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Phospholipid hydroperoxide glutathioneperoxidase: promoter, expression, regulation, genetic manipulation and role in the pathogenesis of atherosclerosis

Objectif

Objectives
The objectives of the application are to understand what factors control phospholipid hydroperoxide glutathione peroxidase (PHGPX EC.1.11.1.12) expression at transcriptional level, what factors are necessary for the overexpression of PHGPX and what is the effect of PHGPX on the development of atherosclerotic lesions in rabbit and mouse arteries. PHGPX will be studied at gene and promoter level, in stably transfected cell culture systems, and in normal and atherosclerotic NZW and WHHL rabbit blood vessels using intraarterial gene transfer.

Our application focuses on the understanding of the mechanisms of selenium incorporation into selenoproteins, especially those that control phospholipid hydroperoxide glutathione peroxidase (PHGPX, EC.1.11.1.12) expression and activity. Furthermore, the role of PHGPX in atherogenesis will be studied. Atherosclerosis-related diseases (myocardial infarction, stroke and peripheral vascular disease) cause nearly half of the deaths in the European Union. They have an enormous impact on health and economics. There is much evidence that LDL oxidation and lipid peroxidation play key roles in the pathogenesis of atherosclerosis. For its ability to reduce phospholipid, cholesterol and cholesterol ester hydroperoxides, PHPGX is an essential part of the antioxidative defense against lipid hydroperoxide and radical-mediated injuries. It is anticipated that better understanding of the physiological significance of this enzyme will help us to define new treatment strategies for atherosclerosis. The objectives of the application are to understand what factors control PHGPX expression at transcriptional and translational level, what factors are necessary for the over-expression of PHGPX and what is the effect of PHGPX on the development of atherosclerotic lesions in rabbit and mouse arteries. PHGPX will be studied at gene and promoter level, in stably transfected cell culture systems, in normal and atherosclerotic NZW and WHHL rabbit blood vessels using intraarterial gene transfer and constructing a knock-out mouse model for PHGPX.The results may lead to new therapeutic approaches where PHGPX itself or factors affecting its expression could be used as specific targets for treatment either by molecular means or by gene therapy.

Our proposal has been divided into the following tasks:
1. Identification of conditions and factors at posttranscriptional level necessary for the overexpression of one single selenoprotein (PHGPX) in selected cells and analysis of consequences of an additional PHGPX activity on endogenous membrane hydroperoxide content and on cellular signalling events relevant for atherogenesis;
2. Identification of promoter sequences affecting the expression of PHGPX and other GPXs at transcriptional level and mechanisms involved, in order to acquire ability to manipulate an individual GPX at the cellular level. Lipid hydroperoxide measurement in biological samples;
3. Identification of the hierarchy of selenium supply to selenoproteins and of the effects of over expression of a selenoprotein on the cellular selenoprotein pattern;
4. Construction of retroviral and adenoviral gene transfer vectors suitable for the generation of stably transfected PHGPX cell lines (as models for PHGPX studies) and in vivo gene transfer experiments in normo- and hypercholesterolemic rabbit carotid and iliac arteries;
5. Construction and characterization of PHGPX knock-out mouse and testing the effect of the absence of PHGPX on the development of atherosclerosis in atherosclerosis-susceptible mice strains. The resultant discoveries and potential targets for treatment will be transferred to the European pharmaceutical industry.

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UNIVERSITY OF KUOPIO
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Adresse
2,Neulaniementie 2
70211 KUOPIO
Finlande

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