Immunopathology and artherosclerosis

Research project P 14741 Immunpathology of atherosclerosis Georg WICK 27.11.2000 During the last decade we have developed a new "autoimmune" hypothesis for the development of atherosclerosis. In principle, this concept postulates that when classical atherosclerosis risk factors exert their effect on the endothelium. of arteries, this leads to the expression of the stress protein heat shock protein 60 (HSP 60) entailing a cross-reaction of pre-existing humoral and cellular immunity against microbial HSP 60. Thus, via antigenic mimicry" an autoimmune reaction ensues that leads to the earliest inflammatory stage of atherosclerosis. HSPs with a molecular weight of 60 kD are phylogenetically highly conserved reflected, e.g., by an over 55 % homology at the DNA and protein level between HSP 65 of Mycobacterium tuberculosis and human HSP 60. According to our concept, we have to "`pay" for our protective immunity against microbial HSP 60 by the danger of a cross- reactivity with our own HSP 60 expressing arterial endothelial cells, if these are maltreated by atherogenic stress factors (high blood pressure, high serum cholesterol levels, smoking, etc.). At those areas of the arterial tree that are subjected to major haemodynamic (turbulent) stress, e.g. the aortic arch or the branching of larger vessels, intimal infiltrations by mononuclear cells can already be found in healthy children. We have tentatively denoted these accumulations as the vascular associated lymphoid tissue (VALT). These are the areas where later on, due to the livelong mechanical pre-stressing by the arterial blood pressure, the earliest inflammatory reactions develop in response to classical atherogenic risk factors. In experimental animals, we were able to show that lymphocytes found in early atherosclerotic lesions preferentially react with HSP 60. In addition, circulating antibodies against HSP 60 antibodies are present. Immunization of rabbits or mice with bacterial HSP 60 leads to the development of atherosclerotic lesions that can be significantly augmented by simultaneous feeding of a cholesterol-rich diet. Within the framework of`a longitudinal atherosclerosis prevention study in humans (the so-called Bruneck-Study) we found that the titer of antibodies against bacterial HSP 60 is significantly correlated with the presence of sonographically demonstrable atherosclerotic lesions in the carotid arteries. Furthermore, we were able to show that the immune reaction against HSP 60 most probably represents the common denominator for the numerous infectious agents that have been discussed in the literature in, connection with atherosclerosis (e.g. Chlamydiae). This idea is also supported by our most recent data that the odds ratio to develop atherosclerosis runs in parallel with the total infectious load to which an individual has been subjected. In summary, atherosclerosis starts as early inflammatory infiltrate in the intima when pre-existing humoral and/or cellular immune-reactims against microbial HSP 60 recognize human HSP 60 on the surface of stressed endothelial cells. The present project is aimed at answering the following main questions: 1. Studies in experimental animals: Using our immunological atherosclerosis model in mice, we aim at answering the question, if HSP 60 reactive T cells or antibodies initiate the disease. Furthermore, we will investigate if the development of atherosclerosis can be prevented or mitigated by immunosuppressive measures or the induction of immune tolerance against HSP 60. 2. Studies in humans: After having established an association of atherosclerosis with the presence and titer of humoral antibodies against HSP 60, we will now focus our endeavours on the elucidation of the cellular immune reaction against this antigen within the framework of the BruneckStudy-. 3. Studies in cell culture: An essential factor in the development of atherosclerosis lies in the fact, that arteries are subjected to higher blood pressure than veins. We will therefore identify the sensors" for mechanical (shear) stress in endothelial cells as well as the signal transduction pathways triggered via these sensors.

In the industrialized world - including Austria - atherosclerosis is the cause of death number one. The disease is manifested by a thickening and hardening (sclerosis) of the arterial wall, the appearance of lipid-rich protrusions at the innervascular surface (so called fatty streaks) and finally progressive, often exulcerated and even calcified lesions, so-called atherosclerotic plaques. A consequence of these alterations is vascular stenosis with attachment of blood clots and finally complete occlusion entailing myocardial infarction, stroke, and decreased blood flow in peripheral vessels, e.g. of the legs. Although these changes only become clinically manifest at older age and atherosclerosis is, therefore, one of the age-associated diseases, it has become evident since many years that the vascular changes already begin to develop early in life. The scientific focus of the research group of G.Wick in the Division of Experimental Pathophysiology and Immunology, Biocenter, Medical University of Innsbruck, Austria, is mainly on these earliest stages of atherosclerosis. It is the goal of the group to identify the first disease-causing (pathogenetic) processes. During the last 20 years it became clear that an inflammation of the arterial wall occurs in early atherosclerosis due to the immigration of cells of the immune system to this site. The main goal of the FWF- funded project was to find out why and when during life this early inflammatory process is started. We made the unexpected observation that the first stage of the disease consists in the attack of the immune system on the cells that line the body`s own arteries (the so-called endothelial cells). This finding did contradict current dogma that considered the above-mentioned fatty streaks to represent the earliest atherosclerotic lesions. Diseases based on a reaction of the immune system against autologous cells and tissues are called self-aggressive or autoimmune diseases. We were able to show that subjection of arterial endothelial cells (Ecs) to classical atherosclerosis risk- factors, such as high blood-pressure, high serum cholesterol levels, smoking, etc. leads to the production of so- called stressproteins that are also transported to the surface of these cells. Such stressproteins were first identified in more primitive organisms (flies) when these were treated with heat. Hence, they are also designated as heat shock proteins (HSPs). HSPs exist in various families that differ in molecular weight. In the present context, HSPs with a molecular weight of 60.000 (HSP60) are of special importance. Classical atherosclerosis risk-factors lead to the production of HSP60 in ECs. HSP60 is an evolutionary very old molecule that is produced in all living organisms, from bacteria to humans. All humans elicit an immune reaction against microbial HSP60 already shortly after birth that - in combination with immune reactions against other microbial components - protects from infection. Since human HSP60 has a very similar chemical structure as HSP60 of bacteria and parasites we "pay" for the maltreatment of our arteries with atherosclerosis risk-factors by the danger of an immunologic error reaction, a so-called crossreaction. This means that a principally beneficial protective immune reaction against microbial HSP60 turns against HSP60 appearing on our own stressed ECs. In this respect, we made the following interesting and important observations during the last period of the present FWF-funded research project: a) Cigarette smoke also acts as a stress factor and leads to the production of HSP60 by ECs b) Using a modified version of the very sensitive atomic force microscopy (AFM) that allows for the identification of single molecules on the cell surface, we were able to show that classical atherosclerosis risk-factors really do induce the expression of HSP60 on the surface of human ECs. These molecules are not randomly distributed but rather aggregate into molecular clusters. c) In order to stimulate and perpetuate an immune reaction, the immune-stimulating substances (so-called antigens) have to be decomposed and presented to the cells of the immune system in an appropriate fashion. To this end, an additional type of cells, so-called antigen presenting cells (also called dendritic cells) is necessary. Surprisingly, we could show that a sofar unknown complex network of such dendritic cells is present in the innermost layer of the arteries thus providing the prerequisite for the initiation of a local immune reaction against HSP60 at this site. d) It has been known for a long time that one artery of the human body, the internal mammary artery (IMA) does not develop atherosclerosis even when the disease is present at other arterial territories. This fact seems to be due to the special flow conditions in the IMA. This observation also has practical relevance since the IMA is preferably used to bridge stenosed coronary arteries in bypass operations. We therefore started a cooperation with colleagues from the Kantonshospital Basel, Switzerland, where we investigate age-dependent changes in the IMA applying the so-called tissue microarray method. With this technique it is possible to analyse aging processes in a normal artery without interference of concomitant atherosclerosis. In the tissue microarrays method, a large number of very small microscopical tissue sections are applied to glass slides - similar to an electronic microchip - that are then analysed with the most modern microscopic equipment. e) In a study of 17 to 18 year old male recruits (Atherosclerosis Risk-Factors in Male Youngsters - ARMY - Study) we were able to show that 28 % of this young, clinically healthy cohort already display thickened arterial walls at at least one of eight sonographically measured sites, pointing to incipient atherosclerosis. These pathologic changes corrolate significantly with the demonstration of an immune reaction against HSP60 in the peripheral blood. f) In a just started, but not yet completed, analogous study in young women (Atherosclerosis Risk-Factors in Female Youngsters-ARFY - Study) these alarming pathologic alterations in young men seem to be present in females, too.