Group Leaders: Wendy Jessup and Len Kritharides Overview: Our major research objective is to develop knowledge of the mechanism, nature and impact of sterol accumulation in human disease, with particular emphasis on the generation and biology of the macrophage foam cell in atherosclerosis. We have ongoing interests in the processes which mediate lipid accumulation by macrophages, including lipoprotein modifications that promote uptake of lipid (such as oxidation), and processes by which macrophages export cholesterol, and by which atherosclerosis may be reversed. Knowledge gained from these studies addresses fundamental issues of cholesterol traffic within the macrophage foam cell. Extensions of these studies into the areas of inflammation and leucocyte activation will allow us to investigate how cholesterol and inflammation are linked. 
Scanning electron microscopy image
of a mouse peritoneal macrophage
(Jessup, unpublished data). | 
Immunocytochemical demonstration of human macrophage apo E distribution. The arrows depict strong perinuclear staining of the Golgi and peripheral staining of the plasma membrane, N; nucleus (Garner, unpublished data). |
Projects: Mechanism of intimal lipoprotein oxidation and its prevention
Oxidised lipids are present in human atherosclerotic lesions and thought to contribute to initiation and progression of atherosclerosis. Evidence points strongly to local oxidation of intimal lipoproteins, in which the cells almost certainly play a controlling role. Cell-mediated oxidation is absolutely dependent on trace levels of extracellular transition metals and we have found that the cellular process involved is a plasma membrane electron transport system that maintains the metals in a reduced state. This provides a selective target for intervention and current studies are investigating how to identify and control expression of the gene(s) involved in collaboration with Prof Ian Dawes (Molecular Genetics, UNSW). Metabolism of oxidized proteins and lipoproteins by macrophages
Oxidised lipids and lipoproteins exist within the atherosclerotic intima and have been implicated in macrophage foam cell formation via increased uptake, impaired catabolism, and restricted export. We have measured oxysterols in human lesions and in foam cells isolated from them, and have generated model foam cells containing comparable amounts of defined oxysterols (such as 7-ketocholesterol, 7KC). Using such models we have shown specific inhibitory effects of 7KC on sterol transport (see below). Together with Dr Andrew Brown (UNSW Biochemistry) we have also recently identified a pathway for metabolism/elimination of 7KC by macrophages. This involves a mitochondrial cytochrome P450 enzyme, sterol 27-hydroyxylase (CYP27A).pathway, which is also functional in the liver. It appears to operate sub-optimally in foam cells and is a target for stimulation to enhance cholesterol and oxysterol disposal. We are currently studying regulation of this pathway in macrophages. Plasma membrane heterogeneity in macrophages
Cholesterol and glycosphingolipids and other sphingolipids (predominantly sphingomyelin) form liquid-ordered microdomains or 'lipid rafts' in the plasma membrane of cells. These domains have been generally viewed as ordered clusters of lipids that float, like rafts, in a sea of liquid-disordered lipids; predominantly phospholipids. It is becoming increasingly recognised that such domains play important roles in many cellular functions, including signal transduction and membrane trafficking . We are currently studying how lipid raft composition, abundance and function are controlled in the macrophage, particularly in response to development of the foam cell phenotype. Mechanisms for removal of sterols from foam cells
Foam cell formation depends on a net excess of cholesterol import/synthesis over export which is mediated by so called "good cholesterol" high density lipoprotein (HDL), and its main protein component apo A-I. We are currently studying the importance of lipid rafts in apoA-1 cell interactions and are investigating how cholesterol accumulation affects macrophage raft structure and function. Cholesterol export is inhibited by 7KC, implying that overcoming this inhibition is potentially therapeutic for atherosclerosis. 7KC inhibits cholesterol export at the level of the plasma membrane, with concurrent inhibition of phospholipid export and changes to lipid rafts Excess cholesterol is stored within foam cells as cholesteryl ester. The rate of clearance of CE from foam cells is believed to be rate limiting for cholesterol removal from foam cell macrophages. Using a combination of biochemical, structural and proteomic techniques we are investigating factors and proteins regulating droplet stability and turnover in foam cells. Influence of lipids and apolipoproteins on macrophage function
Macrophage persistence and proliferation in lesions may be mediated by the accumulation of oxidized lipids and the persistence of indigestible components within the cells, based on our recent collaborative work with Professor John Hamilton (Melbourne University). One of the most important regulatory proteins in atherosclerosis is apolipoprotein E (apo E). We identified that during stimulation of cholesterol efflux, apolipoprotein A-I also stimulates the secretion of apo E from macrophages. Ongoing studies together with Kerry Anne Rye (Heart Research Institute Sydney) have identified that the structural properties of apo A-I required for stimulation of apo E secretion differ from those involved in cholesterol efflux. Studies of the cellular mechanisms of apo A-I-stimulated apo E secretion are in progress. Neutrophil and monocyte activation after cardiac surgery
Cardiac surgery induces massive inflammatory and pro-coagulant stimuli which are associated with substantial patient morbidity. Understanding the mechanisms by which leucocytes are activated during this procedure may to lead to useful therapies, and will also provide a powerful tool with which to investigate regulation of leucocyte activity in man. We have found that neutrophil integrin expression is unexpectedly down-regulated during bypass surgery. Together with Carolyn Geczy (Cytokine Research Unit, UNSW) and Paul Bannon (Cardiothoracic Surgical Unit RPAH) we will investigate the mechanism of this down regulation and its significance in relation to functional activities of neutrophils and monocytes. Glycobiology of Atherosclerosis
Research into the functions of specific glycoconjugates, including glycoproteins and glycosphingolipids (GSLs), which play a role in atherosclerosis is led by Dr Brett Garner. Current work is focused on two areas: 1. the role of the carbohydrate moiety of apolipoproteins; including apo (a), apo B100 and apo E, in regulating apolipoprotein function and metabolism; and 2. the role of GSLs in macrophage foam cell development and atherogenesis. Recent studies utilising direct carbohydrate sequencing technology have helped to define several basic biochemical characteristics of apolipoprotein structure and metabolism including the role that apo (a) glycans play in regulating its proteolytic degradation. In vitro work has shed new light on the potential for GSLs to modulate important macrophage functions, including cholesterol accumulation and apo E secretion. This work is currently being extended in a study of the mechanisms by which specific GSLs may alter macrophage "lipid raft" (see above) structure and function. The work on GSLs has also been extended into studies of glycosphingolipid metabolism in humans and in the apo E gene knockout mouse, an animal model widely used to study mechanisms of atherosclerosis. The study of atherogenesis from a glycobiology perspective will provide novel insights into disease mechanisms and potential therapeutic strategies. Key References: Rees D, Sloane T, Jessup W, Dean RT, Kritharides L. (1999)
Apolipoprotein A-I stimulates secretion of apolipoprotein E by foam cell macrophages. J Biol Chem 274: 27925-27933. Jessup W, Kritharides L. (2000)
Metabolism of oxidized LDL by macrophages. Curr Opin Lipidol 11: 473-481. Brown AJ, Watts GF, Burnett JR, Dean RT, Jessup W. (2000)
Sterol 27-hydroxylase acts on 7-ketocholesterol in human atherosclerotic lesions and macrophages in culture. J Biol Chem 275: 27627-27633. Baoutina A, Dean RT, Jessup W. (2000)
Macrophages can decrease the level of cholesteryl ester hydroperoxides in low density lipoprotein. J Biol Chem 275: 1635-1644. Gaus K, Gooding JJ, Dean RT, Kritharides L, Jessup W. (2001)
A kinetic model to evaluate cholesterol efflux from THP-1 macrophages to apolipoprotein A-1. Biochemistry 40: 9363-9373. Baoutina A, Dean RT, Jessup W. (2001)
Trans-plasma membrane electron transport induces macrophage-mediated low density lipoprotein oxidation. FASEB J 15: 1580-1582. Garner B., Merry A.H., Royle L., Harvey D.J., Rudd. P.M. Thillet J. (2001)
Structural elucidation of the N- and O-glycans of human apolipoprotein(a). Role of O-glycans in conferring protease resistance. J Biol Chem. 276: 22200-22208. Garner B., Priestman D.A., Stocker R., Harvey D.J., Butters, T.D., Platt F.M. (2002)
Increased glycosphingolipid levels in serum and aortae of apolipoprotein E gene knockout mice. J Lipid Res 43: 204-215. Kritharides L, Stocker R. (2002) The use of antioxidant supplements in coronary heart disease.
Atherosclerosis, 164, 211-219. M. Vallely, P. Bannon, C. Hughes, M Bayfield, L. Kritharides. (2002)
Endothelial expression of ICAM-1 and VCAM-1 is suppressed by post bypass plasma containing increased soluble ICAM-1 and VCAM-1 J. Thorac. Cardiovasc. Surg., in press. Group Members: | 
