Diabetic Complications

Dr Judy De Haan
Head, Oxidative Stress
Judy.DeHaan@bakeridi.edu.au

Laboratory Research Overview

Oxidative stress has been linked to the development of diabetic complications such as atherosclerosis, the accumulation of fatty deposits in the lining of blood vessels, as well as diabetic nephropathy, a debilitating kidney disease that affects 25-40% of diabetic patients. This group is investigating the genetic component of oxidative stress in these often linked diabetic co-morbidities. They are working on the development of dietary interventions or drug compounds to increase the activity of antioxidant enzymes, thereby reducing the effects of diabetic complications. This novel approach is highlighting the feasibility of addressing two diabetic complications with one treatment regimen through the approach of targeted antioxidant therapy.

Judy started her research in the area of oxidative stress and Down Syndrome. Oxidative stress describes a state characterised by an excess of unstable elements, known as free radicals, in the body. Free radicals can be overproduced as a result of exposure to some environmental toxins and are believed to be involved in the premature ageing of organs and in cellular damage.

Beginning her studies by examining the presence of certain enzymes in the normal ageing process, Judy's research team developed a mouse model lacking an important antioxidant enzyme, one that is usually present to remove an excess of free radicals. Experiments showed that these mice could function quite normally until they were stressed in some way. From there studies moved on to trying to better understand the link between oxidative stress, injury and disease.

Since moving her research team to the Baker IDI in 2004, Judy's research initially focused on atherosclerosis, both within and outside the setting of diabetes, and the role that oxidative stress plays in the development of atherosclerotic disease. Studying atherosclerotic diabetic mice that are missing an antioxidant enzyme, which would otherwise mop up the free radicals, this lab has shown a direct link between reduced levels of this antioxidant and increased components of atherosclerosis in these animals, for example, increased lesions. Recent findings have established that similar mechanisms are responsible for accelerated diabetic nephropathy in these mice.

These studies have established the framework for the next important phase of Judy's research: the development of novel compounds that mimic the action of this antioxidant, to reduce these diabetic complications.

Research Focus

• The role of oxidative stress in diabetes-associated atherosclerosis
• The role of oxidative stress in diabetic nephropathy
• A focus on pro-inflammatory pathways and oxidative stress
• In vitro and in vivo models of diabetic complications
• Novel antioxidant drugs to reduce diabetic complications

Research Projects

Can Gpx1-mimetics reduce diabetes associated atherosclerosis?

This project will investigate whether a novel synthetic antioxidant with GPx-like activity reduces diabetes-associated-atherosclerosis (DAA). It will investigate the mechanisms leading to this reduction with particular focus on inflammatory pathways. We have exciting data using the parent compound ebselen in support of our hypothesis that GPx-mimetics reduce DAA. 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 an analogue of ebselen with improved GPx-like activity to ascertain its potential as a novel targeted antioxidant therapy 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.

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, 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.

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. 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:
(i) 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.
(ii) 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.
(iii) 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.

Lab Head Profile

Dr Judy de Haan is Head of the Oxidative Stress Laboratory at Baker IDI Heart and Diabetes Institute. She holds an Adjunct Senior Lectureship in the Department of Biochemistry and Molecular Biology at Monash University.

Dr de Haan has made significant contributions to the field of oxidative-stress driven pathogenesis, initially investigating Down Syndrome, premature aging and stroke, and more recently diabetes-associated atherosclerosis and kidney disease. This has given her tremendous insight into targeting of specific ROS that contribute to pathogenesis. She has recently shown an important role for the antioxidant, GPx1, in limiting two often linked co-morbidities, diabetes associated atherosclerosis and diabetic nephropathy. Her recent discovery that a GPx1-mimetic, ebselen, reduces diabetic complications holds tremendous potential as a novel therapeutic strategy for the treatment of diabetic complications.

Dr de Haan has been an invited speaker at numerous national and international conferences, institutes and universities. She was an invited session speaker at the Pharmacia Corporation Inaugural Bioscience Foundation Meeting, Sydney, the CSANZ meeting 2009, and the Australian Atherosclerosis Society meeting, Cairns, 2010. In 2006, she received the NSW Ministry of Science and Medicine Award for Basic Science in Vascular Biology.

Dr de Haan has received numerous peer-reviewed grants from organisations including NHMRC, NHF, JDRF, as well as attracting industry support. Her current NHMRC grants investigate novel antioxidant therapies to prevent diabetes-associated atherosclerosis.

Dr de Haan holds positions on various working committees at Baker IDI, most notably, the PAC Animal Ethics Committee where for a period she was Deputy-Chair. She is also the Baker IDI Seminar Co-ordinator and most recently has been appointed as Newsletter Editor of the Australian Atherosclerosis Society.

Achievements/Awards

Publication Highlights

• De Haan JB, Witting PK, Stefanovic N, Pete J, Daskalakis M, Kola I, Stocker R, Smolich JJ. Deficiency in the antioxidant enzyme glutathione peroxidase-1 (GPx1) does not increase atherosclerosis in C57BL/J6 mice fed a high fat diet. J Lipid Res 2006;47:1157-67.
• 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 (GPx1) accelerates atherosclerosis in diabetic apolipoprotein E-deficient mice. Circulation 2007;115:2178-2187.
• Chew P, Yuen DYC, Koh P, Stefanovic N, Febbraio MA, Kola I, Cooper ME, De Haan JB. Site-specific antiatherogenic effect of the antioxidant ebselen in the diabetic apolipoprotein E-deficient mouse. ATVB 2009;29:823-830.
• Chew P, Yuen DYC, Stefanovic N, Pete J, Coughlan MT, Jandeleit-Dahm KA, Thomas MC, Rosenfeldt F, Cooper ME and DE HAAN JB. Anti-atherosclerotic and renoprotective effects of Ebselen in the diabetic Apolipoprotein E/GPx1-double knockout mouse. Diabetes Sep 7 2010 (Epub ahead of print).
• De Haan JB and Cooper ME. Targeted antioxidant therapies in hyperglycemia-mediated endothelial dysfunction. Frontiers in Bioscience 2010, in press.

Key Staff

Contact

Direct: +613 8532 1520
Email: judy.dehaan@bakeridi.edu.au