Benjamin S. Rayner

Hydrogen peroxide promotes endothelial dysfunction by stimulating multiple sources of superoxide anion radical production and decreasing nitric oxide bioavailability.

Hydrogen peroxide (H₂O₂) is an oxidant implicated in cell signalling and various pathologies, yet relatively little is known about its impact on endothelial cell function. Herein we studied the functional and biochemical changes in aortic vessels and cultured porcine aortic endothelial cells (PAEC) exposed to H₂O₂. Exposure of aortic rings to 25 or 50 µM, but not 10 µM, H₂O₂ for 60 min prior to constriction significantly decreased subsequent relaxation in response to acetylcholine (ACh), but not the nitric oxide (^·NO) donor sodium nitroprusside. Treatment of PAEC with 50 µM H₂O₂ significantly decreased ACh-induced accumulation of ^·NO, as measured with a ^·NO-selective electrode, yet such treatment increased nitric oxide synthase activity ∼3-fold, as assessed by conversion of L-arginine to L-citrulline. Decreased ^·NO bioavailability was reflected in decreased cellular cGMP content, associated with increased superoxide anion radical (O₂^–·), and overcome by addition of polyethylene glycol superoxide dismutase. Increased cellular O₂^–· production was inhibited by allopurinol, diphenyliodonium and rotenone in an additive manner. The results show that exposure of endothelial cells to H₂O₂ decreases the bioavailability of agonist-induced ^·NO as a result of increased production of O₂^–· likely derived from xanthine oxidase, NADPH-oxidasse and mitochondria. These processes could contribute to H₂O₂-induced vascular dysfunction that may be relevant under conditions of oxidative stress such as inflammation.

Comparative reactivity of myeloperoxidase-derived oxidants with mammalian cells.

Myeloperoxidase is an important heme enzyme released by activated leukocytes that catalyzes the reaction of hydrogen peroxide with halide and pseudo-halide ions to form various hypohalous acids. Hypohalous acids are chemical oxidants that have potent antibacterial, antiviral, and antifungal properties and, as such, play key roles in the human immune system. However, increasing evidence supports an alternative role for myeloperoxidase-derived oxidants in the development of disease. Excessive production of hypohalous acids, particularly during chronic inflammation, leads to the initiation and accumulation of cellular damage that has been implicated in many human pathologies including atherosclerosis, neurodegenerative disease, lung disease, arthritis, inflammatory cancers, and kidney disease. This has sparked a significant interest in developing a greater understanding of the mechanisms involved in myeloperoxidase-derived oxidant-induced mammalian cell damage. This article reviews recent developments in our understanding of the cellular reactivity of hypochlorous acid, hypobromous acid, and hypothiocyanous acid, the major oxidants produced by myeloperoxidase under physiological conditions.

Human S-Nitroso Oxymyoglobin Is a Store of Vasoactive Nitric Oxide

Nitric oxide (·NO) regulates vascular function, and myoglobin (Mb) is a heme protein present in skeletal, cardiac, and smooth muscle, where it facilitates O2 transfer. Human ferric Mb binds ·NO to yield nitrosylheme and S-nitroso (S-NO) Mb (Witting, P. K., Douglas, D. J., and Mauk, A. G. (2001) J. Biol. Chem. 276, 3991–3998). Here we show that human ferrous oxy-myoglobin (oxyMb) oxidizes ·NO, with a second order rate constant k = 2.8 ± 0.1 × 107 m–1·s–1 as determined by stopped-flow spectroscopy. Mixtures containing oxyMb and S-nitrosoglutathione or S-nitrosocysteine added at 1.5–2 moles of S-nitrosothiol/mol oxyMb yielded S-NO oxyMb through trans-nitrosation equilibria as confirmed with mass spectrometry. Rate constants for the equilibrium reactions were kforward = 110 ± 3 and kreverse = 16 ± 3 m–1·s–1 for S-nitrosoglutathione and kforward = 293 ± 5 and kreverse = 20 ± 2 m–1·s–1 for S-nitrosocysteine. Incubation of S-NO oxyMb with Cu2+ ions stimulated ·NO release as measured with a ·NO electrode. Similarly, Cu2+ released ·NO from Mb immunoprecipitated from cultured human vascular smooth muscle cells (VSMCs) that were pre-treated with diethylaminenonoate. No ·NO release was observed from VSMCs treated with vehicle alone or immunoprecipitates obtained from porcine aortic endothelial cells with and without diethylaminenonoate treatment. Importantly, pre-constricted aortic rings relaxed in the presence of S-NO oxyMb in a cyclic GMP-dependent process. These data indicate that human oxyMb rapidly oxidizes ·NO and that biologically relevant S-nitrosothiols can trans-(S)nitrosate human oxyMb. Furthermore, S-NO oxyMb can be isolated from cultured human VSMCs exposed to an exogenous ·NO donor at physiologic concentration. The potential biologic implications of S-NO oxyMb acting as a source of ·NO are discussed.

Oxidative stress in myocardial ischaemia reperfusion injury: a renewed focus on a long-standing area of heart research.

Abstract Advances in the treatment of coronary artery disease have seen a significant drop in mortality and morbidity particularly amongst patients with acute myocardial infarction (MI). In particular, percutaneous trans-luminal balloon angioplasty (PTCA) with stenting to re-open atherosclerotic coronary arteries has yielded marked improvement in clinical outcome for patients with acute MI. Furthermore, with the advent of drug-eluting stents occurrence rates for coronary artery restenosis, one common clinical problem associated with angioplasty and stent deployment, have declined markedly. However, coronary restenosis in diabetic patients remains an on-going problem. The success of drug-eluting stents has seen a renewed focus on myocardial ischaemia reperfusion (IR) injury as this represents one area of research where many questions remain unanswered. In particular, the relationship between myocardial IR injury and decreased myocardial micro-vasculature re-flow post PTCA (that ultimately leads to poor clinical outcome and myocardial damage/dysfunction) is one area of research with the potential to decrease current complication rates further in patients suffering myocardial IR injury sustained during MI. This review discusses the role for oxidative stress, oxidant source(s) and both gene regulation and stem-cell therapy as potential strategic targets in the ischaemic myocardium, with the ultimate aim of providing significant cardioprotection in the setting of acute MI.

Comparative reactivity of the myeloperoxidase-derived oxidants hypochlorous acid and hypothiocyanous acid with human coronary artery endothelial cells.

In the immune response, hypohalous acids are generated by activated leukocytes via the release of myeloperoxidase and the formation of H2O2. Although these oxidants have important bactericidal properties, they have also been implicated in causing tissue damage in inflammatory diseases, including atherosclerosis. Hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN) are the major oxidants formed by myeloperoxidase under physiological conditions, with the ratio of these oxidants dependent on diet and smoking status. HOCl is highly reactive and causes marked cellular damage, but few data are available on the effects of HOSCN on mammalian cells. In this study, we have compared the actions of HOCl and HOSCN on human coronary artery endothelial cells (HCAEC). HOCl reacts rapidly with the cells, resulting in extensive cell death by both apoptosis and necrosis, with necrosis dominating at higher oxidant doses. In contrast, HOSCN is consumed more slowly, with cell death occurring only by apoptosis. Exposure of HCAEC to HOCl and HOSCN induces changes in mitochondrial membrane permeability, which, in the case of HOSCN, is associated with mitochondrial release of proapoptotic factors, including cytochrome c, apoptosis-inducing factor, and endonuclease G. With each oxidant, apoptosis appears to be caspase-independent, with the inactivation of caspases 3/7 observed, and pretreatment of the cells with the caspase inhibitor Z-VAD-fmk having no effect on the extent of cell death. Loss of cellular thiols, depletion of glutathione, and the inactivation of thiol-dependent enzymes, including glyceraldehyde-3-phosphate dehydrogenase, were seen with both oxidants, though to a much greater extent with HOCl. The ability of myeloperoxidase-derived oxidants to induce endothelial cell apoptosis may contribute to the formation of unstable lesions in atherosclerosis. The results with HOSCN may be particularly significant for smokers, who have elevated plasma levels of SCN(-), the precursor of this oxidant.

Oxidant Stress and Damage in Post-Ischemic Mouse Hearts: Effects of Adenosine

Despite the general understanding that ischemia-reperfusion (I/R) promotes oxidant stress, specific contributions of oxidant stress or damage to myocardial I/R injury remain poorly defined. Moreover, whether endogenous ‘cardioprotectants’ such as adenosine act via limiting this oxidant injury is unclear. Herein we characterized effects of 20 min ischemia and 45 min reperfusion on cardiovascular function, oxidative stress and damage in isolated perfused mouse hearts (with glucose or pyruvate as substrate), and examined whether 10 μM adenosine modified these processes. In glucose-perfused hearts post-ischemic contractile function was markedly impaired (< 50% of pre-ischemia), cell damage assessed by lactate dehydrogenase (LDH) release was increased (12 ± 2 IU/g vs. 0.2 ± 0.1 IU/g in normoxic hearts), endothelial-dependent dilation in response to ADP was impaired while endothelial-independent dilation in response to nitroprusside was unaltered. Myocardial oxidative stress increased significantly, based on decreased glutathione redox status ([GSSG]/[GSG + GSSH] = 7.8 ± 0.3% vs. 1.3 ± 0.1% in normoxic hearts). Tissue cholesterol, native cholesteryl esters (CE) and the lipid-soluble antioxidant α-tocopherol (α-TOH, the most biologically active form of vitamin E) were unaffected by I/R, whereas markers of primary lipid peroxidation (CE-derived lipid hydroperoxides and hydroxides; CE-O(O)H) increased significantly (14 ± 2 vs. 2 ± 1 pmol/mg in normoxic hearts). Myocardial α -tocopherylquinone (α-TQ; an oxidation product of α -TOH) also increased (10.3 ± 1.0 vs. 1.7 ± 0.2 pmol/mg in normoxic hearts). Adenosine treatment improved functional recovery and vascular function, and limited LDH efflux. These effects were associated with an anti-oxidant effect of adenosine, as judged by inhibition of I/R-mediated changes in glutathione redox status (by 60%), α-TQ (80%) and CE-O(O)H (100%). Provision of 10 mM pyruvate as sole substrate (to by-pass glycolysis) modestly reduced I/R injury and changes in glutathione redox status and α-TQ, but not CE-O(O)H. Adenosine exerted further protection and anti-oxidant actions in these hearts. Functional recoveries and LDH efflux correlated inversely with oxidative stress and α -TQ (but not CE-O(O)H) levels. Collectively, our data reveal selective oxidative events in post-ischemic murine hearts, which are effectively limited by adenosine (independent of substrate). Correlation of post-ischemic cardiovascular outcomes with specific oxidative events (glutathione redox state, α-TQ) supports an important anti-oxidant component to adenosinergic protection.

Trichomonas vaginalis: underdiagnosis in urban Australia could facilitate re-emergence

Objectives Trichomonas vaginalis (TV) has a low profile in urban sexually transmitted infection (STI) clinics in many developed countries. The objective of this study was to determine the true prevalence of TV in an Australian urban sexual health setting using sensitive molecular diagnostic techniques. Methods A cross-sectional study investigating the aetiology of cervicitis in women attending two urban sexual health clinics in Sydney, Australia, enrolled 356 consecutive eligible women from 2006 to 2008. The diagnostic yield from the standard clinical practice of discretionary high vaginal wet preparation microscopy in women with suspicious vaginal discharge was compared with universal use of nested PCR for TV of cervical samples. Results TV was detected by PCR in 17/356 women (4.8%, 95% CI 2.8 to 7.5%), whereas only four cases (1.1%, 95% CI 0.3 to 2.8%) were detected by discretionary wet preparation microscopy. Eleven of the 17 women (p=0.003) were of culturally and linguistically diverse background. Additionally, cervicitis was found to be significantly associated with TV, RR 1.66 (1.14 to 2.42), p=0.034. Conclusions Traditional TV-detection methods underestimate TV prevalence in urban Australia. The TV prevalence of 4.8% by PCR testing in this study exceeds previously reported urban Australian TV rates of <1%. An increase in trichomoniasis-associated adverse reproductive outcomes and enhanced HIV transmission poses a salient public health threat. Accordingly, TV warrants a higher profile in urban STI clinic settings in developed countries, and we suggest that priority be given to development of standardised molecular TV detection techniques and that these become part of routine STI testing.

Remote ischemic preconditioning attenuates EGR-1 expression following myocardial ischemia reperfusion injury through activation of the JAK-STAT pathway

BACKGROUND/OBJECTIVES Remote ischemic preconditioning (RIPC) protects the myocardium from ischemia/reperfusion (I/R) injury however the molecular pathways involved in cardioprotection are yet to be fully delineated. Transcription factor Early growth response-1 (Egr-1) is a key upstream activator in a variety of cardiovascular diseases. In this study, we elucidated the role of RIPC in modulating the regulation of Egr-1. METHODS This study subjected rats to transient blockade of the left anterior descending (LAD) coronary artery with or without prior RIPC of the hind-limb muscle and thereafter excised the heart 24h following surgical intervention. In vitro, rat cardiac myoblast H9c2 cells were exposed to ischemic preconditioning by subjecting them to 3cycles of alternating nitrogen-flushed hypoxia and normoxia. These preconditioned media were added to recipient H9c2 cells which were then subjected to 30min of hypoxia followed by 30min of normoxia to simulate myocardial I/R injury. Thereafter, the effects of RIPC on cell viability, apoptosis and inflammatory markers were assessed. RESULTS We showed reduced infarct size and suppressed Egr-1 in the heart of rats when RIPC was administered to the hind leg. In vitro, we showed that RIPC improved cell viability, reduced apoptosis and attenuated Egr-1 in recipient cells. CONCLUSIONS Selective inhibition of intracellular signaling pathways confirmed that RIPC increased production of intracellular nitric oxide (NO) and reactive oxygen species (ROS) via activation of the JAK-STAT pathway which then inactivated I/R-induced ERK 1/2 signaling pathways, ultimately leading to the suppression of Egr-1.

Regio- and stereo-chemical oxidation of linoleic acid by human myoglobin and hydrogen peroxide: Tyr103 affects rate and product distribution

Mb (myoglobin) plus H2O2 catalyses the oxidation of various substrates via a peroxidase-like activity. A Y103F (Tyr103-->Phe) variant of human Mb has been constructed to assess the effect of exchanging an electron-rich oxidizable amino acid on the peroxidase activity of human Mb. Steady-state analyses of reaction mixtures containing Y103F Mb, purified linoleic acid and H2O2 revealed a lower total yield of lipid oxidation products than mixtures containing the wild-type protein, consistent with the reported decrease in the rate constant for reaction of Y103F Mb with H2O2 [Witting, Mauk and Lay (2002) Biochemistry 41, 11495-11503]. Irrespective of the Mb employed, lipid oxidation yielded 9(R/S)-HODE [9(R,S)-hydroxy-10E,12Z-octadecadienoic acid] in preference to 13(R/S)-HODE [13(R,S)-hydroxy-9Z,11E-octadecadienoic acid], while 9- and 13-keto-octadecadienoic acid were formed in trace amounts. However, lipid oxidation by the Y103F variant of Mb proceeded with a lower V(max) value and an increased K(m) value relative to the wild-type control. Consistent with the increased K(m), the product distribution from reactions with Y103F Mb showed decreased selectivity compared with the wild-type protein, as judged by the decreased yield of 9(S)-relative to 9(R)-HODE. Together, these data verify that Tyr103 plays a significant role in substrate binding and orientation in the haem pocket of human Mb. Also, the midpoint potential for the Fe(III)/(II) one-electron reduction was shifted slightly, but significantly, to a higher potential, confirming the importance of Tyr103 to the hydrogen-bonding network involving residues that line the haem crevice of human Mb.

Polymorphonuclear leukocyte phagocytic function increases in plasminogen knockout mice

BACKGROUND Mice lacking plasminogen (PG-/-) require alternative pathways of fibrinolysis for survival. This may depend on polymorphonuclear leukocytes (PMN) that can clear soluble and insoluble fibrin(ogen) through PG-independent processes. Our objective was to demonstrate that PMNs from PG-/- mice exhibit increased Mac-1 dependent phagocytic activity, which may explain their increased fibrin(ogen)lytic activity compared with wild type (PG+/+) mice. METHODS Phagocytic activity of PMNs from PG-/- and PG+/+ mice was compared following exposure to Staphylococcus aureus (S. aureus) particles and the expression of Mac-1 was assessed in parallel by flow cytometric analysis. Resistance to phorbol-12-myristate-13-acetate (PMA)-induced cell death was compared between PMNs from the different genotypes. RESULTS Stimulation of PG-/- PMNs by opsonized S. aureus diluted in PG-/- plasma significantly increased phagocytosis (15-fold) compared with stimulation of PG+/+ PMNs in PG+/+ plasma. Incubation of PG-/- PMNs with PG+/+ plasma (control) or PG-/- plasma supplemented with human PG inhibited this increased phagocytic activity. Mac-1 cell surface density increased 6.2+/-1.0-fold in PG-/- PMNs versus 2.9+/-0.6-fold in PG+/+ PMNs (P < 0.01) indicating that Mac-1 may be associated with increased phagocytic activity. Supporting this, treatment of PG-/- PMNs with an anti-Mac-1 antibody in PG-/- plasma inhibited phagocytic activity. In addition, physiologic PG blocked Mac-1 accessibility at the surface of PMNs. Addition of PMA resulted in 33% death of PMNs from PG-/- mice versus 68% in PG+/+ controls (P < 0.001). CONCLUSIONS PMNs from PG-/- mice exhibit a Mac-1 dependent increase in phagocytic activity that is suppressed with human PG, an anti-Mac-1 antibody or the plasma from PG+/+ mice. The propensity for PMNs from PG-/- mice to be activated in response to PMA together with their relative resistance to PMA-toxicity may contribute to increased PMN half-life and enhanced fibrin(ogen) clearance in the setting of PG deficiency.