Postgraduate Studies Projects

Lab Head:   Associate Prof Peter Meikle
Phone:   8532 1770
Email:   peter.meikle@bakeridi.edu.au

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 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 - lipid profiling: early detection of unstable heart disease

Atherosclerosis is the single most common cause of cardiovascular disease and is the major contributor to the development of angina, heart attacks, coronary heart disease and stroke. Identification of individuals prior to the development of unstable heart disease will enable early intervention and will have a profound effect on the health of the Australian population.

While it has long been recognized that cholesterol levels are useful predictors of heart disease, it is now becoming clear that many other lipid types are altered during, and exert an effect upon atherosclerosis.  A number of lipids and lipid ratios have been proposed as useful indicators and predictors of atherosclerosis.  However, these provide only a restricted picture and a limited interpretation of the atherosclerosis status of an individual.  In this project we will use a novel lipidomic approach to generate lipid profiles from patients with stable and unstable heart disease to identify those changes that are associated with progression from stable to unstable disease

Hypothesis

Changes in lipid metabolism leading to and resulting from unstable heart disease will be reflected in the plasma lipid profile.

Project Aim

• Identify and quantify changes to the plasma lipid profile associated with different stages of cardiovascular disease

Outcomes

The primary outcome of this project will be the development of plasma lipid profiling technology to enable the early detection of “unstable” patients in addition to methods for classifying atherosclerosis patients and monitoring treatment.

Project 1 Techniques

• LDL/HDL preparation from plasma - Tandem mass spectrometry 
• Lipid extraction - Statistical analysis.

Project 2 - Lysosomal dysfunction in foam cell formation

One of the early events leading to atherosclerosis is the formation of “fatty streaks”; deposits of monocytes, macrophages, foam cells and lipids within the intima of the arterial wall.  Most/all adults have fatty streaks within their arterial walls, however not all fatty streak lesions progress to form a fibrolipid plaque the precursor to the more clinically relevant complicated/unstable plaque.  Associated with the progression of fatty streaks to the fibrolipid plaque is the appearance of foam cells where the cholesterol accumulates primarily within lysosomes rather than the cytoplasm.  The concomitant activation of macrophages contributes to the inflammatory process of atherosclerosis and the subsequent apoptosis of macrophage/foam cells leads to the necrotic core of the atherosclerotic plaque. 

Our laboratory has developed a model of foam cell formation whereby macrophages derived from the differentiation of murine THP-1 monocytes are fed modified forms of LDL in culture.  This model allows us to study the early cellular events which contribute to foam cell formation.  Using cell fractionation, fluorescence microscopy and state of the art tandem mass spectrometry, we have detected an accumulation of both cholesterol and ceramide within the lysosomes of macrophages cultured with copper-oxidised LDL.  Thus, lysosomal dysfunction seems to be an early event in lesion progression. However, it is unclear whether these cellular changes also occur in human 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.

Hypothesis

The alterations in the lipid composition of lysosomes resulting from the uptake of physiologically oxidised low density lipoprotein (LDL) leads to lysosomal dysfunction and secondary alterations of lipid metabolism in human macrophages.  

Project Aim

To define the effects of myeloperoxidase-oxidised LDL uptake on the composition, structure and function of lysosomes and characterise the secondary changes in lipid metabolism in human macrophages.

Outcomes

Characterisation of the mechanism of lysosomal dysfunction is the will help to identify new sites for therapeutic intervention to halt or reverse the process of atherosclerosis. 

Project 2 Techniques

• Cell culture - SDS-PAGE / Western analysis 
• Tandem mass spectrometry - Cell fractionation
• Fluorescence microscopy - Pulse labelling techniques.

Project 3 - Plasma lipid profiling: unravelling the relationship between obesity, insulin resistance and diabetes

Austrailia, and most of the western world, are facing an obesity epidemic; associated with this is an increase in the levels of insulin resistance and type 2 diabetes.  While it is clear that obesity can increase the risk of developing insulin resistance and diabetes, not all obese people progress to these disease endpoints.  The reasons for this are still largely unknown and importantly there is no definitive test to differentiate healthy individuals from those headed towards insulin resistance and diabetes.
Lipidomics (a sub-set of metabolomics) is the systematic study of the unique lipid fingerprints of biological systems.   Using “state of the art” tandem mass spectrometry we are able to quantify 100s of lipids from just a few l of blood.   This powerful approach provides us with a detailed view of the changes in lipid metabolism associated with different disease states.

Hypothesis

Changes in lipid metabolism in some obese individuals result in a pro-inflammatory state that ultimately leads to insulin resistance and type 2 diabetes.  These changes in lipid metabolism and the resulting inflammation are reflected in the plasma lipid profile.

Project Aim

To characterise the changes in plasma lipids that are associated with the development of insulin resistance and type 2 diabetes. 

Outcomes

The successful completion of this project will provide the basis for a new diagnostic test to identify those individuals at greatest risk of developing type 2 diabetes. In addition the results generated will improve our understanding of the changes in lipid metabolism leading to insulin resistance and type 2 diabetes.  This is an important first step in the identification of new sites for therapeutic intervention to halt disease progression.

Project 3 Techniques

• Statistical analysis of large databases
  
• Fast Phase Liquid Chromatography
• Lipid extraction
  
• Tandem mass spectrometry
• Ultracentrifugation.