonged cardiac repolarisation that was greatly reduced by zingerone treatment. In addition, the reduction in PR interval attested that zingerone improved AV delay in diabetic rats. The fibrogenic transforming growth factor ß1 upregulation in diabetic hearts was suppressed by zingerone. The marked glycogen deposition and muscle degeneration seen in diabetic heart sections were also alleviated by zingerone. Furthermore, zingerone prevented the decrease in of the serum anti-inflammatory cytokine adiponectin in diabetics. The heightened levels of oxidative stress markers 8-isoprostane and uric acid in diabetic rats were suppressed. In the diabetic heart, the reduced catalase activity was improved and the excessive expression of angiotensin receptor 1 was inhibited by zingerone. CONCLUSION: Cardiac delayed repolarisation and AV conduction in rats with diabetes were halted by zingerone. It appears that inhibition of cardiac fibrosis and associated inflammation-oxidative stress signalling underpins the zingerone effect.

Leukocyte navigation mechanisms as targets in airway diseases.

Respiratory diseases, including asthma and chronic obstructive pulmonary disease, are among the most significant diseases in terms of their disabling effects and healthcare burden. A characteristic feature of almost all respiratory diseases is the accumulation and activation of inflammatory leukocytes in the lung or airway. Recent advances in the understanding of the molecules and intracellular signalling events controlling these processes are now translating to new therapeutic entities. In this article, the process of leukocyte accumulation is summarized, together with the preclinical and clinical evidence supporting the utility of the individual components of this process as targets for disease therapy.

Differential modulation of COX-2 expression in A549 airway epithelial cells by structurally distinct PPAR(gamma) agonists: evidence for disparate functional effects which are independent of NF-(kappa)B and PPAR(gamma).

Ligands of peroxisome proliferator-activated receptor-gamma (PPAR(gamma)) are thought to possess anti-inflammatory properties mediated via both PPAR(gamma) dependent and independent mechanisms. This work investigates the effects of PPAR(gamma) ligands on the regulation of cyclooxygenase-2 (COX-2) in the human lung epithelial cell line, A549. The synthetic ligand troglitazone activated the phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase pathway (MAPK), whereas the endogenous ligand, 15-deoxy-Delta(12,14)-prostaglandin J2 (15d-PGJ2), only activated the PI3K pathway. 15d-PGJ2 had no detectable effects on COX-2, mPGES expression, or PGE2 production. However, troglitazone induced time-dependent COX-2 expression, which was insensitive to PPAR(gamma) antagonists, but was abrogated by inhibitors of PI3K and the ERK MAP kinase pathway. Furthermore, troglitazone induced mPGES expression and PGE2 production. Neither troglitazone nor 15d-PGJ2 was able to convincingly activate NF-kappaB in A549 cells. Further heterogeneity in the responses to troglitazone and 15d-PGJ2 was observed in the regulation of gene expression as assessed by microarray analysis. In summary, this study provides compelling evidence that troglitazone (like 15d-PGJ2) can exert functional effects independently of actions via PPAR(gamma). Moreover, we have identified unique biochemical and functional actions of troglitazone that are not shared by 15d-PGJ2, which may influence the therapeutic potential of this compound in inflammatory settings.

Protective effect of zingerone on increased vascular contractility in diabetic rat aorta.

The aim of the present study was to investigate the effect and possible mechanism of action of zingerone, the main constituent of ginger, on vascular reactivity in isolated aorta from diabetic rats. The results show that incubation of aortae with zingerone alleviates the exaggerated vasoconstriction of diabetic aortae to phenylephrine, as well as the impaired relaxatory response to acetylcholine in a concentration-dependent manner. Furthermore, Zingerone directly relax phenylephrine-precontracted aortae. The vasorelaxatory response is significantly attenuated by the nitric oxide synthase inhibitor Nω-nitro-l-arginine methyl ester hydrochloride and the guanylate cyclase inhibitor methylene blue but no effect of either the potassium channels blocker tetraethylammonium chloride, or the cyclooxygenase inhibitor indomethacin was observed. Zingerone had no effect on advanced glycation end product formation as well. In conclusion, zingerone ameliorates enhanced vascular contraction in diabetic aortae which may be mediated by its vasodilator effect through NO- and guanylate cyclase stimulation.

6-Gingerol alleviates exaggerated vasoconstriction in diabetic rat aorta through direct vasodilation and nitric oxide generation.

The aim of the present study is to investigate the effect and potential mechanism of action of 6-gingerol on alterations of vascular reactivity in the isolated aorta from diabetic rats. Male Wistar rats were divided into two experimental groups, control and diabetics. Diabetes was induced by a single intraperitoneal injection of streptozotocin (50 mg kg(-1)), and the rats were left for 10 weeks to develop vascular complications. The effect of in vitro incubation with 6-gingerol (0.3-3 µM) on the vasoconstrictor response of the isolated diabetic aortae to phenylephrine and the vasodilator response to acetylcholine was examined. Effect of 6-gingerol was also examined on aortae incubated with methylglyoxal as an advanced glycation end product (AGE). To investigate the mechanism of action of 6-gingerol, the nitric oxide synthase inhibitor Nω-nitro-l-arginine methyl ester hydrochloride (100 µM), guanylate cyclase inhibitor methylene blue (5 µM), calcium-activated potassium channel blocker tetraethylammonium chloride (10 mM), and cyclooxygenase inhibitor indomethacin (5 µM) were added 30 minutes before assessing the direct vasorelaxant effect of 6-gingerol. Moreover, in vitro effects of 6-gingerol on NO release and the effect of 6-gingerol on AGE production were examined. Results showed that incubation of aortae with 6-gingerol (0.3-10 µM) alleviated the exaggerated vasoconstriction of diabetic aortae to phenylephrine in a concentration-dependent manner with no significant effect on the impaired relaxatory response to acetylcholine. Similar results were seen in the aortae exposed to methylglyoxal. In addition, 6-gingerol induced a direct vasodilation effect that was significantly inhibited by Nω-nitro-l-arginine methyl ester hydrochloride and methylene blue. Furthermore, 6-gingerol stimulated aortic NO generation but had no effect on AGE formation. In conclusion, 6-gingerol ameliorates enhanced vascular contraction in diabetic aortae, which may be partially attributed to its ability to increase the production of NO and stimulation of cyclic guanosine monophosphate.

Nitric oxide-induced potentiation of the killing of Burkholderia cepacia by reactive oxygen species: implications for cystic fibrosis.

Burkholderia (formerly Pseudomonas) cepacia has emerged as an important pulmonary pathogen in cystic fibrosis, and survives within the lung despite a vigorous neutrophil-dominated immune response. Nitric oxide (NO) contributes to the antimicrobial activity of reactive oxygen species in the normal lung, but recent evidence suggests that inducible NO synthase is not expressed in the airway epithelial cells of cystic fibrosis (CF) patients. This may explain the failure of the neutrophil response to eliminate B. cepacia. To test this hypothesis, the present study examined the combined effect of NO, superoxide and H2O2 against B. cepacia. There was no killing of a highly transmissible strain by either superoxide or NO alone, but their combination reduced the bacterial count by >1000-fold over 75 min. This bactericidal activity was not sensitive to addition of superoxide dismutase, but was abrogated completely by catalase, suggesting that NO and hydrogen peroxide were the bactericidal mediators. Increased killing by NO in combination with H2O2 was seen for seven of a further 11 strains examined. The lack of NO in the lungs of CF patients may contribute to the survival of B. cepacia.

Phosphoinositide 3-kinase alpha-dependent regulation of branching morphogenesis in murine embryonic lung: evidence for a role in determining morphogenic properties of FGF7.

Branching morphogenesis is a critical step in the development of many epithelial organs. The phosphoinositide-3-kinase (PI3K) pathway has been identified as a central component of this process but the precise role has not been fully established. Herein we sought to determine the role of PI3K in murine lung branching using a series of pharmacological inhibitors directed at this pathway. The pan-class I PI3K inhibitor ZSTK474 greatly enhanced the branching potential of whole murine lung explants as measured by an increase in the number of terminal branches compared with controls over 48 hours. This enhancement of branching was also observed following inhibition of the downstream signalling components of PI3K, Akt and mTOR. Isoform selective inhibitors of PI3K identified that the alpha isoform of PI3K is a key driver in branching morphogenesis. To determine if the effect of PI3K inhibition on branching was specific to the lung epithelium or secondary to an effect on the mesenchyme we assessed the impact of PI3K inhibition in cultures of mesenchyme-free lung epithelium. Isolated lung epithelium cultured with FGF7 formed large cyst-like structures, whereas co-culture with FGF7 and ZSTK474 induced the formation of defined branches with an intact lumen. Together these data suggest a novel role for PI3K in the branching program of the murine embryonic lung contradictory to that reported in other branching organs. Our observations also point towards PI3K acting as a morphogenic switch for FGF7 signalling.

Arginase inhibition alleviates hypertension in the metabolic syndrome.

BACKGROUND AND PURPOSE: We have previously shown that arginase inhibition alleviates hypertension associated with in a diabetic animal model. Here, we investigated the protective effect of arginase inhibition on hypertension in metabolic syndrome. EXPERIMENTAL APPROACH: Metabolic syndrome was induced in rats by administration of fructose (10% in drinking water) for 12 weeks to induce vascular dysfunction. Three arginase inhibitors (citrulline, norvaline and ornithine) were administered daily in the last 6 weeks of study before and tail BP was recorded in conscious animals. Concentration response curves for phenylephrine (PE), KCl and ACh in addition to ACh-induced NO generation were obtained in thoracic aorta rings. Serum glucose, insulin, uric acid and lipid profile were determined as well as reactive oxygen species (ROS) and arginase activity. KEY RESULTS: Arginase activity was elevated in metabolic syndrome while significantly inhibited by citrulline, norvaline or ornithine treatment. Metabolic syndrome was associated with elevations in systolic and diastolic BP, while arginase inhibition significantly reduced elevations in diastolic and systolic BP. Metabolic syndrome increased vasoconstriction responses of aorta to PE and KCl and decreased vasorelaxation to ACh, while arginase inhibition completely prevented impaired responses to ACh. In addition, arginase inhibition prevented impaired NO generation and exaggerated ROS formation in metabolic syndrome. Furthermore, arginase inhibition significantly reduced hyperinsulinaemia and hypertriglyceridaemia without affecting hyperuricaemia or hypercholesterolaemia associated with metabolic syndrome. CONCLUSIONS AND IMPLICATIONS: Arginase inhibition alleviates hypertension in metabolic syndrome directly through endothelial-dependent relaxation/NO signalling protection and indirectly through inhibition of insulin resistance and hypertriglyceridaemia.

Voltage-independent calcium channels mediate lipopolysaccharide-induced hyporeactivity to endothelin-1 in the rat aorta.

The roles of intracellular calcium concentration ([Ca(2+)](i)) and Ca(2+) sensitization in lipopolysaccharide (LPS)-induced vascular smooth muscle (VSM) hyporesponsiveness are incompletely understood. To investigate these roles, contraction responses to endothelin-1 (ET-1) and 80 mM KCl; relaxation responses to nifedipine; the expression levels of mRNAs of ET-1 and its receptors (ET(A) or ET(B)); the expression levels of protein kinase C (PKC) and phosphorylation of Rho kinase (ROKalpha), CPI-17, and myosin phosphatase target subunit-1 (MYPT1); and changes in aortic VSM cell [Ca(2+)](i) were measured in LPS-treated aortic rings from male Wistar rats (250-300 g). LPS (10 mug/ml, 20 h) decreased contraction induced by ET-1 (0.3-100 nM) or 80 mM KCl. LPS-induced hypocontractility was not observed in the absence of external Ca(2+), but LPS-treated aorta remained hypocontractile on subsequent stepwise restoration of extracellular Ca(2+) (0.01-10 mM). Vascular relaxation to nifedipine; mRNA expression levels of ET-1, ET(A), or ET(B); protein expression levels of PKC; and phosphorylation levels of ROKalpha, CPI-17, and MYPT1 were not affected by LPS. In isolated aortic VSM cells, ET-1 caused a transient initial increase in [Ca(2+)](i), followed by a maintained tonic increase in [Ca(2+)](i), which was decreased by LPS pretreatment and was dependent on external Ca(2+). Subsequent restoration of extracellular Ca(2+) increased [Ca(2+)](i), but this increase was lower in the LPS-treated group. This difference in response to extracellular Ca(2+) addition was not affected by diltiazem, but was abolished by SKF-96365. Therefore, LPS induces hyporeactivity to ET-1 in rat aorta that depends on external Ca(2+) influx through non-voltage-operated Ca(2+) channels, but not on ET-1 receptor expression or Ca(2+) sensitization.

Haem oxygenase-1 induction protects against tumour necrosis factor alpha impairment of endothelial-dependent relaxation in rat isolated pulmonary artery.

BACKGROUND AND PURPOSE: Disturbances in pulmonary vascular reactivity are important components of inflammatory lung disease. Haem oxygenase-1 (HO-1) is an important homeostatic enzyme upregulated in inflammation. Here we have investigated the potentially protective effect of HO-1 against cytokine-induced impairment in pulmonary artery relaxation. EXPERIMENTAL APPROACH: Haem oxygenase-1 protein levels were assessed by immunofluorescence. HO activity was assessed by conversion of haemin to bilirubin. Rings of rat isolated pulmonary artery in organ baths were used to measure relaxant responses to the endothelium-dependent agent ACh and the endothelium-independent agent sodium nitroprusside (SNP). Production of nitric oxide (NO) and reactive oxygen species (ROS) was assessed by confocal fluorescence microscopy and fluorescent probes. KEY RESULTS: Haem oxygenase-1 protein expression was strongly induced in pulmonary artery after 24-h incubation with either haemin (5 microM) or curcumin (2 microM), accompanied by a significant increase in HO activity. Incubation with tumour necrosis factor alpha (TNFalpha, 1 ng.mL(-1), 2 h) significantly decreased relaxation of arterial rings to ACh, without affecting responses to SNP. Induction of HO-1 by curcumin or haemin protected against TNFalpha-induced hyporesponsiveness to ACh. The competitive HO inhibitor, tin protoporphyrin (20 microM), abolished the protective effect of haemin. HO-1 induction prevented a TNFalpha-induced increase in NO generation without affecting the TNFalpha-induced increase in ROS generation. HO-1 induction prevented the TNFalpha-induced decrease in ACh-stimulated NO generation. CONCLUSIONS AND IMPLICATIONS: Induction of HO-1 protected against TNFalpha impairment of endothelium-dependent relaxation in pulmonary artery, by a mechanism involving a reduction in inducible NO synthase-derived NO production.

Phosphoinositide 3-kinase signalling in lung disease: leucocytes and beyond.

The family of lipid kinases termed phosphoinositide-3-kinase (PI3K) is known to contribute at multiple levels to innate and adaptive immune responses, and is hence an attractive target for drug discovery in inflammatory and autoimmune disease, including respiratory diseases. The development of isoform-selective pharmacological inhibitors, targeted gene manipulation and short interfering RNA (siRNA) target validation have facilitated a better understanding of the role that each member of this family of kinases plays in the physiology and pathology of the respiratory system. In this review, we will evaluate the evidence for the roles of specific PI3K isoforms in the lung and airways, and discuss their potential as targets for novel drug therapies.