Centre for Vascular Research University of Sydney - Honours projects available in 2010

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

This group focuses on understanding how oxidative processes contribute to the regulation of blood vessel function in health and atherosclerosis and related cardiovascular and diabetic disease. IN their research they use various animal models of disease; human tissue; intact blood vessels; tissue culture; advanced microscopy; genetic, molecular and pharmacological tools; gene expression analysis; histology; immunohistochemistry; HPLC, LC-MS and biochemical approaches.

Recent interesting publications:

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

Maghzal G, Thomas SR, Hunt NH, Stocker R. Cytochrome b5, not superoxide anion radical, is a major reductant of indoleamine 2,3‑dioxygenase in human cells. J Biol Chem 2008;283:12014-12025

Wu BJ, Midwinter R, Cassano C, Beck K, Wang Y, Changsiri B, Gamble JR, Stocker R. Heme oxygenase-1 increases endothelial progenitor cells. Arterioscl Thromb Vasc Biol 2009;in press

1. Unravelling the function of heme oxygenase-1 using a genetic approach and Biochemistry

Supervisor + contact details:

• Professor Roland Stocker
• Ms Emma Collinson

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 additional activities that translate into protection against diseases associated with increased oxidative stress, such as cardiovascular, neurodegenerative and inflammatory conditions. The mechanisms underlying these protective effects however, are largely unknown, although antioxidant protection has been put forward as one likely possibility. Recently, a functionally active yeast heme oxygenase has been identified. Work in the laboratory has shown that the yeast homolog of heme oxygenase-1, Hmx1p, also protects against oxidative stress. Using a global transcriptional approach, we have demonstrated that, contrary to present dogma, this protection is achieved via up-regulation of transcripts encoding enzymes involved in cellular antioxidant defense. This project will use standard yeast molecular techniques and biochemistry to further elucidate this novel antioxidant protection.

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

Supervisor + contact details:

• Professor Roland Stocker
• Dr Ghassan Maghzal

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. This project will investigate how cytochrome b₅ activates 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. This project will use standard cell culture, molecular biology, Western blotting, protein chemistry and biochemical techniques.

3. Regulating vascular cells: Induction of Apoptosis by Heme oxygenase-1

Supervisor + contact details:

• Dr Robyn Midwinter
• Professor Roland Stocker

Atherosclerosis is a major cause of heart attacks and stroke in Australia. There is an urgent need to develop novel drugs with anti-inflammatory and anti-oxidant properties to both prevent the progression of the disease and to help repair any damage to the vessel after it has occurred.

Our group identified the protein heme oxygenase-1 (HO-1) as a target of the anti-atherosclerotic action of an old lipid lowering drug, probucol, and are developing novel probucol analogs that share the beneficial effects of probucol. Induction of HO-1 by probucol, in animals, can provide protection by inhibiting the excessive proliferation of vascular smooth muscle (SMC) cells, while promoting the growth of endothelial cells after the vessel has been damaged. While in vitro probucol analogs induce HO-1 mRNA and protein and increase the proliferation of endothelial cells while inhibiting vascular cell growth.

This project aims to increase our knowledge on how HO-1 exerts this striking cell specific effect, by looking more closely at the specific proliferation and apoptotic signalling pathways in both endothelial and SMC that are altered when HO-1 is induced. The techniques involved will include cell culture, and analysing by Western blot, the specific cell signalling pathways altered by HO-1 induction, along with looking at various apoptotic pathways by flow cytometry.

4. Contribution of indoleamine 2,3-dioxygenase to the regulation of vascular tone

Supervisor + contact details:

• Professor Roland Stocker
• Dr Yutang Wang
• 

High blood pressure or hypertension is a major cause for morbidity and mortality in society. Research into the regulation of vascular tone will provide insight into the pathogenesis of hypertension and hence produce new therapies.

We have shown recently that during systemic experimental inflammation such as malaria and endotoxemic shock, endothelial cells express the enzyme indoleamine 2,3-dioxygeanse (IDO) that converts tryptophan to kynurenine. Kynurenine itself relaxes smooth muscle via activation of soluble guanylate and adenylate cyclases, and this can result in a decrease in blood pressure. This project investigates the role of IDO in the regulation of vascular tone in human placenta, and its potential relevance to intra-uterine growth. The project requires close interaction with staff at the Royal Prince Alfred Hospital, from where placental material will be obtained. The work will involve vessel function studies, in combination with cellular and mechanistic studies, and the integration of the experimental work with clinical outcomes.

5. Antioxidant drugs as novel treatments for diabetes and atherosclerosis associated with diabetes

Supervisor + contact details:

• Professor Roland Stocker
• Dr Neil Hime

Diabetes is characterized by an inability of the body to regulate glucose metabolism. This can be the result of damaged endocrine cells in the pancreas having poor insulin secretion (type 1 diabetes) and/or the inability of tissues to utilize insulin and take up glucose from the blood (type 2 diabetes). Type 2 diabetes accounts for more than 95% of cases of diabetes in Australia and its incidence is increasing rapidly. Both types of diabetes can lead to severe health problems, particularly those associated with atherosclerosis of large blood vessels (such as heart disease) and small arteries (blindness and gangrene).

Probucol is an old antioxidant drug that lowers blood cholesterol and reduces atherosclerosis but is no longer used clinically in Australia because of detrimental side effects. We use probucol experimentally as it has been shown to improve the ability of the pancreas to secrete insulin. A derivative of probucol, called succinobucol, does not have the side effects of probucol and has been shown in a human clinical trial to delay the onset of type 2-diabetes. We have shown that the anti-atherosclerotic action of probucol and probucol derivatives, including succinobucol, depends on the induction of the enzyme heme-oxygenase-1 (HO-1). We are using animal models of both type 1 and type 2-diabetes to determine whether the anti-diabetic effects of probucol and succinobucol are dependent on induction of HO-1, and whether these drugs are effective in treating atherosclerosis that is associated with diabetes. Other derivatives of probucol will also to be tested. This project will use techniques including: in vivo tests of glucose metabolism, immunohistochemistry, ELISA, quantification of atherosclerosis and quantitative real time PCR to determine the effectiveness and mechanism of these novel drugs.

These studies will determine whether HO-1 induction is a legitimate target for the treatment of diabetes and atherosclerosis associated with diabetes and is likely to show which derivatives of probucol are favourable to be tested in clinical trials.

6. Unravelling the molecular mechanism behind heme oxygenase 1 induced adaptive response.

Supervisor + contact details:

• Professor Roland Stocker
• Dr Maria Lonn

Atherosclerosis is the major cause of cardiovascular disease and the primary cause of death in the western world. It is a progressive disease, which eventually can lead to a reduction in blood supply to vital organs. Heme oxygenase was identified in 1968 as a heme degrading protein. However, its role has since expanded and the enzyme is now also regarded to protect against a number of pathologies, including atherosclerosis. Several pharmacological agents used against cardiovascular disease induce heme oxygenase. However, it remains unclear how precisely induction of heme oxygenase protects against diseases like atherosclerosis. We hypothesise that the protective effect originates from an adaptive response of cells to prolonged increases in the heme oxygenase activity rather than its metabolic activity per se. Indeed, we have observed that stable, but not transient, expression of heme oxygenase‑1 profoundly affects cellular iron homeostasis. This is accompanied by an increase in superoxide production, yet surprisingly these cells show an increased ability to withstand an oxidant challenge. The aim of this project is to unravel the molecular mechanism underlying this adaptive response, with a focus on the increased superoxide production. During this project we will use standard methods in cell culture, molecular biology, protein chemistry and various biochemical techniques.