Centre for Vascular Research University of Sydney - Honours projects available in 2009

An Honours project undertaken in this lab would be administered by the Discipline of Pathology.

This group uses mouse, rat, and rabbit models of disease, in vivo and in vitro blood vessel function assays, tissue culture, flow cytometry, gene expression analysis, transfection, interference RNA, histology and immunohistochemistry, pharmacologic and biochemical approaches, HPLC, mass spectrometry and other analytical techniques, as well as chemical synthesis in their research.

Recent interesting publications:

Lau A, Leichtweis SB, Hume P, Mashima R, Hou JY, Chaufour X, Wilkinson B, Hunt NH, Celermajer DS, Stocker R. Probucol promotes functional re-endothelialization in balloon-injured rabbit aortas. Circulation 2003;107:2031-2036

Deng YM, Wu B, Witting PK, Stocker R. Probucol protects against smooth muscle cell proliferation by up-regulating heme oxygenase-1. Circulation 2004;110:1861-1866

Stocker R, Keaney JF, Jnr. The role of oxidative modifications in atherosclerosis. Physiol Rev 2004;84:1381-1478

Rayner BS, Wu BJ, Raftery M, Stocker R, Witting PK. Human S-nitroso-myoglobin is a store of vasoactive nitric oxide. J Biol Chem 2005;280:9985-9993

Witting PK, Wu BJ, Raftery M, Southwell-Keely P, Stocker R. Probucol protects against hypochlorite-induced endothelial dysfunction: identification of a novel pathway of probucol oxidation to a biologically active intermediate. J Biol Chem 2005;280:15612-15618

Choy K, Beck K, Png FY, Leichtweis SB, Wu BJ, Thomas SR, Hou JY, Croft KD, Mori TA, Stocker R. Processes involved in the site-specific effect of probucol on atherosclerosis in apolipoprotein E gene knockout mice. Arterioscl Thromb Vasc Biol 2005;25:1684-1690

Wu BJ, Kathir K, Witting PK, Beck K, Choy K, Li C, Croft KD, Mori TA, Tanous D, Adams MR, Lau AK, Stocker R. Antioxidants protect from atherosclerosis by a heme oxygenase-1 pathway that is independent of free radical scavenging. J Exp Med 2006;203:1117-1127

Tanous DJ, Br?sen JH, Choy K, Wu BJ, Kathir K, Lau A, Celermajer DS, Stocker R. Probucol promotes re-endothelialization and inhibits in-stent neointimal hyperplasia and thrombosis. Atherosclerosis 2006;189:432-349

Stocker R, Perrella MA. Heme oxygenase-1: a novel drug target for atherosclerotic diseases? Circulation 2006;114:2178-2189

Wu BJ, Di Girolamo N, Beck K, Hanratty CG, Choy K, Hou JY, Ward MR and Stocker R. Probucol [4,4'-[(1-methylethylidene)bis(thio)]bis-[2,6-bis(1,1-dimethylethyl)phenol]] inhibits compensatory remodeling and promotes lumen loss associated with atherosclerosis in apolipoprotein E-deficient mice. J Pharmacol Exp Ther 2007;321:477-484

Hunt NH, Stocker R. Heme moves to center stage in cerebral malaria. Nature Med 2007;13:667-669

Thomas SR, Terentis AC, Cai H, Takikawa O, Levina A, Lay PA, Freewan M, Stocker R. Post-translational regulation of human indoleamine 2,3-dioxygenase activity by nitric oxide. J Biol Chem 2007;282:23778-23787

1. Role of indoleamine 2,3-dioxygenase in control of blood vessel tone in inflammation

Supervisor + contact details:

• Professor Roland Stocker
• Professor Nicholas Hunt

Using a mouse model we are studying the role of the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO) in various aspects of the pathogenesis and related physiological changes of cerebral malaria infection. We were the first to show that endothelial cells located in small blood vessels of the brain and other tissues express IDO during malaria infection, in a process strictly dependent on interferon gamma. We recently (unpublished) observed that IDO contributes to the control of vascular tone (blood pressure) in malaria-infected mice.

In this joint project between the two laboratories, the blood vessel relaxing properties of IDO-derived products will be studied, with the aim of establishing the active component derived from tryptophan and the mode of action of this compound.

We also will examine the role of IDO in the regulation of vascular tone in other mouse models of inflammation, including atherosclerosis. The techniques used will include measurement of blood pressure in and isolation of blood vessels from mice, and in vitro vascular function studies using physiological myobath systems.

2. The role of heme oxygenase as an antioxidant defense

Supervisor + contact details:

• Professor Roland Stocker

Heme oxygenase catalyzes the oxidative degradation of heme and plays a key role in iron homeostasis by facilitating the 'return' of heme-derived iron to the bone marrow where it can be used for hematopoiesis. A large body of recent literature suggests that one of the isozymes of heme oxygenase (heme oxygenase-1) has a number of protective activities in diseases associated with increased oxidative stress, such as cardiovascular, neurodegenerative, and inflammatory conditions. The mechanisms underlying these protective effects are largely unknown, although antioxidant protection has been put forward as one likely possibility.

Recently, a functionally active yeast heme oxygenase has been identified. The role of HMX1 in controlling intracellular heme levels has been elucidated. However, little is known about whether it offers protection against oxidative stress and whether its expression is redox regulated. This project will use standard yeast molecular techniques and biochemistry to address these questions.

3. Control of growth of vascular cells by heme oxygenase1

Supervisor + contact details:

• Dr Konstanze Beck
• Professor Roland Stocker

Atherosclerosis is the major single cause of cardiovascular disease (CVD) that itself remains the single major cause of death in Western countries including Australia. While lipid-lowering drugs (e.g., statins) have been extremely successful in lowering CVD, there nevertheless is an urgent need for the development of novel drugs that protect against atherosclerotic vascular disease by means other than lipid lowering. We recently identified heme oxygenase1 (HO1) as a target of the anti-atherosclerotic action of an old, now uncommonly used, drug (probucol) and are developing novel probucol analogs that share the beneficial but not undesirable side effects of probucol. Regulation of HO1 by probucol and the novel drugs we are developing provides several protective effects in vivo, including the promotion of re-endothelialization and the inhibition of excessive proliferation of vascular smooth muscle cells. In vitro studies confirm that the novel drugs induce HO1 mRNA, protein and activity in vascular smooth muscle cells, and this is directly responsible for the inhibition of excessive growth of these cells. However, at present, we know much less about how the novel drugs exert this striking cell-specific effect, i.e., they promote (rather than inhibit) the growth of endothelial cells. As hydrogen peroxide (H2O2) is the only agent known to have such cell type-specific effect, this project will investigate the effect of the novel drugs on enzymes involved in H2O2 synthesis and metabolism. This will be examined in both endothelial and vascular smooth muscle cells. The techniques involved include cell culture, molecular techniques (e.g., to increase and decrease HO1 expression) and biochemical methods (to relate differences in HO1 activity to differences in peroxidase activity).

4. The role of heme oxygenase-1 in cerebral malaria infection

Supervisor + contact details:

• Professor Roland Stocker
• Professor Nicholas Hunt

High activity of heme oxygenase1 (HO1) in host brain and in tissue macrophages has recently been reported to protect against experimental cerebral malaria, and this protective effect could be recapitulated by administration of carbon monoxide (CO), a metabolic product of heme oxygenase activity. It is thought that CO binds to hemoglobin released from erythrocytes undergoing rupture during the blood stage of the parasite life cycle, and by doing so prevents hemoglobin from releasing the toxic heme that otherwise damages endothelial cells and contributes to the adherence to, and hence accumulation of, CD8+ T cells in cerebral blood vessels. The latter event has been intimately linked to the pathogenesis of cerebral malaria. High endogenous activity of heme oxygenase, or induction of HO-1 by pharmaceutical agents, is thought to protect against cerebral malaria because it results in enhanced generation of CO.

As heme plays a central role in the proposed new function of HO-1, this project will examine (i) the potential role of the heme-binding protein hemopexin in cerebral malaria, (ii) where precisely HO-1 is induced during infection, and (iii) whether metabolic products in addition to CO may impact on disease outcome. For this, the mouse model of cerebral malaria established in the laboratory of Professor Hunt will be used in conjunction with techniques available in the laboratory of Professor Stocker. The outcome of malarial infection will be assessed by survival, neurological and biochemical parameters.

5. Imaging redox regulation of cellular signaling systems

Supervisor + contact details:

• Dr Sabine Wimmer-Kleikamp
• Professor Roland Stocker

If you chose this project you will become familiar with a number of cutting edge microscopy and imaging technologies, in combination with biochemical and molecular biology methods. Receptor tyrosine kinases (RTKs) regulate key cellular processes like cell migration, differentiation, and proliferation. Abnormal signaling of many family members has been linked to diseases, such as cancer and vascular disease. The aim of this project is to identify oxidants and enzymes that affect the redox state and redox processes following receptor tyrosine kinase activation on endothelial cells. We will approach this question from both a cellular biology and biochemical perspective and test the functional relevance of our findings in experimental animal models. This multifaceted project will provide a better understanding of these complex processes, which may allow the development of novel therapies to target abnormal redox-modulated pathways in endothelial disease.

6. Role of cytochrome b₅ in the reductive activation of indoleamine 2,3-dioxygenase

Supervisor + contact details:

• Dr Ghassan Maghzal
• Professor Roland Stocker

Human indoleamine 2,3-dioxygenase (IDO) is an intracellular heme enzyme that catalyzes the oxidative metabolism of LTryptophan (L-Trp) along the kynurenine (kyn) pathway. The induction of IDO and the formation of kynurenine pathway metabolites have been implicated in processes such as immune regulation, neuropathology, microbial and tumor defense and more recently by our group in the regulation of vascular tone.

IDO requires to be activated via reduction of its ferric heme. For the last 30 years, the dogma has been that IDO requires and consumes superoxide anion radical (O2-^.) to metabolise L-Trp to Kyn. We observed that O2^. can also activate recombinant human IDO. However, the extent of this activation is modest, and small changes in the cellular concentration of O2. barely affect IDO activity, suggesting that O2^. plays only a minor role. Instead, we have recently obtained evidence for a role of microsomal cytochrome b5 and NAPDH cytochrome P450 reductase in the activation of cellular IDO. In this project, we will investigate the mechanism of cytochrome b5-induced activation of IDO by examining whether a physical interaction is required. We will also investigate if the mitochondrial form of cytochrome b5 is also involved in the activation of IDO.