2011 Student Research P ono urs - Baker Institute Student Research... · We have recently shown in laboratory mice or patients with acute myocardial infarction functional ... The

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Cardiology & Therapeutics 3

Experimental Cardiology 4 Heart Failure Research 8 Heart Failure Pharmacology 12 Molecular Cardiology 17

Diabetic Complications 19

Advanced Glycation 20 Human Epigenetics 22 Oxidative Stress 29 Proliferation & Fibrosis 32

Metabolism & Obesity 33

Cardiac Hypertrophy 34 Cellular & Molecular Metabolism 37 Metabolic & Vascular Physiology 40 Muscle Biology & Therapeutics 45 Nutritional Studies 46 Physical Activity 48

Population Studies & Profiling 52

Clinical Diabetes & Epidemiology 53 Genomics & Systems Biology 55 Metabolomics 58

Vascular & Hypertension 61

Atherothrombosis & Vascular Biology Laboratory 62 Neuropharmacology 68 Stroke Epidemiology 70

3 2011Baker IDI Student Projects

Cardiology & Therapeutics


Experimental Cardiology

Xiao‐Jun Du xiao‐[email protected] 8532 1267

Research Focus Our research has focused on mechanisms and novel therapies of pathological components of a failing heart

• Cardiac inflammation and remodeling following acute myocardial‐infarct; • Cardiovascular fibrosis (accumulation of excessive amount of scar tissue) and its reversal • Autonomic nervous control and beta‐adrenergic signaling in heart disease

Project 1: Cellular biomarkers of post‐infarct myocardial inflammation Supervisors: Xiao‐Jun Du, Karen Lu Fang, Xiao‐Ming Gao Acute cardiac ischemia and infarction evokes marked and sustained inflammatory responses of whole body and at compromised cardiac muscle. The extent of inflammation contributes significantly to the extent of cardiac injury and ventricular remodeling. We have recently shown in laboratory mice or patients with acute myocardial infarction functional changes of peripheral blood mononuclear cells (PBMC) indicating activation of PBMC in circulation. We also have experimental evidence that difference in the scale of PBMC activity relates to the degree of cardiac inflammatory responses and subsequent ventricular remodeling. In this project, we plan to address the question whether the functional state of PBMC can be used as a cellular marker for the extent cardiac inflammation and injury after acute myocardial infarction (MI). Our working hypothesis is that PBMCs are activated following acute MI as the whole‐body inflammatory responses. The extent of activation is in proportion to the severity of the disease insult and magnitude of regional inflammation, and contributes to the overall cardiac damage.

Research approaches:

Animal model of MI will be induced by open‐chest surgery in mice to occlude the left coronary artery. At the different time‐points after MI, blood samples will be collected and BPMC prepared following our standard protocol. Cardiac function and remodeling in mice will be determined by non‐invasive echocardiography.

Molecular and biochemical assay: expression at mRNA and protein levels of pro‐inflammatory genes (cytokines, adhesion molecules, matrix metalloproteinases (MMP) by real‐time qPCR, ELISA and western blot;

Whole blood cell sorting (FACS): to determine cell‐cell interactions and expression of pro‐inflammatory ligands/receptors and monocyte‐platelet conjugation as well as subset of monocytes.

Measures of cardiac inflammation will be determined by inflammatory cell infiltration (immunohistochemistry) and expression of inflammatory markers ( zymography, RT‐PCR).


Human PBMC: will be prepared from patients with acute MI and cultured. Similar assays will be performed according to findings from animal model.

Project 2: Reversal of arterial remodeling and stiffening by relaxin therapy Supervisors: Xiao‐Jun Du, Qi Xu, Helen Kiriazis Hypertension and/or ageing result in structural changes (remodelling) of conduit arteries and vessel stiffening, which is an independent risk factor for cardiovascular morbidity and mortality. Thus, reversal of large artery remodelling/stiffening is regarded as an important therapeutic goal. However, current anti‐hypertensive therapies are in general unsatisfactory and slow in achieving this and there has been no report of a drug that can reverse arterial remodelling within a short‐term. Our recent studies have demonstrated that the reproductive peptide hormone relaxin is potent and specific in the reversal of established fibrosis in the heart and large arteries. This project will further these findings and seek for pre‐clinical evaluation of relaxin on its specific ability to reverse large arterial remodelling/stiffening seen in hypertension and ageing. The plan is based on the well‐documented therapeutic actions of relaxin and on our recent findings in senescent spontaneously hypertensive rats (SHR) showing that short‐term relaxin therapy reversed large arterial remodelling and improved arterial compliance. This project would be expected to generate proof‐of‐concept data critical for the design of clinical trials using relaxin either as monotherapy or in combination with other drugs to reverse arterial remodelling/stiffening in ageing and hypertensive patients.

The study will be conducted on aged SHR and normotensive control rats (WKY). Detailed assessment of blood pressure, large artery stiffness and histological changes in the large artery will be performed in rats without and with relaxin therapy for a period of four weeks. The persistence of the efficacy of relaxin will also be examined. In vitro study will examine the action of relaxin on cultured human vascular smooth muscle cells and adventitial fibroblasts focusing on collagen and elastin turnover.

This project is suitable for honours students as well as students wishing to continue for PhD degree.

Project 3: Cardiac phenotype in a transgenic model of Huntington’s disease Supervisors: Xiao‐Jun Du, Helen Kiriazis, Pamela Davern (Neuropharmacology Lab)

Huntington’s disease (HD) is one of the neurodegenerative diseases due to intracellular accumulation of poly‐glutamine (Poly‐Q) aggregates with profound interference in gene expression in the brain tissue. HD represents a single gene mediated neuronal disorder with poor quolity of life and shortened life‐span. Although heart disorder is believed to be the second leading cause of death in HD patients accounting for >30% mortality, the nature of the cardiac abnormalities in HD patients remain largely unexplored either clinically or experimentally.

This proejct will be conducted using a transgenic mouse model of HD, R6/1, due to expression of a human Huntington gene derived from a HD patient. Our recent experiments on this strain of mice have revealed significnat abmormalities in the cardiac regulation by the autonomic nervous system. As the consequence, HD mice showed abbarent levels and regularity in heart rate and contractile function at baseline and during �‐adrenegic activation. Interestingly, these abnormalities occurs at pre‐motor symdrome phase. We will examine the autonomic nervous function by telemetry method in conscious animals and test the cardiac responses to stressors including pressure


overload and ischemic insult to fully explore the cardiac phenotype in the HD mice. We will also test effects of beta‐adrenergic antagonists and muscarinic antagonists on neurocardiac phenotype in HD mice.

This study represents the first to thoroughly investigate cardiac phenotype in a clinically relevant mouse model of HD, an area that has rarely been explored. Whereas HD is not a common disease in Australia, its genetic mechanism is representative of a class of neurodegenerative diseases (e.g. Alzheimer’s, Parkinson’s, familial amyotrophic lateral sclerosis FALS), recently defined as Poly‐Q diseases.

Project 4: Novel inflammation‐independent action of MIF in hypertrophic heart Supervisors: Xiao‐Ming Gao, Xiao‐Jun Du,

Hypertension is a leading cause of heart failure and a major risk factor for cardiovascular diseases. Cardiac hypertrophy is an adaptive response to increased work‐load. However, if hemodynamic stress persists, maladaptive hypertrophy ensues and eventually leads to heart failure through mechanisms that remain poorly understood. Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that regulates inflammatory response and energy metabolism. MIF acts as a proinflammatory cytokine in a variety of inflammatory disorders, including ischemic heart disease. Cardioprotective effects have been reported by inhibition of MIF. However, our recent pilot studies have found that deletion of MIF gene leads to an exacerbated cardiac hypertrophy and dysfunction in response to long‐term pressure overload. This finding raises an interesting question: why MIF has such distinctly actions under different disease stress? We propose that a novel and inflammatory‐independent action of MIF contributes to such benefit in the hypertrophic heart. We will use in vivo and in vitro model to investigate the potential mechanism. Results from this project will shed a light on the novel role of MIF in energy balance and angiogenesis in hypertrophic progression and provide important experimental evidence for developing a new anti‐hypertrophy strategy.

Research approach

1. In vivo animal model of pressure overload induced by microsurgery in genetic‐modified mice. Echocardiography and micromanometry will be performed to monitor the structural and functional changes. Molecular studies to examine alterations of key genes and proteins during the hypertrophy development.

2. In vitro model: Cell culture work to further dissecting the key molecular mechanism.

Project 5: Targeting ventricular remodeling and fibrosis of infarcted heart by anti‐platelet therapy Supervisors: Xiao‐Jun Du, Helen Kiriazis, Anthony Dart

Inflammation following acute myocardial infarction (MI) contributes to geometry change of the ventricle (ventricular remodeling) and fibrosis of non‐infarcted regions, increasing the risk of heart failure. Recent studies, including ours, have shown that platelets contribute significantly to the inflammatory responses of infracted hearts and that anti‐platelet therapy is effective in suppressing acute inflammation and related complications. However, it remains to be


studied whether anti‐platelet therapy given during the acute phase of MI exerts longer term benefits in terms of ventricular remodeling and fibrosis.

This project will be conducted on mice with surgically induced MI. Animals will then be assigned to either no treatment control or anti‐platelet drug treatment. Animals will be then examined by serial echocardiography to estimate ventricular remodeling and function over a period of 4 weeks. Detailed histopathological, molecular and biochemical analyses will be performed on hearts to evaluate the extent of ventricular remodeling, fibrotic healing of infracted region and interstitial fibrosis of non‐infarcted myocardium.

This project is expected to generate novel information on the efficacy of anti‐platelet drugs in the setting of ischemic heart disease. This project is suitable for honours student with strong interest in cardiology.

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Project Title: Mitochondrial L‐arginine transport and its role in the pathogenesis of heart failure Supervisors: Professor David Kaye and Dr. Kylie Vernados The failing heart is characterized metabolically by heightened oxidative stress, abnormal mitochondrial function and altered energy metabolism. Together these processes impact adversely on contractile function and contribute to the progressive nature of heart failure. Mitochondrial metabolism is known to be regulated by nitric oxide (NO), and in this context we have previously shown that myocardial L‐arginine metabolism and expression of the L‐arginine CAT1 transporter appears to be altered in heart failure. Whilst a mitochondrial specific isoform of nitric oxide synthase (NOS) has been demonstrated, little is known about mitochondrial L‐arginine transport and its role in mitochondrial biology.

In this study we aim to characterise the biochemical properties and regulation of the mitochondrial L‐arginine transporter, determine its role in mitochondrial biology and test the novel hypothesis that impaired mitochondrial L‐arginine transport plays a key role in the pathogenesis of heart failure and other cardiovascular disease states. We will also examine the biochemical and physiological effects of augmented mitochondrial L‐arginine transport. This project is suitable for a Honours or PhD student and can be tailored to the students interests or abilities. It will provide the opportunity for learning a range of techniques including the isolation of respiring mitochondria from healthy hearts and various disease models such as heart failure and diabetic models; cell culture; biochemical measures (western blot, L‐arginine uptake, markers of oxidative/nitrosative damage); confocal microscopy; and molecular biology (cell transfection, real‐time PCR).

Understanding the nature of mitochondrial L‐arginine transport and its role in mitochondrial biology will provide new insights into potential therapies for heart failure and other myocardial disorders.


Project Title: Wnt signalling pathways and myocardial ischemia‐reperfusion injury Supervisors: Professor David Kaye and Dr. Kylie Vernados

Coronary heart disease remains the greatest killer in the Western world, and given our ageing population, along with increasing risk factors, it is predicted to become an even more significant problem worldwide over the next 20 years. Ischemic heart disease arises when there is an imbalance between the myocardial oxygen demand and blood supply. Faulty functioning of the coronary circulation, most commonly due to fatty atherosclerotic plaques or blood clots,

causes a reduction in blood flow, and subsequently ischemia and/or myocardial infarction (MI). Reperfusion (restoration of blood flow), without doubt is the most effective treatment for ischemic myocardium. However, this produces deleterious effects upon cells, and depending on the severity, may ultimately lead to cell death. The pathogenesis of ischemia‐reperfusion injury is complex including endothelial dysfunction, calcium overload and heightened oxidative stress although the key driving mechanism remains uncertain. Existence of reperfusion injury has clinical implications in the recovery of cardiac function to pre‐ischemic levels, particularly following invasive procedures such as the application of stents, coronary bypass surgery, coronary angioplasty, heart transplantation or following MI. These changes contribute to increased myocardial stiffness, contractile dysfunction, reduced cardiac output, diastolic dysfunction, ventricular fibrillation and left ventricular failure which are seen after MI. Eventually this can lead to ventricular dilatation, heart failure and even sudden death.

The Wnt signalling pathways have been well conserved through evolutional processes among a variety of species and play important roles in embryonic development, cellular proliferation, differentiation, migration and survival. In humans, Wnt‐mediated signalling has been known to play a role in carcinogenesis however accumulating evidence now indicates Wnt signalling is also an important mediator of inflammation and recovery from injury.

In this project we will investigate the role of the Wnt signalling pathways in ischemia‐reperfusion injury of the heart and determine whether manipulation of this system provides cardioprotection. This will be done in both whole hearts and at a cellular level using models of ischemia‐reperfusion injury and hypoxia‐reoxygenation. This project will provide the opportunity for learning a range of techniques including cell culture; langendorff perfusion of isolated hearts including functional analysis; biochemical measures (western blot, markers of cellular injury); and molecular biology (cell transfection, real‐time PCR).


Project Title: Preventing Cardiac Fibrosis in Heart Failure Supervisors: Prof David Kaye and Dr Po‐Yin Chu In addition to a loss of pumping capacity, the failing heart is characterised by increased stiffness due to the accumulation of interstitial collagen (‘fibrosis’). Physiologically this leads to an elevation of the pressure inside the heart during diastole, contributing further to the symptoms experienced by patients living with heart failure. To date, the specific mechanisms that contribute to fibrosis in heart failure have not been clearly determined and accordingly there are no treatments available to specifically address this problem. Recently we have identified that fibrocytes originating in the bone marrow travel to the heart when it fails. This work has extended to the identification of putative homing signals released by the failing heart.

In this study we aim to evaluate the influence of the activation of specific signalling cascade on the recruitment of pro‐fibrotic cells to the heart. Using selective antagonists the study will evaluate the influence of cytokine inhibition on fibroblast proliferation in cell culture model under conditions that mimic those of heart failure. In appropriate circumstances (eg PhD studies) this project will be extended into animal models of cardiac fibrosis, with relevant studies involving chronic administration of antagonists. Techniques to be learned include cell culture, biochemical measures (western blot, markers of cellular proliferation and hypertrophy); and molecular biology (cell transfection, real‐time PCR).


Heart Failure Pharmacology

Dr Rebecca H Ritchie [email protected] 8532 1392

Research Focus The Heart Failure Pharmacology laboratory comprises 2 postdoctoral fellows, 2 research assistant, 1 PhD and 2 Honours students under the leadership of Rebecca Ritchie. Our core activities centre on the prevention and treatment of the cardiac complications of diabetes, hypertension (high blood pressure), myocardial infarction (heart attack) and arthritis, alone and in combination. These complications include structural changes to the heart, specifically cardiac hypertrophy (an enlarged heart and/or cardiac myocytes) and cardiac fibrosis (increased extracellular matrix deposition), which together are known as : left ventricular (LV) remodeling. Cardiac function may also be impaired: LV systolic dysfunction (reduced cardiac contractile function) and/or diastolic dysfunction (impaired cardiac relaxation and delayed LV filling following each contraction) may be evident. These cardiac pathophysiological disorders can otherwise progress to heart failure (and perhaps death) if not well managed.

Project 1: Nitroxyl, a naturally‐occurring molecule for treating diabetic heart disease

Supervisors: Rebecca Ritchie and Jennifer Irvine The leading cause of death of patients with diabetes is cardiovascular complications; the cardiac dysfunction manifest in these patients is a major contributor to morbidity and mortality, often exacerbated by underlying LV fibrosis, hypertrophy of cardiac myocytes, and excess generation of reactive oxygen species (ROS) such superoxide . The nitric oxide (NO•)/cGMP signalling system is as a powerful cardiac antihypertrophic mechanism. Nitroxyl (HNO), a novel redox sibling of NO•, has several therapeutic advantages for the treatment of cardiovascular diseases. We have shown that HNO prevents hypertrophy and generation of superoxide in isolated cardiomyocytes. Excitingly, HNO also potentiates cardiac function, in contrast to NO•, via the cardiac calcium handling proteins, SERCA2a (sarcoplasmic reticulum Ca2+ATPase) and the ryanodine receptor RyR2. The activity and expression of these enzymes is abnormally affected by diabetes, and together with the upregulation of ROS is recognised for their causal role in diabetes‐induced LV dysfunction. HNO thus is likely to be favourable for treating diabetic cardiac disease.

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Our general hypothesis is that HNO REPRESENTS NOVEL PHARMACOTHERAPY FOR THE PREVENTION AND TREATMENT OF DIABETES‐INDUCED MYOCARDIAL DYSFUNCTION. We aim to demonstrate that (i) HNO is cardioprotective in cardiomyocytes isolated from diabetic hearts; (ii) that HNO acutely enhances cardiac function in the diabetic heart, and (iii) chronic HNO rescues myocardial structure and function in the diabetic heart over the longer‐term. The scope of this project will be modified depending on the nature of the project sought (e.g. Honours or PhD project), and may include in vitro techniques (using cardiomyocytes and/or cardiac fibroblasts), ex vivo (Langendorff‐perfused isolated rat hearts from diabetic vs non‐diabetic animals) or in vivo models of diabetic cardiac disease. The project can also be tailored according to the student’s abilities and interests and will provide the opportunity for learning a range of techniques, including cell culture, biochemical (Westerns, ROS detection, ELISA), molecular (real‐time PCR, Northerns) and physiological/pharmacological (e.g. assessment of cardiac function, blood pressure) techniques. The outcome of this project will be definitive information regarding the mechanism(s) and effectiveness of HNO‐mediated rescue of myocardial function specifically in diabetes. Ultimately, HNO‐based strategies may offer new treatment options for diabetic cardiac disease, either alone or on top of standard care.

Project 2: B‐type natriuretic peptide rescues LV fibrosis & dysfunction in type 2 diabetes

Supervisors: Rebecca Ritchie and Marissa Bowden As described above, the leading cause of death of patients with diabetes is cardiovascular complications. Diabetic cardiac disease, or diabetic cardiomyopathy is defined as changes in the myocardium independent of associated coronary vascular disease. The precise cause of diabetic cardiomyopathy in humans is poorly understood, but up to 90% of patients diagnosed with the disorder have type 2 diabetes. Cardiac fibrosis, cardiomyocyte hypertrophy and diastolic dysfunction (often followed by systolic dysfunction) are observed in diabetic cardiac disease, and are associated with alterations in myocardial energy metabolism and microvascular changes. These abnormalities are often worse than predicted from the level of coexistent coronary heart disease or hypertension.

We have previously demonstrated that the cardiac hormone B‐type natriuretic peptide (BNP) has acute antihypertrophic actions in the type 1 diabetic heart. Chronic treatment of type 1 diabetic rats with the related peptide, atrial natriuretic peptide (ANP), markedly improves cardiac function and limits cardiomyocyte hypertrophy, but was not able to reduce cardiac fibrosis (likely a ROS‐triggered event). The efficacy of the ANP/BNP family has not been investigated in type 2 diabetes. As mice lacking BNP exhibit spontaneous cardiac fibrosis, together with BNP’s


ROS‐suppressing actions, endogenous BNP is thought to have exciting anti‐fibrotic properties. We now propose that chronic BNP treatment rescues diabetes‐induced cardiac fibrosis and LV dysfunction in a mouse model of type 2 diabetes in vivo. The project will provide the opportunity for learning biochemical (Westerns, ROS detection, ELISA), histological/immunohistochemical, molecular (real‐time PCR, Northerns) and physiological/pharmacological (e.g. assessment of cardiac function, blood pressure) techniques. We predict that the effectiveness of BNP will resemble a combination of its sibling peptide, ANP, and an antioxidant. BNP‐based therapies alone or in combination with standard care may provide a more effective means to limit diabetes‐induced cardiac fibrosis and hence rescue diabetic cardiac function.

Project 3: Targeting the anti‐inflammatory protein Annexin‐A1 for protection from myocardial infarction (heart attack)

Supervisor: Rebecca Ritchie Myocardial ischaemia, in which coronary blood flow is reduced, causes anginal chest pain, myocardial infarction (MI, also known as heart attack), and death. Indeed, MI is Australia’s major cause of morbidity and mortality, and hence new treatments are thus essential. The primary determinant of outcome from MI is the extent of cell death during and after ischaemia, from necrosis, apoptosis and/or autophagy. Restoration of blood flow (reperfusion) however is

associated with the development of further cell death and impaired recovery of cardiac function, referred to as “reperfusion injury”. Myocardial ischaemia‐reperfusion induces an inflammatory response, with damage resulting from both infiltration of circulating inflammatory cells (neutrophils, monocytes), as well as neutrophil‐independent direct actions on myocardium and endothelium (including Ca2+ overload, ROS generation and impaired mitochondrial regulation all contributing mechanisms to cell death. In addition, there is incomplete recovery of left ventricular (LV) function. Together these phenomena contribute to increased risk of ischaemic cardiomyopathy, heart failure and death. Novel treatment strategies that

protect against multiple mechanisms of MI injury, both neutrophil‐mediated and myocardial‐directed, will have major clinical impact.

We have previously shown that the anti‐inflammatory protein Annexin‐A1 preserves cardiomyocyte viability and contractile function in vitro, rescues post‐ischaemic recovery of myocardial function in the heart, targets neutrophil and non‐neutrophil causes of myocardial dysfunction, and has powerful systemic anti‐inflammatory actions. Building on this previous work with Annexin‐A1, we now propose that novel synthetic Annexin‐A1 mimetics are a novel lead for rescuing myocardial viability and contractile function in the intact heart post MI. We aim to demonstrate that our new Annexin‐A1 mimetics are cardioprotective in (i) ischaemic cardiomyocytes in vitro; (ii) the intact heart ex vivo, and (iii) with chronic treatment in mice subjected to MI in vivo. The scope of this project will be modified depending on the nature of the project sought (e.g. Honours or PhD project) and can also be tailored according to the student’s abilities and interests. It will provide the opportunity for learning a range of techniques, including cell culture, biochemical (Westerns, ELISA, cardiac injury assays), histological/immunohistochemical, molecular (real‐time PCR,

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Heart Failure Pharmacology Laboratory 2010


Molecular Cardiology

Associate Professor Liz Woodcock [email protected] 8532 1255 The central role of PLC 1b in atrial pathology We have shown that PLC 1b is the major PLC subtype in heart and that this PLC is elevated in atria from valvular heart disease patients and in a mouse model with dilated cardiomyopathy. We have prepared adenoviruses expressing the active PLC and these viruses can be used in studies with isolated cardiomyocytes. In addition, we have designed and made an inhibitory mini‐gene that selectively inhibits PLC 1b activity. This mini‐gene is currently being incorporated into a transgenic mouse line by our collaborators in St Louis.

The proposed studies will involve the following:

Examining signalling responses in these mini gene expressing mice.

Crossing these mini‐gene mice with the mice with dilated cardiomyopathy to evaluate the contribution of PLC 1b to the disease.

Examining arrhythmogenic responses in the mice to define the involvement of PLC1b to this pathological response.

The relationship between PLC activity and atrial dilatation Atrial dilatation is a major contributor to cardiac disease, especially atrial fibrillation. We have shown that there is a correlation between the size of the left atrium of both humans and mice and the activity of PLC. We now need to show whether increased PLC activity causes dilatation or whether dilatation causes increased PLC activity. We have also shown that overexpression of PLC 1b in cardiomyocytes causes cell death and death of cardiomyocytes is a major cause of dilatation.

The proposed experiments are as follows:

Overexpress PLC 1b in hearts or atria of mice and show that this causes dilatation.

A number of different models will be sued that show atrial dilatation and PLC 1b expression and activity will be measured.


How does PLC 1b bind to the cardiomyocyte membrane to be selectively active and cause pathological problems? We have shown that the unique C‐terminal tail of PLC1b is responsible for its binding to the sarcolemma. However, in order to develop selective drugs to prevent this binding and thus inhibit PLC�1b activity in heart, we need to establish exactly how this interaction occurs.

The studies will involve:

Molecular studies to identify amino acid residues involved. This involves mutagenesis followed by preparation of adenoviruses for use with cardiomyocytes in culture.

Identification of the interacting proteins. We have identified a possible candidate using yeast‐2‐hybrid technology, a protein called abLIM‐1. The project will involve using si‐RNA technology to lower the expression of abLIM1 to demonstrate its requirement for PLC 1b responses. We have developed adenoviral vectors for this purpose. Co‐immunoprecipitation and localization studies will also be preformed.

The significance of phospholipase C in atrial dilatation Phospholipase C (PLC is a newly described PLC subtype that is expressed in heart. PLC is a very large protein with a MW of 250 kDa comprised of a number of different functional modules. Currently unpublished data from one of our major collaborators Dr Joan Heller Brown, Department of Pharmacology, UCSD, has shown that PLC mediates responses to thrombin in cardiomyocytes.

Recent studies from our laboratory show that PLC expression is increased in the dilated atria of patients with valvular heart disease and also in a mouse model that has dilated atria. This suggests a relationship to atrial disease, especially dilatation. The dilated human and mouse atria contain thrombi and these will provide a local source of thrombin to activate the heightened PLC. Therefore we think that thrombin/PLC may contribute to atrial pathology directly.

The proposed studies will involve the following:

Measuring thrombin activated PLC responses in human atrial tissue from valve patients and controls.

Similar experiments involving atria from the mice with dilated cardiomyopathy.

Measuring the changes in PLC expression in relation to the progression of disease in the mouse model.

Assessing the effect of the thrombus, by measuring PLC activity in mouse atria wit and without thrombus.

Crossing the mouse cardiomyopathy model with PLC ‐/‐ mice to establish the contribution to disease.


Diabetic Complications


Advanced Glycation

Associate Professor Josephine Forbes [email protected] 8532 1456 Are you interested in why organs turn into toffee with diabetes? What about finding a real cure for diabetes and its complications?

Advanced glycation is the biochemical reaction which occurs in our bodies to make proteins (and us) grow old. Although it is a natural process, food chemists also use this reaction to make our food taste better, perhaps most easily recognised as brewed beer, freshly baked bread crusts and vegemite and other golden delights!

Our laboratory uses a variety of approaches including cell culture, molecular biology, genetically modified mice and human clinical studies. We currently have 11 personnel including 2 post‐doctoral scientists and 3 PhD students. We have an extremely accomplished track record where our work is published in the very best journals in medical research and attracts regular research funding from both national and international sources.

Research Projects Mitochondrial dysfunction as a therapeutic target for diabetes Dysfunction of the small power stations or the “mitochondria”, within our cells is an extremely important step towards irreversible kidney and islet damage, which affects up to 40% of persons with diabetes. As our cellular energy levels are depleted, the cells slow down and eventually die. We have discovered that damage to other cell compartments by advanced glycation (AGEs) can cause and intiate mitochondrial damage and energy depletion from the outside. Advanced glycation end products (AGEs) are sticky sugar/protein complexes and are abundant with sugar excesses such as that seen with diabetes. These AGEs interact with receptors and cause the generation of free oxygen radicals within cells, which directly interrupt energy production by mitochondria. As current therapies do not address this pathway it is a pertinent new target for therapies in diabetes and its complications.


A marriage between lifestyle and genetic factors which affect the development of type 2 diabetes It is well known that certain lifestyle choices such as high dietary fat and processed foods contribute to the development of diabetes. Indeed, bad lifestyle choices have worse effects in some individuals which may be related to susceptibility to diabetes which is present within their genetic make up.

Dietary fat and processed foods are extremely high in a group of sugar modifications known as advanced glycation end products (AGEs). These molecules make our food taste better in addition to providing food chemists and manufacturers with benefits such as longer storage times and less food spoilage. Excessive dietary intake of AGEs has recently been shown to contribute to the development of type 2 diabetes, especially in the context of a high fat diet. Once in the blood stream dietary AGEs can cause inflammation and free oxygen radical production by binding to specific receptors, including the receptor for AGEs, RAGE. Indeed, it is even now predicted that defects in the RAGE gene confer susceptibility to diabetes per se.

Increased dietary AGE intake, directly contributes to elevated levels of circulating AGEs which have been implicated as factors which sustain inflammation in humans. We hypothesise that a reduction in dietary AGE intake will improve insulin sensitivity and first phase insulin secretion in overweight individuals by lowering chronic low‐grade activation of the immune system (CLAIS). This project therefore aims to determine the impact of an ~80% change in dietary AGE intake over a period of two weeks in a randomised blinded cross‐over study design on circulating AGEs, insulin sensitivity and insulin secretory function in young, healthy obese individuals and also establish whether chronic activation of the immune system plays an intermediary role in the association between AGE intake, insulin sensitivity and secretion.


Human Epigenetics

Associate Professor Sam El‐Osta assam.el‐[email protected] 8532 1 Hons and PhD Program and Projects Program 1: Understanding the MeCP2‐associated regulatory complex in disease Supervisor: Assam El‐Osta

Introduction

Alterations in the controls of DNA methylation and histone deacetylation play a profound role in human disease. We are interested in examining epigenetic events such as DNA methylation, histone modification and the determinants involved on chromatin to further our understanding of endogenous gene transcription. These studies are now challenging the way we view gene regulation beyond our simple understanding of "textbook" operations. Our desire to dissect the molecular details allows us to determine the roles of transcriptional regulators and provide us with a greater understanding of how they are involved in transcriptional regulation.

Aims

Our laboratory has a focus on endogenous gene regulation. We have generally relied on genetic and biochemical systems to study gene regulation, because, genetics excels in identifying transcription factors and regulatory complexes required for expression, whereas biochemical testing transcend what they are capable of in vitro. However, neither discipline can tell us definitely the order of events that occur naturally on endogenous genes. Understanding endogenous mechanisms of transcriptional control allows the biologist to manipulate a biological system often seen as difficult to control. Understanding epigenetic events provides an exciting platform to design transcription factors and control the Fragile X Syndrome and the FMR1 gene, which has historically eluded translational science.

Relevant Reading

Nature 393, 386‐9 (1998) Nature Genetics 19, 187‐191 (1998)

Science 302, 885‐889 (2003) Science 302, 890‐893 (2003)

J Biol Chem 279, 46490‐6 (2004) Nature Genetics 37, 254‐4 (2005)

PNAS 102, 17551‐8 (2005)

Nature Genetics 38, 962‐4 (2006) Nature Genetics 38, 964‐7 (2006)

Aim


Characterizing the MeCP2 corepressor associated SWI/SNF complex; what are the biophysical properties of MeCP2 in the brain? And what is the significance of the complex in mental health (fragile X syndrome) ?

Program 2: Improving molecular targeting and treatments for cancer gene therapy Supervisors A/Professor Sam El‐Osta and Dr Tom Karagiannis Introduction

Given the widespread use of radiotherapy in cancer (about 50% of cancer patients are treated with x‐rays), there has been a longstanding interest in the investigation of chemical compounds that can modify cellular responses to ionizing radiation. We are evaluating histone deacetylase inhibitors (HDACi) as potential radiation sensitizers. Our clonogenic survival experiments and other cell‐growth assays indicate that incubation of cells with the HDAC inhibitors, Trichostatin A and valproic acid enhances radiation induced cell‐death. The aim of this honours project is to further investigate the cellular and molecular basis for the radiosensitizing effect.

This Hons and PhD Program will investigate the effect of HDACi on radiation‐induced DSB repair in transcriptionally active or inactive euchromatin and constitutive heterochromatin analyses of γH2AX accumulation and hyperacetylation of histone H3 using chromatin immunoprecipitation (ChIP). The candidate will investigate pre‐treatment with low concentration of HDACi before irradiation, results in classic hallmarks such as histone hyperacetylation and the stable accumulation of γH2AX that is more pronounced in euchromatic alleles than heterochromatic areas of the genome. At this exciting stage of gene therapy, our combined results suggest that the inhibition of HDACs can potentiate therapy by a mechanism that renders DNA more accessible to treatments by histone hyperacetylation in the absence of cytostasis, apoptosis and/or growth arrest in mammalian cells.

Histone deacetylase inhibitors are emerging as a new class of targeted cancer chemotherapeutics. Several histone deacetylase inhibitors are currently in clinical trials and promising anti‐cancer effects at well‐tolerated doses have been observed for both hematologic and solid cancers. Histone deacetylase inhibitors have been shown to induce cell cycle and growth arrest, differentiation and in certain cases apoptosis in cell cultures and in vivo. There is growing commercial and therapeutic interest in potential clinical use of histone deacetylase inhibitors in combination with conventional cancer therapies. The focus of the Hons and PhD Program is on the different mechanisms by which histone deacetylase inhibitors significantly improve cancer therapy.

Aim Improving molecular targeting and treatments for cancer gene therapy

Methods This will require training in cell model immortalisations and tissue culture, protein extraction and purification. Chromatin immunoprecipitation (ChIP).

Keywords Cancer, chemotherapy, radiotherapy, cancer gene therapy, DSB; double strand break, DNA repair,

chromatin, HDAC; histone deacetylase, HDACi; HDAC inhibitors, γH2AX


Program 3: Identification of new regulatory mechanism of transcriptional repression in response to cancer chemotherapy Supervisor A/Professor Sam El‐Osta

Introduction

The general focus of this project is to investigate the mechanisms by which specific MBD1 regulatory complexes serve to integrate and repress gene in models of cancer. Specifically, the project will define the functional roles of specific components of the co‐repressor complexes in gene repression events and characterise the link to cancer‐associated loci such as the multidrug resistance gene (MDR1) using genetic and genomic approaches. The central focus of this aim takes the Teams latest findings on transcriptional repression to the next logical step and further extends the mechanism, as currently understand and published recently in Nature Genetics (2005) 37:254-264 and Oncogene (2005) 24:8061-7075. The project is aimed to examine the controversial point of MBD1 repression independent of direct DNA methylation.

Human methylation dependent proteins are well characterized and the best characterized is MeCP2 which is strongly associated with transcriptional silencing. However, little is known of the role of other MBD co‐repressor complexes such as MBD1 in gene silencing. Repression of transcription exerts an equally fundamental role in gene regulation as activation. We have functionally defined components of the corepressor and coactivator complexes that are required for regulatory activities, proving that a regulated exchange of coactivator and corepressors dictates the level of expression of specific genes in different disease models. We hypothesize that one of the critical functions of these complexes is to integrate MBD1 recruitment in response to input from multiple signaling pathways. The presence of MBD1 associated components in multiple complexes, coupled with their biological roles in development, presents an ideal model for elucidation of basic principles of regulatory gene expression and the generality of this regulation. In cancer tumor suppressor genes arte often silenced and characterized by profound epigenetic changes. However, it is not known whether an epigenetic program and in particular MBD1 corepressor complex is implicated in gene regulation. The team has exciting experimental evidence that MBD1 corepressor complex is critical in regulating gene activity associated with cancer.

Aim Identification of new regulatory mechanism of transcriptional repression in response to cancer chemotherapy

Methods Chromatin immunoprecipitation (ChIP), High‐resolution separation of proteins according to size using FPLC. This will require training in cell model immortalisations and tissue culture, protein extraction and purification.

Keywords Cancer, chemotherapy, drug resistance, MBD1; methyl‐CpG binding protein 1, FPLC; fast protein liquid chromatography, transcriptional repression, chromatin, heterochromatin, euchromatin


Program 4: Epigenetic memory in diabetes and metabolic syndrome Supervisor A/Professor Sam El‐Osta Introduction

Diabetes and its consequence of accelerated vascular complications is a major global clinical problem. Diabetic complications occur as a result of pathological processes activated by chronic hyperglycaemia. Of particular interest is the phenomenon of “metabolic memory” where diabetic patients, despite improved glycaemic control, develop complications as a result of prior poor glycaemic control. In recent clinical evaluates from the large Diabetes Control and Complications Trial, and its follow‐up, the prospective Epidemiology of Diabetes Interventions and Complications trials, diabetic subjects have continued to develop ongoing end‐organ injury, as a result of previous periods of poor glycaemic control. This has been interpreted as indicating that there is a memory of past glycaemia known as “metabolic memory” which is a major determinant of subsequent development of vascular complications. Several hypotheses have been raised to explain this phenomenon, including the possibility of “epigenetic memory”. However, such epigenetic pathways relevant to diabetic complications have not been previously determined. Such a possibility is further suggested from twin studies where it was shown in identical twins that there is a lack of concordance in complications. Although this could be related to environmental differences, epigenetic mechanisms have been postulated to explain this discordancy between identical twins. It remains unknown as to the mechanism whereby hyperglycaemic memory leads to a program of diabetic vascular complications

This Hons and PhD Program will investigate the epigenetic pathways that act as a bridge linking hyperglycaemia to the central molecular and cellular events, which lead to vascular injury in diabetes. We have identified that in the context of a hyperglycaemic milieu, transcriptional competence is directly linked with epigenetic changes. These findings present a new paradigm for histone methyltransferase function and epigenetic modification that is relevant to our understanding of the transcriptional response to glucose. Finally, these findings will provide new targets for generating end‐organ protective agents for the common and devastating clinical problems of diabetic vascular complications.

Aim The role of the histone code hypothesis and the epigenome on hyperglycemic memory

Methods Endothelial cell models, chromatin immunoprecipitation (ChIP), This will require training in cell model immortalisations and tissue culture, protein extraction and purification.

Keywords Diabetes, hyperglycaemia, histone methyltransferase, histone methylation, histone code hypothesis, transcriptional activation, chromatin


Program 5: Heart disease, Stem cells and Epigenetics Supervisor A/Professor Sam El‐Osta Introduction

An enlarged heart, or cardiac hypertrophy, results from an increase in the thickness of the heart muscle in response to stress on the heart and blood system and is the heart’s mechanism of coping with the increased effort of pumping blood around the body. The stressor is often high blood pressure and prolonged hypertrophy is associated with an increased risk of heart disease. Whereas it has been known for years that epigenetic factors, specifically genomic methylation, function to modulate gene transcription, their role in the gene expression profile of compromised hearts associated with aging, hypertrophy and heart failure, remains unknown. The key issue that this project is designed to address relates to how pathological stimuli are finally converted into an altered gene expression profile that eventually leads to hypertrophy and heart failure. A better understanding of the epigenetic factors that control gene transcription in the compromised heart will advance the current knowledge of the mechanism responsible for molecular remodeling in the diseased organ. The planned studies will generate valuable data to answer important questions including whether modulating genomic methylation can alter hypertrophic growth and whether different gene expression profiles in aging or hypertrophic hearts could be partly attributable to epigenetic mechanisms. Furthermore, these studies could inform on the potential of novel therapeutic targets aimed at modulating epigenetic factors to alter or reverse unwanted gene transcription.

We are offering honours and PhD students the opportunity to join the Human Epigenetics Laboratory at the Baker Heart Research Institute and work towards reducing death and disability arising from heart disease in the community. Stem cells have the unique capacity for self‐renewal and differentiation into specialised cell types. We are offering projects using stem cell cultures to understand how the healthy heart develops and hence how heart problems can arise when development goes awry. We will look at important developmental genes and determine how they are controlled as the heart forms from embryonic, to foetal, to neonatal‐like stages.

Aim 1 The role of epigenetic factors on transcriptional regulation in the failing heart

Aim 2 Defining the impact of the foetal gene‐expression program during the differentiation of stem cell‐derived heart cells.

Methods Cardiac cell models, chromatin immunoprecipitation (ChIP), High‐resolution separation of proteins according to size using FPLC, Stem cell culture, Generation of transgenic stem cells.

Keywords Heart disease, hypertrophy, stem cells, differentiation and epigenetic changes


Program 6: Mechanisms of genetic changes in imprinting disorders Supervisors Dr Christine Gicquel and A/Professor Sam El‐Osta Introduction

Genomic imprinting is a particularly attractive example of epigenetic regulation, since in the same cell one of the two parental alleles is stably repressed by epigenetic modifications, whereas the other allele is maintained in an active state. This allele‐specific regulation is entirely dependant on whether the gene is inherited from the mother or from the father. Imprinted genes are organised in clusters that are regulated by imprinting control regions (ICRs). To achieve allele‐specific expression, ICRs are regulated by epigenetic modifications (DNA methylation and modifications of the histone proteins) that differentially mark the parental alleles as either active or repressed. These epigenetic modifications are controlled by DNA methyltransferases, histone modifying proteins, methyl CpG binding domain proteins, insulator proteins and chromatin‐modifying complexes. Genomic imprinting is a multistep process involving the active erasure of existing epigenetic marks in primordial germ cells and the establishment of novel imprints later during gametogenesis (maternal marks in oocytes and paternal marks in male germ cells). Imprinted genes play key functions in development, and more particularly in foetal growth and their epigenetic deregulation results in different human diseases, including cancer. Two imprinted foetal growth disorders [the Beckwith‐Wiedemann (BWS) and the Silver‐Russell (SRS) syndromes] display opposite phenotypes: overgrowth in BWS and growth retardation in SRS. These two disorders are caused by disruption of imprinting of the 11p15 region by epigenetic mechanisms that are opposite: gain of methylation in BWS and loss of methylation in SRS of the same 11p15 ICR. Although abnormal methylation of ICRs have been demonstrated in various imprinting disorders, the exact molecular mechanisms resulting in abnormal methylation of ICRs remains unknown.

Aim 1 ‐ Elucidating the mechanism(s) resulting in loss of imprinting in patients with epigenetic defects by analysing key trans‐acting regulatory factors involved in the maintenance of imprinting during early foetal development.

Aim 2 ‐ Evaluating if biochemical modification of core histones may represent an alternative and/or complementary mechanism to methylation defects in patients with imprinting disorders.

Recommended reading (Nat Genet 2005; 37: 1003) (Bioessays 2006; 28: 453) (Best Pract Res Clin Endocrinol Metab 2008; 22: 1)

Keywords Foetal growth, imprinting disorders, reproduction, development and epigenetic changes


Program 7: Genome wide approaches to the study of gene‐environment interactions and chromatin modifications Supervisor A/Professor Sam El‐Osta

Introduction

The two metre human genome is a dynamic physical structure comprised of protein‐DNA associations, all of which participate in nuclear functions such as transcriptional regulation, replication and repair and epigenetic inheritance. Therefore it is imperative we look beyond mRNA expression profiling as an endpoint measure. Understanding when DNA binding proteins are bound or released from gene sequences is just as important as where this occurs on the genome. The availability of large‐scale high throughput epigenomic strategies and more specifically, next generation sequencing coupled with chromatin immunopurification (ChIP) approaches will allow us to explore these questions of spatial and temporal landscapes with exquisite detail.

Examples of the application the El‐Osta team are currently interested include but not limited to genome wide mapping of DNA methylation using bisulfite sequencing protocols, determination of histone modifications and nucleosome positioning, chromatin accessibility for the determination and monitoring of changes in chromatin structures and remodeling, chromasome capture conformation and long range interaction and chromatin communication (ChIP‐loop, 4C) and mapping of transcription factor binding sites.

Aim 1 ‐ Development of computational tools that integrate genome‐wide data sets for proximal and distal transcription binding sites and histone modification mapping.

Aim 2 ‐ Development of ChIP approaches and strategies to the broad application of determining epigenomic signatures in models of human health and disease.

Aim 3 ‐ Development of epigenomic protocols and strategies for low cell numbers from clinical isolates.

Recommended reading (Nature Review Genetics 2008) (Mol Biotechnol 2008) (Front Biosci 2008)

Keywords Chromatin immunoprecipitation, bisulfite sequencing, chromatin conformation, next generation sequencing, bioinformatics


Oxidative Stress

Dr. Judy de Haan [email protected] 8532 1520 Oxidative Stress, Antioxidants, Diabetes‐Associated Atherosclerosis (DAA)

Atherosclerosis, the accumulation of fatty deposits in the lining of blood vessels, is a major cause of death due to heart attacks and strokes. A major risk factor for the development of atherosclerosis is diabetes. Atherosclerosis is accelerated in people with diabetes, leading to cardiovascular disease occurring earlier in diabetic patients. In addition, oxidative stress has been linked to cardiovascular disease and in particular to atherosclerosis.1 Evidence suggests that oxidative stress is increased in diabetic patients.2 Two contributing factors lead to increased oxidative stress, namely (i) increased formation of reactive oxygen species (ROS) and/or (ii) reduced removal of ROS by antioxidants. That diabetic patients experience enhanced oxidative stress is most likely due to both factors, since it has been shown that hyperglycaemia (elevated glucose) leads to increased ROS formation, while a recent clinical study has linked reduced expression of glutathione peroxidase‐1 (GPx1), a major ROS‐removing enzyme, and increased risk of macrovascular diseases in type 2 diabetic patients.3 Understanding the mechanisms leading to enhanced oxidative stress in diabetes and its key complications has been a major focus of the Oxidative Stress Laboratory. Recently, our laboratory showed for the first time, a direct link between reduced GPx1 activity and accelerated atherosclerosis in a diabetic ApoE‐/‐ mouse model.4 We also showed that lack of GPx1 affected important inflammatory pathways known to be of significance in diabetes‐associated atherosclerosis (DAA). The Oxidative Stress Laboratory now focuses on antioxidants that mimic the function of this important antioxidant enzyme, with the aim of developing targeted antioxidant therapeutics to reduce DAA.

Project 1: Can Gpx1‐mimetics reduce diabetes associated atherosclerosis? This project will investigate whether several novel antioxidant compounds with GPx‐like activity reduce DAA. It will investigate the mechanisms leading to this reduction with particular focus on inflammatory pathways. We have exciting preliminary data in support of our hypothesis that GPx‐mimetics reduce DAA. Using ebselen [2‐phenyl‐1,2‐benzisoselenazol‐3[2H]‐one], a synthetic lipid soluble seleno‐organic low molecular weight compound with GPx‐like antioxidant activity,5 we have shown significant reductions in atherosclerotic plaque and the attenuation of several important inflammatory pathways in diabetic ApoE‐/‐ mice. This project will focus on several structurally similar compounds (analogues of ebselen) with improved GPx‐like activity to ascertain their potential as novel targeted antioxidant therapies for DAA.

We will utilize the diabetogenic drug streptozotocin to induce diabetes for 10 and 20 weeks within ApoE‐/‐ and ApoE‐/‐Gpx1‐/‐ double knockout mice. Compounds will be administered to the animals by gavage. Techniques will include an assessment of plaque size and histology (analysis of the entire aorta by the enface technique and cryostat sectioning and staining of the aortic sinus), immunohistochemistry and a range of molecular biology techniques such as RT‐PCR to assess expression of inflammatory and other antioxidant genes, and Western blotting to determine protein levels.


Project 2: Use of GPx1‐mimetics to reduce endothelial dysfunction Oxidative stress is known to contribute to endothelial dysfunction, an important early pro‐atherogenic event, and particularly within a diabetic context.6 A number of factors are known to increase superoxide radical production in the diabetic milieu, eg. increased NADPH oxidase activity as a direct consequence of increased glucose levels leads to elevated superoxide production. Superoxide in turn, interacts with bioavailable nitric oxide (NO) to produce highly toxic peroxynitrite radicals. These highly reactive radicals interacts with DNA, proteins and lipids of biomembranes to cause damage, leading to endothelial dysfunction. Endothelial dysfunction in turn is responsible for enhanced inflammatory responses. Increased oxidative stress within endothelial cells thereby initiates a cascade that ultimately leads to accelerated atherosclerosis. Clearly limiting oxidative stress is a desirable aim in the prevention of endothelial dysfunction and DAA.

The antioxidant GPx1 is a potent peroxynitrite reductase, thereby assisting in the removal of peroxynitrite radicals. This project will make use of antioxidants with functions that mimic this important peroxynitrite‐scavenging antioxidant enzyme, to assess whether this approach reduces oxidative stress and limits endothelial cell damage.

Several approaches will be used to achieve this aim:

Human aortic endothelial cells (HAEC) will be used to determine whether ebselen and its analogues protect against known oxidants and pro‐atherogenic stimuli. Cell signalling pathways known to be affected by the diabetic milieu will be explored. In particular, the role of mitogen‐activated protein kinases (MAPKs) and their modulation by these antioxidants will be assessed.

Primary aortic endothelial cells will be isolated from ApoE‐/‐ and ApoE‐/‐GPx1‐/‐ double knockout mice. The consequences of a lack of GPx1 on cell signalling pathways will be assessed. Repletion of GPx function through administration of GPx1‐mimetics will demonstrate specificity and efficacy of these mimetics.

Functional studies in aortic rings derived from ApoE‐/‐ and ApoE‐/‐GPx1‐/‐ mice will determine whether GPx1‐mimetics restore endothelial function.

These studies will determine whether GPx1‐targeted strategies that reduce oxidative stress, improve endothelial function.

Project 3: Intervention studies with the antioxidant ebselen in ApoE‐/‐ mice fed high fat diets

ApoE‐/‐ mice develop severe atherosclerosis when fed a diet rich in fats and cholesterol. Antioxidants have been proposed as potentially therapeutic in limiting oxidative stress and reducing atherosclerosis associated with high‐fat diets. We have already shown the potential usefulness of ebselen [2‐phenyl‐1,2‐benzisoselenazol‐3[2H]‐one], a synthetic lipid soluble seleno‐organic low molecular weight compound with GPx‐like antioxidant activity,5 in reducing atherosclerotic plaque and attenuation of several important pro‐inflammatory pathways in diabetic ApoE‐/‐ mice. We now wish to establish whether this antioxidant is also effective against atherosclerosis and pro‐atherogenic pathways induced by hyperlipidemia. This study will also investigate whether an interventional approach of ebselen administration prevents or retards further lesion formation.


ApoE‐/‐ mice will be fed high fat diets for 7 weeks after which ebselen will be administered by daily gavage for a further 7 weeks. Techniques will include an assessment of plaque size and histology (analysis of the entire aorta by the enface technique and cryostat sectioning and staining of the aortic sinus), immunohistochemistry and a range of molecular biology techniques such as RT‐PCR to assess expression of inflammatory and other antioxidant genes, and Western blotting to determine protein levels.

Honours and PhD student positions are available in the Oxidative Stress Lab. Strong supervision and expertise is available for these projects. Students are encouraged to contact Dr. de Haan for further information.

REFERENCES

1. Witztum JL. The oxidation hypothesis of atherosclerosis. Lancet. 1994;344:793‐795.

2. Baynes JW. Role of oxidative stress in development of complications in diabetes. Diabetes. 1991;40:405‐412.

3. Hamanishi T, Furuta H, Kato H, Doi A, Tamai M, Shimomura H, Sakagashira S, Nishi M, Sasaki H, Sanke T, Nanjo K. Functional variants in the glutathione peroxidase‐1 (GPx‐1) gene are associated with increased intima‐media thickness of carotid arteries and risk of macrovascular diseases in japanese type 2 diabetic patients. Diabetes. 2004;53:2455‐2460.

4. Lewis P, Stefanovic N, Pete J, Calkin AC, Giunti S, Thallas‐Bonke V, Jandeleit‐Dahm KA, Allen TJ, Kola I, Cooper ME, de Haan JB. Lack of the antioxidant enzyme glutathione peroxidase‐1 accelerates atherosclerosis in diabetic apolipoprotein E‐deficient mice. Circulation. 2007;115:2178‐2187.

5. Sies H. Ebselen, a selenoorganic compound as glutathione peroxidase mimic. Free Radic Biol Med. 1993;14:313‐323.

6. Esper RJ, Vilarino JO, Machado RA, Paragano A. Endothelial dysfunction in normal and abnormal glucose metabolism. Adv Cardiol. 2008;45:17‐43.


Proliferation & Fibrosis

Zhonglin Chai and Mark Cooper [email protected] or [email protected] 8532 1231 or 8532 1362 The complication conditions of diabetic patients include macrovascular disease, which directly causes myocardial infarction, stroke and peripheral vascular disease, and microvascular conditions including kidney failure and blindness. These complications are the leading causes of the increased morbidity and mortality in the patients of diabetes.

In the sites of diabetic injury including blood vessels and kidney, a number of key cellular response events occur for the purpose of repairing the damaged tissues. These include the cell differentiation and proliferation at the early stage, followed by excess accumulation of extracellular matrix proteins, a condition called fibrosis or tissue scarring, at the later stage if the damage is too severe to be repaired. The tissue scarring represents a failed repair of damaged tissues or loss of functional tissues/cells, which is seen in heart failure, kidney failure and atherosclerosis.

Cell Division Autoantigen 1 (CDA1) is a molecule recently identified and cloned by our group. It is well established

that CDA1 modulates TGF‐β receptor kinase which activates multiple important intracellular signalling pathways including Smad, MEK/ERK MAPK and p53, ultimately leading to impaired tissue repair and scarring. Elevated CDA1 expression levels are found in diabetes‐accelerated atherosclerosis and advanced diabetic nephropathy, where severe tissue scarring or fibrosis is present. The role of the elevated levels of CDA1 in the development of these diseases is directly shown by the ability of CDA1 to increase gene expression of collagen and other extreacellular

matrix proteins in cultured cells. Targeting CDA1 by siRNA in cells prevent TGF‐β stimulated gene expression of collagen I and III, showing that CDA1 is an attractive molecular target for drug development in order to prevent and treat chronic diabetic complications associated with tissue scarring.

Our research is well funded by Jurvenile Diabetes Research Foundation (USA), Australian National Health & Medical Research Council, National Heart Foundation of Australia, Institute’s Medicinal Chemistry Grant and a commercial partner company.

The research projects available for Honours and/or PhD include 1), Role and molecular mechanism of CDA1 in vascular disease; 2), Role and mechanism of CDA1 in diabetic kidney disease; 3), Regulation of CDA1 gene in physiology and pathology; 4), Development of therapeutic agents by targeting CDA1 for diabetic complications. The techniques used in the lab include most of the molecular biology and cell biology techniques, including:

1. cell culture and recombinant DNA technology; 2. retrovirus and adenovirus directed gene expression; 3. RNA interference technology; 4. yeast 2‐hybrid system for protein‐protein interaction study; 5. Western blot and real‐time RT‐PCR 6. Peptide biology and medicinal chemistry


Metabolism & Obesity


Cardiac Hypertrophy

Dr Julie R. McMullen Email: [email protected] Phone: 8532 1194

Research Focus Targeting novel regulators of exercise induced heart growth to treat heart failure Heart failure is a major clinical problem affecting 1‐2% of Australians. The number of people diagnosed with heart failure is on the rise, due to an ageing population and increased rates of obesity and diabetes, posing a significant healthcare burden. Thus, strategies to protect the heart against insults such as high blood pressure, heart failure, and heart attack are becoming even more critical. My laboratory is focused on identifying genes/proteins that mimic the protective effects of exercise. Growth of the heart (also termed cardiac hypertrophy) can be induced by physiological stimuli (e.g. postnatal development, chronic exercise training) or pathological stimuli (e.g. high blood pressure). Physiological hypertrophy (“good” heart growth) is characterised by a normal organisation of cardiac structure, and normal or enhanced cardiac function; whereas pathological hypertrophy (“bad” heart growth) is associated with cardiac dysfunction, and increased morbidity and mortality.

In an effort to treat patients with heart failure, the majority of investigators have focused on blocking “bad” genes and signalling pathways in the heart, which largely delays heart failure. By contrast, my laboratory are examining the possibility of activating “good” genes and signalling pathways that may normally be activated during the induction of physiological hypertrophy e.g. in the “athlete’s heart”. My group previously reported that the insulin‐like growth factor 1 (IGF‐1)‐phosphoinositide 3‐kinase (PI3K) pathway plays a critical role for the induction of exercise induced heart growth. Thus, activation of PI3K, or novel regulators of this pathway, represents a promising new strategy to treat heart failure.

We have a number of projects that can be tailored for both Honours and PhD students that address our laboratory’s global research aim, each briefly outlined below. Projects utilise genetic mouse models in combination with a number of molecular biology and biochemical techniques (for more detail see “Techniques” below).


Project 1: Targeting PI3K regulated microRNAs and novel genes to treat heart failure Supervisors: Dr Julie McMullen and Dr Bianca Bernardo microRNAs (miRs) are a family of small RNAs that play important roles in the regulation of target genes by interacting/binding with specific sites in 3’untranslated regions of messenger transcripts to repress their translation or regulate degradation. Silencing of miRs in vivo with antagomiRs is a new and expanding area of technology that is considered a powerful approach that may represent a new therapeutic strategy for targeting cardiac disease. Using microarray analysis, we have identified a number of miRs that are differentially regulated in mice with increased or decreased PI3K activity. This project will examine whether inhibition of miRs (i.e. mimicking what happens in a setting of physiological hypertrophy) using an antagomiR can improve cardiac function in vitro and in vivo.

Another area that this particular project can explore is the characterisation of novel genes to treat heart failure. By microarray we have identified a cohort of genes that may be important for the physiological hypertrophic response induced by the PI3K pathway. Avenues that can be undertaken include performing bioinformatics analysis of novel genes to elucidate gene/protein structure and function, characterisation of gene expression in the heart after pathological and physiological stimuli, targeting of novel genes in a setting of heart failure to determine if cardiac function has improved.

Project 2: Novel treatment strategies to protect the heart against atrial fibrillation Supervisors: Dr Julie McMullen and Dr Bianca Bernardo Atrial fibrillation (AF) is a cardiac disorder. It is the most common type of arrhythmia causing an irregular heat beat, weakness, fatigue and dizziness. AF is associated with increased risk of mortality, stroke and heart failure. AF and heart failure may share common triggers and treatment strategies. We have identified activation of PI3K as a novel strategy for the treatment of heart failure. This project will explore whether increasing PI3K(p110α) or novel targets of PI3K in the heart of mouse models with AF (using adeno‐associated viral vectors or novel compounds) will protect the heart against AF.

Project 3: Examining gender differences in mouse models of heart failure Superviors: Dr Julie McMullen and Dr Bianca Bernardo Gender differences exist in the incidence of cardiovascular disease and the response to major cardiovascular drugs. Women typically develop heart disease later than men and this has been attributed to the protective actions of female sex hormones, in particular, estrogen. However, there are exceptions that are yet to be understood. For instance, diabetic women are at greater risk of developing heart failure than men in response to a cardiac insult. This project will explore the importance of the estrogen receptor ERα‐PI3K interaction in female and male hearts utilising mice with cardiac‐specific ERα deletion, and will characterise the phenotype of ERα knockout mouse under basal conditions and in response to pressure overload.

Techniques The above projects involve a number of techniques including characterisation of cardiac specific gene targeted mice, animal work, functional studies in vivo (echocardiography/electrocardiography), Western blot analyses, Northern blot analyses, polymerase chain reaction (PCR), immunoprecipitation, quantitative real time PCR, DNA/RNA/Protein extractions, cell culture, adeno‐associated virus vector technology, microRNAs and microarray.


Previous students Recent Honours students in my laboratory have received First Class Honours and been awarded Australian Postgraduate Award Scholarships to commence PhDs. Students under my supervision have presented their work at conferences (oral and poster presentations), published reviews and original research articles.

Selected references: 1. McMullen JR, Shioi T, Zhang L, Tarnavski O, Sherwood MC, Kang PM, Izumo S. PI3K(p110{alpha}) plays a critical role for the induction of physiological, but not pathological, cardiac hypertrophy. Proc Natl Acad Sci USA. 2003;100(21):12355‐12360.

2. McMullen JR, Shioi T, Huang WY, Zhang L, Tarnavski O, Bisping E, Schinke M, Kong S, Sherwood MC, Brown J, Riggi L, Kang PM, Izumo S. The Insulin‐like Growth Factor 1 Receptor Induces Physiological Heart Growth via the Phosphoinositide 3‐Kinase(p110{alpha}) Pathway. J Biol Chem. 2004;279:4782‐4793

3. McMullen JR, Jennings GL. Differences between pathological and physiological cardiac hypertrophy: novel therapeutic strategies to treat heart failure. Clin Exp Pharmacol Physiol. 2007; 34:255‐62

4. McMullen JR, Amirahmadi F, Woodcock EA, Schinke‐Braun M, Bouwman RD, Hewitt KA, Mollica JP, Zhang L, Zhang Y, Shioi T, Buerger A, Izumo S, Jay PY, Jennings GL. Protective effects of exercise and PI3K(p110{alpha}) signaling in dilated and hypertrophic cardiomyopathy. Proc Natl Acad Sci U S A. 2007;104:612‐7.

5. McMullen JR. The role of IGF1 and PI3K in a setting of heart disease. Clin Exp Pharmacol Physiol 2008;35:349‐54.

6. Pretorius L*, Owen KL*, GL Jennings, McMullen JR. Promoting physiological hypertrophy in the failing heart. Clin Exp Pharmacol Physiol 2008;35:438‐41.

7. Owen K*, Pretorius L*, McMullen JR. PI3K and its protective effects in the failing heart. Clinical Science. 2009;116:365‐75.

8. Pretorius L*, Owen K*, McMullen JR. Role of PI3K in regulating cardiac function. Frontiers in Bioscience. 2009;14:2221‐9.

9. Pretorius L*, Du XJ, Woodcock EA, Kiriazis H, Lin RCY, Marasco S, Medcalf RL, Ming Z, Head GA, Tan J, Cemerlang N, Sadoshima J, Shioi T, Izumo S, Dart AM, Jennings GL, McMullen JR. Reduced PI3K(p110alpha) increases the susceptibility to atrial fibrillation. The American Journal of Pathology 2009; 175(3):998‐1009

10. Huynh, K.*, McMullen, J.R., Julius, T.L., Tan, J.W., Love, J.E.*, Cemerlang, N., Kiriazis, H., Du, X.‐J., and Ritchie, R.H. Cardiac‐specific IGF‐1 receptor transgenic expression protects against cardiac fibrosis and diastolic dysfunction in a mouse model of diabetic cardiomyopathy. Diabetes 2010; 59(6): 1512‐20

11. Lin, R.C.Y., Weeks, K.L.*, Gao, X.‐M., Williams, R.B.H., Bernardo, B.C., Kiriazis, H., Matthews, V.B., Woodcock, E.A., Bouwman, R.D., Mollica, J.P., Speirs, H.J., Dawes, I.W., Daly, R.J., Shioi, T., Izumo, S., Febbraio, M.A., Du, X.‐J., and McMullen, J.R. PI3K(p110 alpha) protects against myocardial infarction‐induced heart failure: identification of PI3K‐regulated miRNA and mRNA. Arterioscler Thromb Vasc Biol 2010; 30(4): 724‐32

12. Bernardo, B.C., Weeks, K.L.*, Pretorius, L.*, and McMullen, J.R. Molecular distinction between physiological and pathological cardiac hypertrophy: Experimental findings and therapeutic strategies. Pharmacology & Therapeutics. 2010. In Press

*previous Honours and/or current PhD students


Cellular & Molecular Metabolism

Professor Mark Febbraio [email protected] 8532 1176 It is now estimated that 10% of the world’s population are overweight or obese. The myriad of disorders associated with obesity, including insulin resistance, glucose intolerance and dyslipidemia eventually lead to the development of type 2 diabetes. Despite major scientific advances in our understanding of the molecular pathways leading to insulin resistance and type 2 diabetes made during the last 10‐15 years, current therapeutic drugs have had limited success. In the Cellular & Molecular Metabolism Laboratory, we aim to determine the key signal transduction pathways that lead to obesity induced insulin resistance in the hope of discovering novel drug targets to treat obesity. Work in our laboratory had centred on the nexus between inflammatory processes and lipid‐induced insulin resistance for several years.

The Cellular and Molecular Metabolism laboratory focuses on:

Inflammation and insulin resistance (Group Leader: Dr Graeme Lancaster)

The role of dietary fat and overnutrition in leading to insulin resistance

(Group Leader: Dr Clinton Bruce)

Our laboratory is at the forefront of research in these areas and we currently comprise 15 personnel including 7 post‐doctoral scientists and 6 post‐graduate students. We use a variety of techniques, ranging from cell biology, through rodent models and eventually to human studies. In this respect we take a classic “bench to bedside” approach. Our work is published in the very best journals in medical research.


Laboratory overview

It is now estimated that 10% of the world’s population are overweight or obese. The myriad of disorders associated with obesity, including insulin resistance, glucose intolerance and dyslipidemia eventually lead to the development of type 2 diabetes. Despite major scientific advances in our understanding of the molecular pathways leading to insulin resistance and type 2 diabetes made during the last 10‐15 years, current therapeutic drugs have had limited success. In the Cellular & Molecular Metabolism Laboratory, we aim to determine the key signal transduction pathways that lead to obesity induced insulin resistance in the hope of discovering novel drug targets to treat obesity. Work in our laboratory had centred on the nexus between inflammatory processes and lipid‐induced insulin resistance for several years.

Accordingly, our laboratory is divided into the following streams:

Inflammation and insulin resistance Group Leader: Dr Graeme Lancaster

In the past decade it has become apparent that hyperlipidemia, a hallmark of obesity, is linked to a state of chronic inflammation. Somehow, lipid‐induced inflammation results in the the activation of key serine threonine kinases namely c‐jun amino terminal kinase (JNK) and inhibitor of κB kinase (IKK) in insulin responsive tissues such as adipose tissue, skeletal muscle and liver. It is known that activation of both JNK and IKK disrupt insulin signalling and cause insulin resistance. It is thought that the mechanisms linking lipid oversupply to inflammation involves increased deposition of lipid species such as diacylglycerol and ceramide which are known to activate JNK and IKK in liver and/or skeletal muscle leading to insulin resistance (see Figure below). However, the precise molecular mechanisms linking these lipid species to upregulation of JNK and IKK and ultimately impaired insulin action are not fully resolved. We have several projects in this group aimed at resolving the role of inflammation in insulin resistance

Peripheral insulin resistance

Decreas

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Increased lipid storage

DAG, ceramide accumulation

JNK IKK

Peripheral insulin resistance

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Increased lipid storage

DAG, ceramide accumulation

JNK IKK


The role of dietary fat and overnutrition in leading to insulin resistance Group Leader: Dr Clinton Bruce

Overnutrition leads to excess lipid deposition in tissues such as skeletal muscle and liver. Since these organs are not normally storage depots for fat they become pathogenic during overfeeding. When lipid is stored in these tissues they become “lipotoxic” and eventually insulin resistant. One strategy to overcome lipid storage is to increase the oxidation of the lipid by enhanicing mitochondrial capacity (see Figure below). In this group we study mechanism by which a) certain lipid species dysregulate muscle metabolism and b) we can enhance lipid oxidation. We hope to derive new drug targets that can reduce fat storage and/or increase fat oxidation when people over eat.


Metabolic & Vascular Physiology

Professor Bronwyn Kingwell [email protected] 8532 1518

Research Focus The Metabolic and Vascular Physiology Laboratory takes a multidisciplinary approach to discovery and translation of

novel molecular mechanisms to clinical application with a focus in the following areas:

• Vascular function including mechanical and endothelium properties and their relationship to cardiovascular risk

• Identification of novel predictors of unstable coronary heart disease

• The role of HDL cholesterol in modulation of glucose and fat metabolism

• Exercise mimetics

• Mechanisms for increasing energy expenditure in obese humans

Laboratory members and key collaborators have a broad collective skill base and range from molecular biologists through to endocrinologists and interventional cardiologists. These skills are integrated to investigate novel diagnostics and therapeutic approaches to the disease continuum linking obesity, type 2 diabetes and cardiovascular disease.


Project: TElmisartan in the management of abdominal aortic aneurYsm (TEDY) Funding agency: MBF Foundation Investigators: Ahimastos, Golledge, Kingwell

Summary Abdominal aortic aneurysm (AAA) is responsible for ∼1,500 deaths and 10,000 hospital admissions per year in Australia. Surgery is the only therapy for AAA but is costly and associated with high morbidity and mortality. No medical therapy has been approved for AAA, highlighting the need for a better understanding of its pathophysiology to implement novel management strategies.

Evolving evidence suggests that angiotensin II (AngII) plays a crucial role in aneurysm formation (1,2). This pro‐aneurysmal pathway can be antagonised by angiotensin II type 1 receptor (AT1) antagonists.

A project is offered in conjunction with a large scale clinical trial which is currently underway with collaborators from Queensland. The aims of the clinical trial are to investigate the efficacy of the AT1 antagonist Telmisartan to:

Reduce AAA growth assessed by computed tomography angiography

Reduce circulating concentrations of pro‐aneurysmal biomarkers.

This clinical trial will be the first to assess the value of a promising medication with significant preliminary data to suggest it can slow aortic destruction, and thus offers the possibility of identifying a new treatment modality for an increasingly common condition.

1. Ahimastos AA, Aggarwal A, D’Orsa KM, Formosa MF, White AJ, Savarirayan R, Dart AM, Kingwell BA. Effect of perindopril on large artery stiffness and aortic root diameter in patients with Marfan syndrome: a randomized controlled trial. JAMA. 298(13):1539-1547, 2007

2. Golledge J, Norman PE. Pathophysiology of abdominal aortic aneurysm relevant to improvements in patients’ management. Curr Opin Cardiol. 24(6):532-8, 2009

Figure 1. The abdominal aorta is the most common extracranial site of aneurysm formation.


Project: Metabolic Actions of HDL

Funding agency: NHMRC

Investigators: Siebel, Duffy, Sviridov, Chin‐Dusting, Steinberg, Carey, Kingwell

Schematic diagram of the mechanisms by which HDL is postulated to modulate plasma glucose: HDL is postulated to increase peripheral glucose uptake and reduce plasma glucose by both increasing plasma insulin (possibly by increased insulin secretion) and via the ABCA1 receptor and stimulation of CaMKK to activate the AMPK pathway in skeletal muscle.

Summary Recent studies in our laboratory have identified novel actions of HDL cholesterol in relation to glucose and fat metabolism which provide new opportunities for therapies to prevent and treat type 2 diabetes.

In a combination of in vitro molecular investigations and a clinical trial of reconstituted HDL (rHDL) infusion, we have recently shown that HDL lowers blood glucose through both stimulation of insulin release from pancreatic

β cells and activation of the key metabolic regulatory enzyme, AMP‐activated protein kinase in skeletal muscle. Our current studies also support a third mechanism involving enhanced insulin sensitivity via anti‐inflammatory actions in both metabolic tissues and macrophages. These findings provide a rationale for therapeutic approaches to raise levels of circulating HDL to manage the metabolic syndrome and type 2 diabetes.

This exciting work is progressing on multiple fronts including:

A drug company collaboration to examine the effects of chronic HDL elevation on glucose metabolism both in vitro and in a clinical trial of type 2 diabetic patients

Examination of novel HDL signalling pathways in human skeletal muscle and macrophage cultures using approaches including gene array and phosphoprotein proteomics.

Determination of metabolic and functional actions of HDL in the heart using cell culture, isolated heart models and human biopsy material.

Both PhD and Honors projects are offered in all the above areas.

Drew BG, et al., Kingwell BA. High‐density lipoprotein modulates glucose metabolism in patients with type 2 diabetes mellitus. Circulation. 2009;119:2103‐11.

Patel S, et al., Kingwell BA. Reconstituted high‐density lipoprotein increases plasma high‐density lipoprotein anti‐inflammatory properties and cholesterol efflux capacity in patients with type 2 diabetes. J Am Coll Cardiol. 2009;53:962‐71.

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HDL

↑ insulin

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CaMKK

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↑ Ca2+

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↑ Glucoseuptake


Project Title: Development of Brown Adipose Tissue for Treatment of Obesity Funding agency: Ramaciotti Foundation Investigators: Carey, Gregorevic, Haviv, Cherk, Duffy, Kingwell

Representative images of cells extracted from muscle from a healthy human via tissue biopsy in the Metabolic and Vascular Physiology Laboratory. Cells were given no treatment (left) or stimulated to differentiate into adipose cells (right). The image on the right demonstrates robust formation of BAT in cells extracted from this healthy patient. The red staining indicates formation of fat droplets characteristic of brown and white adipose tissue, and gene expression patterns (not shown) demonstrate the presence of brown adipose characteristics.

Summary Fundamentally, obesity results from an imbalance between energy intake and expenditure. Current preventative and therapeutic approaches have been either unsustainable or result in significant negative side effects. Increasing brown adipose tissue (BAT) content and activity is currently considered one of the most promising strategies to increase energy expenditure and combat obesity. Projects focusing on the following areas are offered in 2011:

a) gene activation targeted to drive BAT formation in immature stems cells from human muscle and adipose tissues

b) pharmacological activation of brown adipose tissue in humans

c) Examination of the capacity of precursor cells in human tissues to form BAT cells when comparing lean and obese humans, as represented in the figure below.


Project Title: Does a High Fat Diet Initiate an Epigenetic Program for Diabetes? Funding agency: Baker IDI Heart and Diabetes Institute (BIG grant) Investigators: El Osta, Febbraio, Armitage, Head, Duffy, Kingwell

Schematic representation of ongoing rat studies examining the effects of maternal diet on epigenetic control of metabolism in offspring.

Summary

The “pandemic” of metabolic disease is recognized as one that is caused by an interaction between genes and the environment. This study investigates the role of epigenetic modification induced by exposure to a high fat diet (both directly and prenatally) on gene expression and metabolic control. The study incorporates cell, animal (rabbit and rat) and human studies with genome wide histone modification and gene expression analyses. Current projects include studies of offspring from rabbit and rat mothers fed high fat diets. These animals undergo extensive physiological metabolic investigations at the whole body and tissue level as well as epigenetic and gene expression analysis of metabolic tissues (muscle, adipose, liver). Results from our animal investigations will be interpreted in conjunction with parallel data from human vastus lateralis and adipose biopsies (healthy and obese patients with various stages of metabolic disease). A project is offered in relation to both the rat and human aspects of this program in 2010.

Diagram

CHOWCHOW Hi-fatHi-fat

Parental diet (starting 10 days before

mating through to the end of lactation)

Give birth to offspring

Offspring will be placed on CHOW post-

lactation

1 year

Metabolic cage

ITT & GTT

Body composition

Mitochondrial function assessment


Muscle Biology & Therapeutics

Paul Gregorevic [email protected] 8532 1224

Skeletal muscle accounts for almost half a person’s body mass, yet we easily take for granted its role in our health and lifestyle. The reality is that physical frailty caused by a loss of strength is the primary cause of death among a significant proportion of the elderly population, and patients with a host of medical conditions. Moreover, even a moderate decline in muscle strength caused by advancing age, bed rest or inactive livestyle can dramatically increase the incidence and severity of many serious medical conditions. In our lab, the goal is to understand the cellular mechanisms that regulate muscle growth and wasting, so that we can develop new methods of preventing or treating the symptoms of muscle‐related diseases. Also, our work has implications for improving the health of that other important muscle – the heart! The research places a particular emphasis on employing recombinant viral vectors designed and manufactured “in‐house” as a means to selectively alter gene expression in mouse models, and analyses using a host of established and cutting‐edge techniques spanning the disciplines of biological/biomedical science. This approach allows us to interrogate the cellular mechanisms controlling muscle adaptation in vivo with previously unattained precision.

Prominent themes within the lab include:

Programs of protein signalling associated with muscle growth/wasting;

Changes in gene expression implicated in muscle adaptation;

Causes of pathology in models of neuromuscular disease, aging‐, and cancer‐related wasting;

Development of novel genetic and non‐genetic therapies to prevent or reverse the loss of muscle function in disease.

Our major goal is to improve human health through what we learn. By embracing novel technologies in our research, and collaborating with leading laboratories world‐wide, we always strive to be at the leading edge of this field and in the development of new therapeutic approaches. This recent addition to the BakerIDI research family is rapidly growing and looking to accelerate its research program.


Nutritional Studies

Professor Peter Clifton [email protected] 8532 1873

Research Focus

Optimal dietary Interventions

Over the past 20 years, Prof Clifton and his team have examined a variety of weight loss strategies to optimize weight, blood pressure, lipid and glucose outcomes in overweight and obese people with metabolic syndrome, pre diabetes and type 2 diabetes. Much of the focus has been on carbohydrate restriction with protein as the replacement macronutrient

The Nutrition laboratory seeks to undertake novel research to provide new understanding relating long term compliance to energy restricted diets. The Healthy Lifestyle Research centre (an integrated research centre dedicated to the development, refinement and translation of evidence‐based physical activity and nutrition programs for people with or at risk of the complications of obesity) now provides an excellent context for this type of research aimed at translating these exciting findings into public benefit. Some examples of the types of research projects that are available for post‐graduate studies are summarized below.

Project 1: Does informed choice of diet type and the ability to change diets improve long term retention and achieve better 2 year weight outcomes

Supervisors: Professor Peter Clifton and Assoc Prof Jennifer Keogh 400 overweight and obese subjects with type 2 diabetes and pre‐diabetes will be recruited for a 2 year weight loss and exercise study. 4 different dietary strategies will be offered and tested by all participants.

1: a low carbohydrate diet but modified to use lean meats, low fat dairy and abundant poly and monounsaturated fat (South Beach diet). This will be ad libitum with no energy prescription.

2: a high protein, moderate carbohydrate diet similar to the CSIRO Total Well Being Diet with red meat recommended 3‐4 times/week.

3: a high carbohydrate, low fat, moderate protein diet

4. A low protein, high vegetable, high monounsaturated fat, Mediterranean style diet.

Diets 2‐4 will be energy restricted to 6‐7MJ.


Measured end points include weight, body composition (by bioelectric impedance), HbA1c, fasting lipids, glucose, CRP, blood pressure at baseline, 6 months, 12 months, 18 months and 24 months.

All diets will be pretested at the healthy lifestyle research centre over a 6‐8 hour period and rated for palatability and satiety. The volunteer will then select their preferred diet and begin a 3 month weight loss phase aiming to lose 10% of their weight over this period of time. At the end of this period the volunteer will either continue with their first diet or swap to a second diet. Reassessment will occur every 3 months and the volunteer may cycle through all 4 diets or remain on the one diet for the 2 years. Efficacy will be assessed as weight loss/weight gain per 3 month period for each diet averaged over all periods, with covariates of the number of times the diet was used, the total number of diet variations used (diet variability) and the sequence position (eg for weight loss, or for weight maintenance in year 1 or year 2).

Project 2: Does the metabolic response to a diet acutely predict long term success in weight, lipid and glucose outcomes?

Supervisors: Professor Peter Clifton and Assoc Prof Jennifer Keogh All diets in project 1 will be pretested at the healthy lifestyle research centre over a 6‐8 hour period and rated for palatability and satiety. Volunteers will come in fasting on each day and have fasting glucose, insulin and lipids and have glucose and insulin and TG repeated at 2 hours (after breakfast), 4 hours (prior to lunch) and 6 hours (2 hours after lunch). Meal tests will be iso energetic and be performed at weekly intervals. This will assess differing metabolic response to each diet to see if this is a factor in long term success of the diet.

Exercise

During the active weight loss period (first 3 months) walking for at least 30 minutes 4 times/week will be prescribed. Accelerometers will be used in all volunteers to assess compliance. It is hypothesised that as weight loss proceeds activity will spontaneously increase and those most successful at 2 years will have the greatest increase in spontaneous activity.


Physical Activity

Associate Professor David Dunstan [email protected] 8532 1873 Research Focus

Sedentary behavior interventions

Over the past 10 years, A/Prof Dunstan and his team have pioneered an area of research that has provided new perspectives on sedentary behavior and health and disease. This research is widely acknowledged nationally and internationally as a paradigm shift, with prolonged sitting now recognized as an emerging public health concern that may have significant adverse metabolic and health effects, even in people who meet the recommendation of 30 minutes physical activity on most days of the week. Since prolonged sitting is a ubiquitous component of adult’s working, commuting and domestic lives, developing and testing interventions to influence sedentary behavior and ultimately improve health, has become a high public health priority.

The Physical Activity laboratory seeks to undertake novel research to provide new understanding relating to the causal nature of how too much sitting affects health. The Healthy Lifestyle Research centre (an integrated research centre dedicated to the development, refinement and translation of evidence‐based physical activity and nutrition programs for people with or at risk of the complications of obesity) now provides an excellent context for this type of research aimed at translating these exciting findings into public benefit. Some examples of the types of research projects that are available for post‐graduate studies are summarized below.


Project 1: What are the cardio‐metabolic consequences of too much sitting and can breaking up sitting time improve health profiles?

Supervisors: Associate Professor David Dunstan & Professor Bronwyn Kingwell Over the past 2 years the Physical Activity team has dedicated considerable time to developing working protocols and techniques for the establishment of a laboratory‐based ‘sitting time’ experimental model to examine the acute and cumulative effects of prolonged sitting in overweight adults. Building on the earlier epidemiological evidence that brief ‘active breaks’ may be beneficial for health, we are now undertaking a randomised cross‐over experimental trial (IDLE Breaks) to compare the effects on post‐prandial glucose, insulin, lipid and plasma‐free fatty acid levels of prolonged sitting (7‐hours) versus both prolonged sitting + light‐intensity activity bouts (2 min bouts every 20 mins) and prolonged sitting + moderate‐intensity activity bouts (2 mins every 20 mins). In collaboration with Prof Bronwyn Kingwell (Head, Metabolic and Vascular Physiology at Baker IDI) and Prof Marc Hamilton (Pennington Biomedical Research Centre USA), we are also assessing indicators of clotting time, and taking muscle and adipose tissue biopsies in a sub‐sample to explore potential mechanisms. This study will elucidate the extent to which activity interruptions can attenuate the acute postprandial response to prolonged sitting time. This knowledge will potentially identify the optimal ‘dose’ or pattern of behaviour for better health outcomes. The findings relating to the efficacy of interrupting sitting time with light‐intensity versus moderate‐intensity physical activity will also serve to inform the design of a separate randomised cross‐over trial (ABLE breaks) in overweight adults that will investigate the cumulative effects (4 consecutive days) of prolonged sitting, with and without activity bouts on post‐prandial glucose, insulin, lipid, plasma free fatty acid levels and mechanistic outcomes.

Project 2: Can increased standing during the working day benefit metabolic health?

Supervisors: Associate Professor David Dunstan & Professor Bronwyn Kingwell Within the Healthy Lifestyle Research Centre we now have the capacity to investigate the cardio‐metabolic and physiological effects of experimental manipulations of sitting time within ‘simulated office and domestic environments’. Initially, healthy adult computer‐dependent workers will be recruited to examine the effects on cardio‐metabolic and physiological outcomes resulting from systematic manipulations of their daily work time sitting routine. Participants will be given access to a ‘simulated office’ that will feature an electric‐height adjustable desk, and in a systematic manner will have designated sitting time and standing time periods imposed on their working hours. Subsequent studies will explore the effects of less systematic (ad libitum) manipulations of sitting time and extend this to simulations of the domestic setting.


Project 3: Can increased standing during the working day benefit metabolic health? Supervisors: Associate Professor David Dunstan

Building the Stand Up Australia pilot research, we will target sitting time in a new intervention study conducted in the workplace setting among office‐based workers. This study will draw on the experience developed by the group and our national collaborators from previous evaluations in the workplace context, and also through our studies using telephone delivered behavioural intervention approaches. It will have an explicit focus on reducing sedentary time within and outside of the workplace setting (primary outcome). We will also examine the effects of the intervention on cardio‐metabolic biomarkers, to provide further insights into the possible causal nature of the associations of prolonged sitting time at work with health risk.

Research Focus

Physical Activity interventions

A/Prof Dunstan has also established an internationally‐recognised research program focusing on understanding the influence of physical activity in health and disease, particularly in relation to the management of type 2 diabetes. Specifically, his research on the benefits of resistance training in the treatment of type 2 diabetes has contributed extensively to the evidence base supporting the role of resistance training in the management of type 2 diabetes and was pivotal for the incorporation of explicit resistance training recommendations in the American Diabetes Association’s exercise guidelines for people with type 2 diabetes. A/Prof Dunstan is also the creator of the Lift for Life program – a purposefully developed physical activity program designed to facilitate widespread uptake of strength training in community facilities throughout Australia. The new gymnasium and the clinical testing facilities available within the Healthy Lifestyle Research Centre provide increased scope for an expansion of the physical activity research program.

Some examples of the types of research projects that are available for post‐graduate studies are summarized below.


Project 1: Can the health benefits resulting from exercise training be further enhanced by also targeting ‘non‐exercise’ time?

Supervisors: Associate Professor David Dunstan & Professor Bronwyn Kingwell

Exercise is a considered to be one of the cornerstones in the management of type 2 diabetes, obesity and cardiovascular disease. Regular exercise involving aerobic and resistance training is known to have numerous favourable health benefits. However, since ‘exercise time’ constitutes such a small proportion of the waking hours, there have been increased calls to also target the behaviours people undertake during their ‘non‐exercise’ time. In this study, participants attending exercise sessions within the Healthy Lifestyle Research Centre will provided with devices that record physical activity and sedentary time throughout the day. The devices will be used as a key intervention tool to help guide participants to reduce their sedentary time and increase their activity level during the hours in the day that they are not exercising. A control group of participants will undertake exercise sessions in the Healthy Lifestyle Centre, but will not receive any instruction regarding what to do during the ‘non‐exercise’ periods.

Project 2: Does the combination of Vitamin D supplementation and resistance training lead to greater health benefits than resistance training alone?

Supervisors: Associate Professor David Dunstan, Professor Bronwyn Kingwell, Associate Professor Robin Daly (University of Melbourne)

Regular resistance training has been shown to improve insulin sensitivity and glycemic control in people with and at risk of developing type 2 diabetes. An inverse association has been described between insulin sensitivity and vitamin D levels in adults. This project will investigate whether combining Vitamin D supplementation with resistance training will lead to more favourable health benefits than resistance training alone. All participants will undertake resistance training in the Healthy Lifestyle Research Centre, but half will be randomized to receiving Vitamin D and the other half a placebo. Nested sub‐studies involving muscle and adipose tissue biopsies will be undertaken to explore potential molecular mechanisms.


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Projects suitable for post‐graduate students

A wide range of opportunities to examine data relating lifestyle, physiological and social risk factors to diabetes, obesity and kidney disease in a number of population‐based studies exist. There are specific opportunities the following projects:

Environmental pollutants and chronic disease. Evidence is beginning to emerge to suggest that environmental pollutants may be part of the link between modern, westernised lifestyles and chronic diseases such as CVD and diabetes. This project will examine this question at an epidemiological level. Data from environmental surveillance will be matched to data collected in our population‐based surveys to determine if there are any associations between levels of pollutants and risk of CVD, diabetes and related disorders. The broad geographic distribution of our participants provides an ideal framework within which to identify links between environmental toxins and chronic disease.

Diabetes and cancer. It is now clear that people with diabetes are at greater risk of developing and dying from certain cancers. However, most of the data so far accumulated has been from administrative databases, with limited capacity to fully account for other risk factors such as smoking, obesity and physical activity. We are assembling a collaboration of Australian longitudinal epidemiological studies, which will have the capacity to properly investigate the independent effects of diabetes on cancer risk.


Genomics & Systems Biology

Dr. Jeremy Jowett [email protected] 8532 1775

Research Focus There is great diversity among the people you see and meet for example height, complexion or eye colour, characteristics (or traits) that are easy to discern. There are others that are of a more subtle nature and may not be immediately apparent like athletic ability and intelligence. This diversity in humans also extends to our level of predisposition for development of many common non‐infectious diseases such as cancer, type 2 diabetes and heart disease. In fact these diseases "run in families" (or are heritable). Additionally, they are “complex” meaning that an individual’s risk is influenced not only by many genes combined together (polygenic) but also by the environment, such as the types of food we eat and how much exercise we do.

The genomics and systems biology laboratory is working toward providing a better understanding of the process that causes type 2 diabetes, obesity and related cardiovascular disease, (such as heart failure) by identifying disease‐predisposing genes, their products and how those products work at a molecular level inside cells. We are using the latest exciting advances in DNA sequencing and genetic variation detection technology and combining this with gene expression analysis (genomics) in a systems biology approach to discover these genes and their function. Understanding their function and role in disease development will aid in the development of more accurate diagnostic tests and lead to improved therapeutic drugs which will provide better treatments than currently available for these diseases or even prevent them developing altogether.


Project 1: Gene Discovery in Type 2 Diabetes

Supervisor: Dr. Jeremy Jowett Changes in human behaviour and lifestyle over the last century have resulted in a dramatic increase in the incidence of Type 2 Diabetes Mellitus (T2D) worldwide in part driven by increased obesity. Once diagnosed, currently available therapies are inadequate to control disease progression and there are few new therapeutics in the development pipeline due to our limited understanding of the molecular pathogenesis. The complex etiology of T2D involves both environmental and genetic risk factors. A substantial effort has been made to identify the underlying genetic basis of familial predisposition, however currently we have only discovered the basis of about 10% of the risk, so there is a lot more to be identified.

This project will use next generation DNA sequence technology (NGS) to identify all genetic variation in a specific region within a cohort which has been successfully piloted in our laboratory. Comprehensive knowledge of all variation will allow full genetic dissection of the segregating variants contributing to the linkage signal and guarantee identification of the causative gene or genes. This project will focus on cataloguing genetic variation in a large multi‐generation family based cohort at chromosome 12q24, a region that we have previously discovered to contain genes influencing blood glucose levels (a measure upon which type 2 diabetes is diagnosed). Another component will examine specific genes as candidates for their role in controlling glucose levels. Finally, validation experiments using in vitro model systems of glucose homeostasis will also be applied to confirm a role of the candidate genes. Ultimately these findings will translate to an improved understanding of the molecular mechanisms of disease leading to novel and more specific therapeutics to control disease progression in afflicted individuals.


Project 2: Systems Biology Approach Discovers New Genes Controlling Cholesterol and Cardiovascular Disease ‐ But How do they Work?

Supervisor: Dr. Jeremy Jowett

. Many epidemiological studies have shown that a low levels of "good cholesterol", high density lipoprotein cholesterol (HDL‐C) is a strong independent risk factor for development of cardiovascular disease (CVD) including atherosclerosis that causes heart attacks and strokes. Major influences on low HDL‐C levels include obesity, smoking, sedentary behaviour, type 2 diabetes and genetic factors. Current therapies raise HDL‐C levels ineffectively, therefore there is an urgent need for new HDL‐C raising drugs. A full understanding of the mechanisms responsible for the regulation of HDL‐C levels will facilitate the design of new drugs for at risk individuals.

We have used Genome Wide Association Study (GWAS) data and combined this with global gene expression profiling analysis to discover a set of new genes that control HDL‐C levels. This project will investigate the mechanism of action linking these genes to HDL‐C homeostasis. Initially the biological consequences of modulation (over‐expression and knockdown) in tissue culture models of HDL‐C production, remodelling and catabolism will be investigated. Next the upstream genes that were found to control the expression levels of these genes will be validated using functional assays (siRNA knockdown/ vector over‐expression). These validation studies in tissue culture and animal model systems will increase our understanding of the HDL‐C homeostasis mechanisms and reveal multiple new potential therapeutic targets for development of interventions. This will lead to significant improvements in clinical care strategies to combat the rising incidence of cardiovascular disease and reduce its impact on morbidity and mortality in the population.


Metabolomics

Peter Meikle [email protected] 8532 1770

Research Focus Metabolomics is the systematic study of the unique metabolite (small‐molecule) fingerprints of biological systems. The Metabolomics Laboratory uses “state of the art” tandem mass spectrometry to obtain metabolic/lipid profiles from cell and animal models in addition to clinically relevant human samples to develop new approaches to early diagnosis, risk assessment and therapeutic monitoring of diabetes and cardiovascular disease. This approach is also combined with cell biology studies to investigate lipid metabolism and pathogenesis in atherosclerosis and other disease states.

Project 1: High Density Lipoprotein (HDL) composition and functionality

Supervisors: A/Prof Peter Meikle, Prof Bronwyn Kingwell, Dr Dmitri Sviridov

A reduced level of high density lipoprotein (HDL) is a major risk factor for cardiovascular disease. There are a number of mechanisms by which HDL is thought to be protective against cardiovascular disease; HDL is the major component of the reverse cholesterol transport pathway that removes harmful cholesterol from vessel walls and other tissues and transports it back to the liver where it can be excreted from the body. HDL has also been shown to have anti‐inflammatory and antioxidative properties. Importantly, HDL from different individuals displays different levels of functionality.

HDL is a complex lipoprotein particle consisting of protein (primarily Apo AI but also many others) and lipids (phospholipids, triacylglycerol, sphingomyelin and others). The composition of HDL is believed to have a major influence on its ability to function in each of the processes described above.

We hypothesis that: The efficiency of HDL in reverse cholesterol transport is related to the lipid composition of the HDL particles.

Project aim: To characterize the relationship between HDL functionality (reverse cholesterol transport) and its lipid composition.

ApoA‐Iphospholipid

cholesterol

cholesterol ester

triglyceride

ApoA‐I


Individuals with metabolic syndrome, diabetes or cardiovascular disease have HDL particles that are less effective at reverse cholesterol transport. We have clinically defined patient samples which will be used to isolate HDL and measure its functionality. We will then characterize the lipid composition of the HDL, using state of the art tandem mass spectrometry, and determine the relationship between lipid composition and HDL function.

Significance: Therapeutic strategies to increase circulating HDL are currently under development. Knowledge of the relationship between HDL composition and function will have help to direct the development of such therapies.

Project 2: Lysosomal dysfunction in primary human foam cells

Supervisors: Dr Melissa Greeve and A/Prof Peter Meikle

One of the early events leading to atherosclerosis is the accumulation of monocytes, macrophages, foam cells and lipids within the intima of the arterial wall as “fatty streaks” (Fig. 1). While most, if not all, adults have fatty streaks within their arterial walls, not all of these lesions progress to fibrolipid plaques; the precursors of the more clinically relevant complicated/unstable plaque. Associated with the progression of fatty streaks to the fibrolipid plaque is the appearance of macrophage foam cells in which cholesterol accumulates primarily within lysosomes rather than the cytoplasm. These cells contribute to the inflammatory process of atherosclerosis and their death by apoptosis promotes necrotic core formation in atherosclerotic plaque.

Our laboratory has developed a cell culture model which enables us to study the early cellular events involved in foam cell formation. In this model, macrophages derived from the monocytic cell line, THP‐1, are fed modified forms of low density lipoprotein (LDL) and adopt a ‘foamy’ appearance. Using this model, we have observed that the lipids, cholesterol and ceramide, accumulate within the lysosomes of macrophages cultured with copper‐oxidised LDL. However, it is unclear whether these cellular changes also occur in primary macrophages and whether they can be induced by LDL oxidised by physiologically relevant mechanisms (e.g. by myeloperoxidase). This project will provide valuable insight into the cellular mechanisms underlying the progression of human atherosclerotic plaques to instability and help to identify new sites for therapeutic intervention to halt or reverse the process of atherosclerosis

We hypothesise that: the alterations in the lipid composition of lysosomes resulting from the uptake of physiologically oxidised LDL lead to lysosomal dysfunction and secondary alterations of lipid metabolism in human macrophages.

Fig. 1. Fatty streak formation.

monocyte atherosclerotic plaque

lipid

arterial intimamacrophage foam cells

endothelium


Project aim: Using techniques such as cell culture, mass spectrometry, Western blotting and confocal microscopy, we aim to characterise the effects of myeloperoxidase‐oxidised LDL on lipid metabolism and downstream signaling pathways in human peripheral blood‐derived macrophages.

Project 3: Regulation of vascular endothelial permeability by lipids

Supervisors: Dr Melissa Greeve and A/Prof Peter Meikle

The vascular endothelium forms a physical barrier between the blood and underlying tissues and regulates the transport of macromolecules and immune cells into the vessel wall. Atherosclerotic lesions are more permeable than normal arterial tissue and loss of endothelial barrier function within plaques enhances the infiltration of immune cells. Since inflammatory cells determine lesion size and complexity, loss of barrier integrity probably promotes disease progression. However, the underlying causes of endothelial barrier dysfunction are unclear.

Oxidised LDL (oxLDL) and a major lipid constituent of oxLDL, lysophosphatidylcholine (LPC), accumulate in atherosclerotic plaques and promote endothelial permeability (Fig. 1). The permeability of the endothelial cell (EC) layer to macromolecules and immune cells is determined by the relative levels of adherens/tight junction proteins (Fig. 2) and the lipids, cholesterol and ceramide, at cell‐cell junctions. Ceramide has been shown to impair epithelial barrier function and accumulates in macrophage, smooth muscle and endothelial cells in response to oxLDL. However, it is not known if oxLDL promotes endothelial permeability by increasing ceramide levels at cell junctions. This study will establish a novel role for lipids in endothelial barrier function and may cause a paradigm shift in our understanding of endothelial regulation and the progression of atherosclerosis.

We hypothesise that: oxLDL, through the action of the constituent lipid, LPC, promotes endothelial permeability by stimulating the accumulation of ceramide at cell‐cell junctions and causing the redistribution of key junction and signaling proteins to the cytosol.

Project aim: This project will employ a number of techniques, including cell culture, mass spectrometry, Western blotting, confocal microscopy and permeability assays, to determine if oxLDL and LPC regulate endothelial permeability.

Fig. 1. OxLDL and hydrogen peroxide, but not unoxidised LDL (nLDL), increase the permeability of an EC monolayer to dextran.

Tight junction

Adherens junction

Fig. 2. The junctions between ECs contain protein complexes that regulate the permeability of the endothelium to molecules and cells.


Vascular & Hypertension


Atherothrombosis & Vascular Biology Laboratory

Karlheinz Peter [email protected] 8532 1490 Research Focus Research in the Atherothrombosis & Vascular Biology laboratory at Baker IDI spans from basic research in vascular biology to clinical trials in cardiology. This includes studies on the cellular mechanisms of coronary artery disease, encompassing the role of platelets, coagulation and inflammation in atherosclerosis, as well as the mechanisms leading to the rupture of atherosclerotic plaques, which is the final event of atherosclerosis, precipitating myocardial infarction or stroke.

We developed molecular biology techniques that allow the generation of recombinant antibodies for diagnostic and therapeutic use. So‐called “intelligent” drugs, conformation‐specific inhibitors of cell surface receptors are developed as therapeutic agents e.g. as anti‐thrombotic or anti‐inflammatory drugs. In addition, the use of these agents as diagnostic markers for the activation of the coagulation system and the inflammatory system is evaluated. The aim is to diagnose thrombotic disease (e.g. in the heart) and inflammatory diseases (e.g. Multiple Sclerosis) before major non‐reversible health problems occur.

We offer students the unique opportunity to develop recombinant strategies and agents starting from the initial design up to the final in vivo evaluation. The projects are suitable for students with an interest in molecular biology and biotechnological methods. Students will be able to learn a variety of techniques such as recombinant single‐chain antibody production, phage display and cell biology techniques including flow cytometry, flow chamber assays, intravital and confocal microscopy. We also work on the design and generation of diagnostically usable nanoparticles.

We have an excellent record in student‐supervision and we are seeking proactive students that are enjoying the work in a team‐oriented environment but who are willing to develop and test their own independent ideas as well.

Figure Resting platelets (left), activated platelets (right)


Project 1: Recombinant agents for efficient and safe anticoagulation and thrombolysis Supervisors: Dr Christoph Hagemeyer, Dr Jia Fu, Prof Karlheinz Peter

This project is based on the single‐chain antibody (scFv) technology, an emerging class of biotechnologically produced therapeutics. ScFvs are designed to contain the variable regions of a full IgG antibody linked by a small linker. The sequence of scFvs can be encoded on a single plasmid and thus mutations and additional fusions of tags or effector molecules can be performed using molecular biology techniques.

Our aim is the development of novel targeted thrombolytic drugs to treat thrombosis. We recently generated antibodies that specifically bind and block activated GPIIb/IIIa, the platelet receptor for fibrinogen. Its activation represents the final step common to all types of platelet activations. As this receptor’s default state is a non‐activated, resting state, GPIIb/IIIa needs to become activated in order to bind its major ligand, soluble fibrinogen causing platelet aggregation and thrombus formation.

The anti‐GPIIb/IIIa scFvs will be fused to a thrombolytic urokinase type plasminogen‐activator and a third anticoagulation component (Factor Xa inhibition by tick anticoagulant peptide TAP), generating a new class of drugs with thrombolytic and anti‐thrombotic properties in a single molecule. TAP is a highly potent and selective factor Xa inhibitor allowing effective anticoagulation because of its central, up‐stream, and rate‐determining position in the coagulation cascade. Targeting of TAP to clots can decrease systemic anticoagulation and thus bleeding complications.

By accumulating the fibrinolytic drug at the site of the clot, our designed molecules have the potential to improve the efficacy and safeness of the thrombolytic treatment by reducing the overall blood concentration and bleeding complications associated with current drugs. We expect a significant faster reopening of the occluded vessel in the animal model compared to non‐targeted plasminogen activators.

Figure Clot‐targeting allows enrichment at the thrombus

non-targeted:

targeted:

vessel

thrombusanticoagulant agent


Project 2: Single‐chain antibody‐targeted nanoparticles for diagnosis of vascular diseases Supervisors: Dr Christoph Hagemeyer, Dr Hang Ta, Prof Karlheinz Peter

This project aims to develop nanoparticle contrast agents for magnetic resonance imaging (MRI) that selectively target molecular markers of cardiovascular disease (CVD). These imaging agents will be developed for the early detection of unstable, rupture‐prone, vulnerable atherosclerotic lesions, thrombosis and difficult to diagnose vessel occlusions, such as pulmonary embolism.

We have generated specific single‐chain antibodies (scFv) that can selectively target markers associated with atherosclerosis and thrombosis. These include 1) MAN‐1, which targets the activated form of the Mac‐1 integrin on monocytes and macrophages and thus is specific for these leukocytes in the activated state; 2) scFv59D8, which targets fibrin, the end product of humoral coagulation; and 3) scFvanti‐LIBS, which targets the platelet surface glycoprotein integrin receptor IIb/IIIa in its activated, ligand bound form. We have already demonstrated that non‐covalent coupling of superparamagnetic iron oxide (SPIO)‐beads to scFvanti‐LIBS can target and image activated platelets in vessels in vitro and in vivo by MRI.

We will use Gadolinium (Gd)‐loaded dendrimers to give contrast in MRI. Dendrimers have a highly branched, three‐dimensional, nanoscale architecture, low polydispersity and multivalent surfaces that allow simultaneous binding of Gd for imaging and scFvs for targeting. The antigen‐specific binding capacity of the scFv‐dendrimer constructs will be confirmed using fluorescence‐labelled dendrimers in flow cytometry. Optimal size and Gd‐loading will be determined via in vitro MRI of human thrombi. Finally, efficacy studies in a mouse model of atherosclerosis and a rabbit model of pulmonary embolism will provide in vivo proof of the imaging capability of the constructs.

Figure Gd-Dendrimer scFv construct


Project 3: Targeted virus particles for genetic transfer of fusion proteins to inhibit atherosclerosis Supervisors: Dr Christoph Hagemeyer, Prof Karlheinz Peter

The adhesion of leukocytes to endothelium plays a central role in the development of atherosclerosis and thus represents an attractive therapeutic target for anti‐atherosclerotic therapies. Single‐chain antibodies against epitopes associated with vascular inflammation/dysfunction provide a unique tool for specific targeting of virus particles with inhibitory fusion proteins to areas of need. This specific targeting provides a new therapeutic approach for atherosclerotic disease.

Recently, we could show that interference with the cytoskeletal anchorage of adhesion molecules through co‐expression of inert fusion proteins, can reduce cell‐cell interactions, leading to the disruption of monocyte adhesion to inflamed endothelial cells. We achieved this with a new inhibitory fusion protein containing the intracellular part of vascular cell adhesion molecule‐1 (VCAM‐1) and the extracellular and transmembrane part of CD7 as an inert marker.

This project aims to develop novel biotechnological approaches that are based on our expertise in single‐chain antibodies generation, modification and fusion to effector molecules and nanoparticles. The biological effects of this novel virus targeting approach will be evaluated in a mouse model of advanced atherosclerosis.

A successful outcome of this study will be highly significant. We would have demonstrated the ability of an inhibitory CD7‐VCAM‐1 construct to block cell adhesion in vivo to reduce atherosclerosis. In addition, we would have developed several methods for single‐chain targeted delivery of virus particles that might be generally applicable in basic research and human diseases treatment.

Figure Inhibition of monocytes adhesion and migration by a competing fusionmolecule.

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PROJECT 5: Targeting (homing) stem cell for the treatment of atherosclerosis

Supervisors: Dr Denijal Topcic, Dr. Christoph Hagemeyer, Prof Karlheinz Peter

Single‐chain antibodies against epitopes associated with vascular dysfunction/ injury provide a unique tool for specific targeting of stem cells in vascular repair and regeneration processes e.g. in atherosclerosis. We will construct and test different cell targeting approaches using single‐chain antibodies directed against molecular markers of atherosclerotic plaques. In vitro proof of successful targeting will be done in flow chamber models, and in vivo models with proof of targeting efficacy in a mouse model of wire‐induced endothelial injury. Finally, we will use animal methods of atherosclerosis and plaque rupture to provide proof of concept that targeted stem cell therapy can be used to treat atherosclerosis and prevent plaque rupture and vessel closure.

As part of the project we have already established methods to obtain human adult stem cells (endothelial precursor cells) and mouse stem cells. We have a whole repertoire of single‐chain antibodies that are directed towards the epitopes (antigens) listed in the Figure below. The different epitopes are associated with different stages of atherosclerotic disease.

healthy inflamed injured atheroscleroticendothelium endothelium endothelium plaque

P-selectin VCAM-1 Mac-1 GPIIb/IIIa-LIBS fibrin

healthy inflamed injured atheroscleroticendothelium endothelium endothelium plaque

P-selectin VCAM-1 Mac-1 GPIIb/IIIa-LIBS fibrin


Neuropharmacology

Associate Professor Geoff Head [email protected] 8532 1332 Project 1: Effect of resveratrol, a natural antioxidant on the blood pressure of a spontaneously hypertensive mouse Supervisors: Associate Professor Geoffrey Head & Pamela Davern Our recent studies of the spontaneously hypertensive (BPH/2J) Schlager mouse, using radio telemetry for measuring blood pressure, have described for the first time that the hypertension is characterised by markedly exaggerated changes in blood pressure when the mouse goes from the light to the dark (active) period. Furthermore these mice have a very exaggerated increase in blood pressure in response to stress. Since we have also shown that stress involves the production of superoxide in the hypothalamus (brain) in other studies, we now wish to see whether chronic treatment with a naturally occurring antioxidant, resveratrol can restore blood pressure and reduce the response to stress. The project therefore will systematically investigate the effect of antioxidant treatment of the blood pressure and cardiovascular response to stress in conscious mice implanted with radiotelemetry devices. The experimental work for the project will be carried out at Baker IDI Institute in Prahran.

Project 2: Role of brain pathways in chronic stress Supervisors: Associate Professor Geoffrey Head and Pamela Davern

There is now strong evidence to suggest that the sympathetic nervous system (SNS) makes a significant contribution to certain forms of hypertension. One of the suggested mechanisms through which the SNS may be “activated” in hypertension is through the renin‐angiotensin system. We have recently demonstrated that a very low “subpressor” dose of Angiotensin given for several months to rabbits can produce a very modest increase in blood pressure but appears to amplify the effects of chronic stress. Using immuno‐histochemistry, we have found that specific areas of the brain are activated by infusing angiotensin, an action which is mediated through areas of the brain that does not have a blood brain barrier. These central pathways appear to be “sensitised”, reflecting a type of positive feed forward CNS plasticity. We now wish to determine which areas of the brain and in particular the hypothalamus may be responsible for this effect on chronic stress. The project will involve some animal surgery, experiments to measure blood pressure before and during a relatively mild air jet stress and then perfusion fixation of the brain to process for immunohistochemistry. The use of specific antibodies, fluorescence and confocal imaging will then be used to identify brain regions and specific neurochemical involved in the response to acute and chronic stress.


Project 3: Which parts of the brain are affected in obesity related hypertension? Supervisors: Dr James Armitage and Associate Professor Geoffrey Head Cardiovascular disease and its ultimate consequences are a significant burden on health systems in Australia and around the developed world. It is increasingly apparent that the high rate of hypertension in many societies can be attributed to an equally alarming rate of obesity. Despite this link, there is a less than complete understanding of the manner by which obesity alters central nervous system function to result in hypertension. It is known that the sympathetic nervous system (SNS), responsible in part for the control of blood pressure and is overactive in obese humans. It is also known that several peripheral signalling chemicals act at specific brain sites to modulate both appetite and also the activity of the SNS . For example, leptin, produced by fat and responsible for reducing appetite by acting at specific sites in the brain may act also to either stimulate or inhibit SNS activity depending on which neurons it acts at in the hypothalamus. Nonetheless, despite a comprehensive understanding of changes in appetite systems in the brain in obesity, and a known link between some of these appetite centres and other nuclei that control SNS outflow there is little understanding of how the sympathetic nervous system is activated during the development of obesity.

We seek to determine the manner by which various brain centres respond in obesity by studying patterns of neural activation and measuring SNS activity and blood pressure during the development of obesity. This project will involve surgical implantation of telemetric probes (for measurement of renal sympathetic nerve activity and haemodynamic variables) and guide cannulae into distinct brain regions to allow injection of drugs. Students will also learn immunohistochemical techniques in order to identify by immunofluorescent labelling those neurons that are responsible for the genesis of obesity related hypertension

Project 4: The developmental programming of obesity and metabolic disease; what are the consequences of maternal fat intake? Supervisors: Dr James Armitage and Associate Professor Geoffrey Head Maternal fat intake during pregnancy has been associated with the programming of obesity in the offspring. We have recently developed a new model of maternal obesity where female rabbits are fed a 13% fat diet (normally 3 %) for several weeks prior to falling pregnant. The offspring are also exposed during the pre‐weening period. We hypothesize that the foetal exposure to a high fat environment will have detrimental effects on the cardiovascular system in adulthood. The aim of the project is assess blood pressure, sympathetic activity and other cardiovascular indices in the adult rabbits. The cardiovascular state of the offspring will be examined using a rabbit model to study neuronal activation in the brain, renal sympathetic nerve activity, blood pressure, baroreceptor function and the response to appetite modulating factors such as leptin and ghrelin. This project will require students to learn surgical techniques, analysis of physiological data and immunohistochemistry.

We seek to determine the manner by which various brain centres respond in obesity by studying patterns of neural activation and measuring SNS activity and blood pressure during the development of obesity. This project will involve surgical implantation of telemetric probes (for measurement of renal sympathetic nerve activity and haemodynamic variables) and guide cannulae into distinct brain regions to allow injection of drugs. Students will also learn immunohistochemical techniques in order to identify by immunofluorescent labelling those neurons that are responsible for the genesis of obesity related hypertension.


Stroke Epidemiology

Mandy Thrift [email protected] 8532 1636

Research Focus Our research focus is to use epidemiological and clinical trial methods to reduce the burden of stroke. We have a range of projects that we are undertaking in Australia and overseas. The particular projects are outline below:

Project 1: Improving Outcome after Stroke Supervisor: A/Prof Amanda Thrift Approximately 50,000 Australians suffer a stroke each year. Survivors of stroke are more likely to suffer another vascular event (stroke or heart attack) than people who have not had a stroke. Risk factors for development of further stroke include elevations in blood pressure, blood glucose and cholesterol; smoking; and being overweight and inactive. Most stroke patients have at least two of these preventable risk factors. To reduce the likelihood of having another stroke, we must find simple, and efficient approaches to reduce risk factors. Proven therapies for preventing stroke recurrence include strategies such as blood pressure and lipid lowering, but uptake of these therapies is poor.

We aim to encourage General Practitioners to use individualised plans to improve risk factor management in stroke patients. Using a randomised controlled trial design we will allocate stroke patients to either an Intervention group (nurse‐led individually designed plan) or Control group (usual care), and then assess differences in control of risk factors between groups. It is anticipated that stroke patients undertaking a comprehensive management plan in the community will have greater reductions in blood pressure than stroke patients undergoing usual care.

Project 2: North East Melbourne Stroke Incidence Study (NEMESIS) Supervisor: A/Prof Amanda Thrift Over the past 10 years the health of 1686 people aged between 2 and 102 years have been tracked through a series of questionnaires conducted during patient‐nurse interviews. During this time we have assessed the costs of stroke, and physical and mental wellbeing. The data have all been collected and we are looking for students interested in developing their own hypotheses about long‐term outcome, and applying these to the study. For example, little is known about how differently men and women survive in the long‐term after their stroke and whether their quality of life also differs. These, and many other questions, can be answered using the data already collected.


Project 3: International Health in India and Vietnam Supervisor: A/Prof Amanda Thrift There may also be opportunities for students to contribute to some of the international health projects that are being undertaken within the unit.

Our team is committed to increasing understanding of stroke prevention and in providing a supportive environment for students to achieve their full research potential. We are seeking motivated and enthusiastic students (PhD and Hons) who are eager to develop their research, problem‐solving and communication skills. Further details on some of our projects are available at:

http://www.bakeridi.edu.au/research/stroke_epidemiology/

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2011 Student Research P ono urs - Baker Institute Student Research... · We have recently shown in laboratory mice or patients with acute myocardial infarction functional ... The