Free Radical Pharmacology and Oxidative Stress

Group heads

• Dr Grant Raymond Drummond
• Dr Stavros Selemidis

Background

Cardiovascular disease — atherosclerosis, coronary artery disease, hypertension, heart failure and stroke — claims more lives worldwide than any other cause, which is likely to increase in the future together with a huge burden on medical and disability costs. It is timely to reduce this anticipated increase in cardiovascular disease through both the development of more efficacious therapeutic and ideally preventive strategies for early diagnosis of patients at risk using new pharmacological tools, plasma parameters and innovative imaging techniques. A widely accepted hypothesis suggests the occurrence of oxygen free radicals in the vascular wall as a major cause of disease. Importantly, free radical-driven pathology is not only involved in cardiovascular disease. Such processes underlie neurodegenerative diseases (Alzheimer’s disease and Parkinson’s disease), chronic inflammation (COPD, asthma) and cancer. Thus we are dealing with a major disease mechanism. Currently no technology is available in the clinic to measure these early markers of disease. Would they become available, the identification of risk patients would be revolutionised towards a much more individualised and effective form of medicine.

Free radicals in biology

Free radicals are molecules with at least one unpaired electron. In biology, oxygen can undergo univalent reduction to form superoxide radical by means of enzymes such as NADPH oxidase, xanthine oxidase and nitric oxide synthase or nonenzymatically during oxidative phosphorylation (i.e. aerobic respiration) in the mitochondria. Superoxide is then enzymatically converted to hydrogen peroxide through the actions of superoxide dismutase (SOD), which is further reduced to water by catalase or glutathione peroxidase (GPX). In the presence of transition metals such as Fe2+, hydrogen peroxide can be converted into the extremely reactive hydroxyl radical. To compensate for this, cells have adapted ways to protect themselves against oxidants through the expression of antioxidant systems. These include the abovementioned enzymes SOD, catalase and GPX as well as nonenzymatic low molecular weight antioxidants such as glutathione, ascorbate (vitamin C) and -tocopherol (vitamin E). However, under certain circumstances, the amount of reactive oxygen species generated exceeds and overwhelms the antioxidant removal processes and as a result damage cells by oxidising proteins, lipids and DNA.

Sources of ROS

To analyse and pinpoint the molecular role of ROS in health and disease, anti-oxidants have been used. Many of them are ineffective in scavenging all ROS or just alter the superoxide/H2O2 ratio of ROS. We pursue the strategy of identifying and validating relevant enzymatic sources of ROS, understanding their regulation and possibly interfering with them pharmacologically. One of the internationally most intensively validated candidates in that respect are NADPH oxidases, the first dedicated enzymatic source of superoxide in vascular cells. They represent hetero-oligomeric enzyme complexes with regulatory subunits such as p47phox, (or homologues such as NOXO1), p67 phox, (or homologues such as NOXA1) and p40phox, a small G protein Rac1, and a membrane-bound cytochrome b558 reductase domain.

The superoxide forming catalytic cytochrome b558 is a heterodimeric protein complex made up of a small -subunit, p22phox, as well as a larger catalytic -subunit called Nox. Upon assembly of its regulatory subunits to the membrane, the NADPH oxidase complex generates superoxide by one-electron reduction of oxygen via its Nox subunit using reduced -nicotinamide adenine dinucleotide phosphate (NADPH) as the electron donor. The nature of Nox appears to vary between different cell types. Nox2 (formerly termed gp91phox) is essential for NADPH oxidase activity in phagocytic cells, vascular cells also partly rely on homologues of this protein, such as Nox1 and Nox4, both derived from distinct genes. In endothelial cells, both Nox2 and Nox4 containing NADPH oxidases are largely responsible for basal superoxide production. Oscillatory shear stress, humoral factors such as angiotensin II and hypercholesterolaemia may up-regulate Nox1. By contrast, vascular smooth muscle cells express both Nox1 and Nox4 and are unlikely to express Nox2 containing NADPH oxidases. Vascular NADPH oxidase is involved in the pathogenesis of intimal hyperplasia induced by periarterial collars, cholesterol-induced atherosclerosis, as well as bypass graft intimal hyperplasia. Importantly, gene disruption of p47phox significantly reduces superoxide production by vascular smooth muscle cells (VSMCs) and retards the development of atherosclerotic lesions in apolipoprotein E-deficient mice. Furthermore, Nox1-derived superoxide is likely to contribute to the pressor and hypertensive effects of angiotensin II by reducing NO bioavailability and endothelial function.

We believe that an increase in Nox activity is not only a symptom but also a causative factor in arterial hypertension and atherogenesis. Elucidating the mechanisms of Nox signalling in vascular cells is important, for this may unravel new approaches to suppressing oxidative stress and the initiation and progression of cardiovascular disease.

Specific research projects

1. Defining the role of NADPH oxidases in vascular remodelling and arterial hypertension

Cardiovascular risk factors including hypertension are chracterised by oxidative stress, which triggers events such as endothelial dysfunction, vascular remodelling and inflammation and ultimately leads to the onset and progression of vascular disease. NADPH oxidases are upregulated during the early stages of arterial hypertension and may thus represent a key trigger for the onset of disease. Three isoforms of Nox (1, 2 and 4) are expressed in blood vessels. We utilize the first colonies of Nox1 and Nox4 knockout mice together with the commercially available Nox2-KO line to conclusively establish the role of each individual Nox isoform in the vascular pathophysiology of hypertension. We anticipate that these studies will reveal innovative clinical approaches for the treatment of cardiovascular disease, which go beyond the current symptomatic treatment of hypertension.

Vascular oxidative stress and endothelial dysfunction in human hypertension: A cross-sectional study

In this study we have formed a multidisciplinary team of biomedical scientists and clinicians (Monash Medical Centre and Southern Health) to be the first to directly quantify ROS production in blood vessels from humans at risk (patients with white coat hypertension) of, or with established hypertension (patients with hypertension with and without vascular disease). By characterizing the source of ROS production in vessels taken from individuals across the spectrum of normotension to hypertension these studies should pave the way for development of therapies aimed at inhibiting vascular oxidative stress by directly targeting the relevant enzymatic source of ROS.

3. Designing the first selective NADPH oxidase inhibitors

Free radicals are the oldest signalling and defence system of cells. The recent discovery of Nox, a major enzyme source, was a land-mark discovery. This Nox enzyme family regulates cell growth, motility or kills invading cells in plants, fungi, invertebrates and vertebrates. In humans, they cause ageing, neuro-degeneration, cardiovascular and chronic inflammatory disease and carcinogenesis. We have developed cutting edge, innovative technologies to express, analyse, and quantify these enzymes to be in a world-wide leading position with two independent small molecular weight modulator compounds that we now apply to a wide range of biological applications.

4. Mechanisms of cross talk between NADPH oxidase and nitric oxide-cGMP signalling pathways in vascular cells

Besides hormones and neurotransmitters, free radicals exert key physiological effects in vascular cells. Nitric oxide (NO), for example, possesses multiple vaso- and athero-protective effects including vasodilatation, inhibition of lipid peroxidation as well as vascular smooth muscle cell proliferation and migration. Because of these properties, NO donors continue to be important drugs for the management of different arterial disease states. Endogenously, NO is synthesised by a family of NO synthases (NOS) and many of its cellular effects are mediated by stimulation of soluble guanylyl cyclase (sGC), leading to the generation of cGMP and activation of cGMP-dependent protein kinase (cGK). Steady state cGMP levels are also regulated by its degradation rate catalysed a family of phosphodiesterases (PDE) and vasodilatation by inhibition of the PDE5A isoform is therapeutically applied in pulmonary hypertension and erectile dysfunction. Whilst the NO-cGMP pathway is tightly regulated through a variety of autofeedback control mechanisms, it is also sensitive to interference by concomitant occurrence of reactive oxygen species (ROS) such as superoxide and hydrogen peroxide (H2O2). NADPH oxidase regulation is also redox sensitive, e.g. stimulated by ROS in a self-propagating manner further augmenting chronic oxidative stress. By removing NO in a diffusion-limited reaction, superoxide compromises the vasoprotective actions of NO-cGMP, encompassed by ROS influencing several components of the NO-cGMP pathway. Importantly, the reverse regulation appears to occur as well: both NO and cGMP decrease Nox activity and ROS production in a sustainable fashion in vascular cells. Thus, it becomes apparent that signaling by two highly conserved radicals, NO and superoxide, cross-talks in a complex manner that is both relevant to physiology, pathophysiology and therapeutic intervention.

We propose to elucidate functionally relevant mechanisms of positive feedforward regulation within the Nox-ROS signalling pathway; elucidate physiologically and pathophysiologically relevant mechanisms of cross-talk from the NO-cGMP pathway towards the Nox-ROS signalling pathway; elucidate the cross-talk of Nox-ROS formation towards NO-cGMP signalling.

NO-cGMP signalling and ROS formation are essential processes in blood vessels, neurons and immune cells and of high clinical therapeutic relevance. Given that three life-saving, evidence-based drugs, nitrovasodilator (NO) drugs, PDE inhibitors and statins all act via these NO-cGMP and ROS systems, this study is likely to provide new insights into their mechanisms of actions, future therapeutic monitoring and better drugs.

Staff

Laboratory heads

• Dr Grant Raymond Drummond
• Dr Stavros Selemidis

Research Officers

• Dr Courtney Judkins

Research Assistant

• Mr Janahan Dharmarajah

Honours Students

• Ms Jennifer Rivera
• Mr Craig Harrison
• Ms Helen Chan Hing Chai
• Ms Poay Sian Sabrina Lee

Collaborators

1. Professor Harald Schmidt, Dept. of Pharmacology, Monash University, Clayton, Australia.
2. Associate Professor Chris Sobey, Dept. of Pharmacology, Monash University, Clayton, Australia.
3. Professor Barry McGrath, Dept. of Medicine, Monash Medical Centre, Faculty of Medicine, Nursing and Health Sciences, Monash University.
4. Associate Professor James Cameron, Department of Vascular Medicine, Monash Medical Centre, Clayton, Australia.
5. Dr Sophia Zoungas, Diabetes, Southern Health Dandenong, Victoria Australia.
6. Professor Helena Teede, Monash Institute of Health Services Research, Monash Medical Centre, Clayton, Australia.
7. Dr Marianne Tare, Dept. of Physiology, Faculty of Medicine, Nursing and Health Sciences, Monash University, Clayton, Australia.
8. Dr Russell Brown, Dept. of Physiology, Faculty of Medicine, Nursing and Health Sciences, Monash University, Clayton, Australia.
9. Dr Kellie Tuck, School of Chemistry, Faculty of Science, Monash University, Clayton, Australia.
10. Professor Gregory Dusting, Bernard O Brien Institute of Microsurgery, Melbourne University, Fitzroy, Australia.
11. Dr Ross Vlahos, Dept. of Pharmacology, Melbourne University, Parkville, Australia.
12. Dr Anna Walduck, Dept. of Microbiology, Melbourne University, Parkville, Australia.

Funding

• NHMRC Project Grants
• Monash University Category Type I (Logan) Fellowship
• NHMRC Peter Doherty Research Fellowship

Recent key references

1. JUDKINS CP, SOBEY CG, DANG TT, MILLER AA, DUSTING GJ, DRUMMOND GR (2006) NADPH-induced contractions of mouse aorta do not involve NADPH oxidase: A role for P2X receptors. J Pharmacol Exp Ther.
2. PARAVICINI TM, MILLER AA, DRUMMOND GR, SOBEY CG (2005) Flow-induced cerebral vasodilatation in vivo involves activation of phosphatidylinositol-3 kinase, NADPH-oxidase, and nitric oxide synthase. J Cereb Blood Flow Metab.
3. LOB H, ROSENKRANZ AC, BREITENBACH T, BERKELS R, DRUMMOND GR, ROESEN R (2005) Antioxidant and nitric oxide-sparing actions of dihydropyridines and ACE inhibitors differ in human endothelial cells. Pharmacology. 76: 8-18.
4. MILLER AA, DRUMMOND GR, SCHMIDT HH, SOBEY CG (2005) NADPH Oxidase Activity and Function Are Profoundly Greater in Cerebral Versus Systemic Arteries. Circ Res. 97: 1055-62
5. MILLER AA, DRUMMOND GR, SOBEY CG (2005) Selective inhibition of NADPH-oxidase isoforms as a therapeutic strategy in hypertension. Drug Discovery Today. 2: 187 - 92
6. KAIRUZ EM, BARBER MN, ANDERSON CR, KANAGASUNDARAM M, DRUMMOND GR, WOODS RL (2005) C-type natriuretic peptide (CNP) suppresses plasminogen activator inhibitor-1 (PAI-1) in vivo. Cardiovasc. Res. 66: 574-82
7. NICHOLLS SJ, DRUMMOND GR, RYE K, DUSTING GJ & BARTER PJ (2005) High density lipoproteins inhibit the pro-oxidant and pro-inflammatory changes induced by a periarterial collar in normocholesterolaemic rabbits. Circulation. 111: 1543-50
8. ELLMARK SH, DUSTING GJ, NG TANG FUI M, GUZZO-PERNELL N, DRUMMOND GR. (2005) The contribution of Nox4 to NADPH oxidase activity in mouse vascular smooth muscle. Cardiovasc Res. 65: 495-504
9. PARAVICINI TM, DRUMMOND GR, SOBEY CG (2004) Reactive oxygen species in the cerebral circulation: Physiological roles and therapeutic implications for hypertension and stroke. Drugs. 64: 2143-2157
10. JIANG F, DRUMMOND GR, DUSTING GJ (2004) Suppression of oxidative stress in the endothelium and vascular wall. Endothelium. 11: 79-88
11. PARAVICINI TM, CHRISSOBOLIS S, DRUMMOND GR & SOBEY CG (2004) Increased NADPH-oxidase activity and Nox4 expression during chronic hypertension is associated with enhanced cerebral vasodilatation to NADPH in vivo. Stroke. 35: 584-589
12. CHAN E.C.H., DRUMMOND G.R., WOODMAN O.L. (2003) 3',4-dihydroxyflavonol enhanced nitric oxide activity and improved vascular function after hindquarters ischaemia and reperfusion injury in the rat. J. Cardiovasc. Pharmacol. Submitted March 2003
13. PARAVICINI T.M., GULLUYAN L.M, DUSTING G.J., DRUMMOND, G.R. (2002) Increased NADPH-oxidase activity, gp91phox expression and endothelium-dependent vasorelaxation during neointima formation in rabbits. Circ. Res. 91: 54-61
14. DAVIS M.E., CAI H., DRUMMOND G.R. & HARRISON D.G. (2001). Shear stress regulates endothelial nitric oxide synthase expression through c-Src by divergent signaling pathways. Circ. Res. 89: 1073-80
15. CAI H., DAVIS M.E. DRUMMOND G.R. & HARRISON D.G. (2001) Induction of endothelial NO synthase by hydrogen peroxide via a Ca2+/calmodulin-dependent protein kinase II/janus kinase 2-dependent pathway. Arteroscl. Thromb. Vasc. Biol. 21: 1571-6
16. DRUMMOND G.R., CAI H., DAVIS M.E., RAMASAMY S. & HARRISON D.G. (2000) Transcriptional and posttranscriptional induction of endothelial nitric oxide synthase by hydrogen peroxide. Circ. Res. 86: 347-354.
17. SELEMIDIS S, SCHMIDT HHHW, WINGLER K & DRUMMOND GR. (2006). Pharmacology of NADPH oxidase inhibitors. Pharmacology and Therapeutics. Invited review.
18. DUSTING GJ, SELEMIDIS S. & JIANG F. (2005) Mechanisms for suppressing NADPH oxidase in the vascular wall. Mem Inst Oswaldo Cruz. Suppl 1:97-103
19. SELEMIDIS S. & COCKS T.M. (2002). Endothelium-dependent hyperpolarization as a remote anti-atherogenic mechanism. Trends Pharmacol. Sci., 23: 213-20
20. DRUMMOND G.R., SELEMIDIS S. & COCKS T.M. (2000) Apamin-senitive, non-nitric oxide (NO) endothelium,-dependent relaxations to bradykinin in the bovine isolated coronary artery: No role for cytochrome P450 and K+. Br. J. Pharmacol. 129: 811-819