Summary of Work: Components of the inflammatory pathways have been theorized to play a significant role in the initiation, progression and maturation of atherosclerotic plaques in animal and human studies. The deposition of inflammatory cells in arterial vessel walls as a result of endothelial injury following exposure to the major risk factors for stroke leads to a potential pro-inflammatory tissue which can result in increased intravascular thrombosis at the site of plaque formation resulting in heart attack and stroke. However, despite the presence of these prerequisite vascular changes, the pathophysiologic mechanisms that result in a symptomatic or prothrombotic state are not well defined. Hypothesis: I hypothesize that the increased expression of inflammatory mediators on the vessel luminal endothelial surface and within the body of the plaque, result in the subsequent intensified interaction between perivascular monocytes/macrophages and the local endothelium, thus predisposing the atherosclerotic plaque to convert from an asymptomatic to a symptomatic state. Specifically I hypothesize that 1) patients with atherosclerotic disease will have an increased expression of inflammatory mediators measurable in the peripheral blood as compared to those without vascular disease; 2) Levels of inflammatory mediators will be highest in patients having had a prior history of stroke, TIA or unstable angina; 3) I further hypothesize that the differential inflammatory expression among patients is influenced by the following factors: a) clonal T-cell population expansion with the plaque activated by endogenous and exogenous antigens, b) infectious agents identified in carotid atherosclerotic plaque resulting in antigen mediated T-cell proliferation, c) gene polymorphisms associated with functional changes of inflammatory cytokine, chemokine or adhesion molecule, and d) a differential profile of gene. Review of Results: Patients with symptomatic atherosclerotic plaque were found to have a pro-inflammatory profile both in the plaque and in the peripheral blood. A 2-fold increase of intercellular adhesion molecule-1 (ICAM-1) expression was seen on the endothelial surface of carotid atherosclerotic plaques in patients with symptomatic vs asymptomatic cerebrovascular disease. (Stroke 1998;29(7):1405-1410.) Additionally, serum sICAM levels are significantly elevated in patients with atherosclerosis vs age-matched controls. To study the association between infectious mediators and symptomatic carotid disease, plaques from 37 symptomatic and 57 asymptomatic consenting patients undergoing carotid endarterectomy were analyzed by PCR for Chlamydia pneumoniae as per the IRB approved protocol. The overall rate of plaques positive for C. pneumoniae was 14.82% with 5/37 (13.5%) of plaques from symptomatic patients and 9/57 (16%) from asymptomatic patients. There was no correlation between C. pneumoniae presence and symptomatic disease, (Chi-squared=0.091) or severity of stenosis. Serum testing for IgG, IgA, and IgM anti-chlamydia antibodies did not correlate with the identification of chlamydia in the plaques but IgA levels were associated with the occurrence of symptomatic disease (Chi-squared=0.04) (Stroke 2001;32:855-860). Further, we studied 14 plaques (5 symptomatic and 9 asymptomatic) positive for C. pneumoniae confirmed by PCR and 14 plaques (6 symptomatic and 8 asymptomatic) from age, sex, and risk-factor matched patients negative for Chlamydia. T-cells and their subpopulations of T-helper, T-cytotoxic and T-memory lymphocytes were identified by indirect enzyme immunohistochemistry using anti- CD3, CD4, CD8, and CD45RO monoclonal antibodies. In the absence of C. pneumoniae there was a modest but significant increase (cells per square mm) of CD3+ (89.6 vs. 55.3, p=0.01), CD4+ (57.3 vs. 32.7, p=0.008), and CD45RO+ (82.8 vs. 43.7, p=0.005), but not CD8+ T-cells (28.5 vs. 25.5, p=0.282) in symptomatic compared to asymptomatic plaques. However, in the presence of C. pneumoniae there was significant increase of all T-lymphocyte subtypes including CD8+ (76.8 vs. 30.3, p=0.03) cells in symptomatic versus asymptomatic plaques. A preferential increase in CD8+ class I restricted T-cells in symptomatic carotid plaque positive for C. pneumoniae suggests that thromboembolic carotid disease is associated with an antigen specific activation of inflammatory cells. (Stroke 2001 Sep 32(9):1966-72). To evaluate the mechanisms of T-cell activation and its role in plaque destabilization, we studied CD40-CD154 receptor ligand interaction and atherosclerotic plaques of symptomatic and asymptomatic patients. In a study of atherosclerotic plaque from 28 patients (11 symptomatic, 17 asymptomatic), 11,176 images from 168 sections were analyzed utilizing immunohistochemical staining of CD40 and CD154. There was a significant increase in the percentage of CD154 positive areas in symptomatic compared to asymptomatic plaques (p less than 0.01). Further, a statistically significant association between memory T-cells and CD154 expression was identified. These findings support our central hypothesis that T-cell dependent mechanisms play a role in destabilization of atherosclerotic plaque. (Neurology 2001 (Apr);56 (3):A462 [abstract]). A preliminary study utilizing cDNA microarray technology examined the differential profile of gene expression in atherosclerotic plaques from symptomatic and asymptomatic patients. Eight carotid endarterectomy plaque specimens (5 symptomatic, 3 asymptomatic) were analyzed utilizing a 1152 gene microarray designed by the National Institute of Aging to study genes related to the immune and inflammatory pathways. A distinct pattern of gene expression was revealed in symptomatic patients, including CCR-5 (chemokine C-C motif receptor-5), CD4, integrin alpha-9, and caspase-1. Genes found to be increased predominantly in asymptomatic patients included interleukin-10 receptor and fibroblast growth factor-5. Numerous genes were found in common. This study supports the feasibility of using cDNA microarray in atherosclerotic plaque tissue in order to identify the profile of genes in the symptomatic state. (Neurology 2001 (Apr): 56(3);A368 [abstract]). IL-1ra, allele 2, VNTR polymorphism: Preliminary data reported in last year?s review revealed an increase allele frequency of allele 2 polymorphisms in symptomatic (n=52) and asymptomatic (n=29) atherosclerosis patients versus risk factor free controls (n=28). Expansion of the data in the past year now allows for analysis of 91 patients with symptomatic atherosclerosis; 106 patients with asymptomatic atherosclerosis, and 113 subjects with no atherosclerosis. The data reveals a non-carriage of allele 2 was associated with reduced likelihood of atherosclerosis (OR=0.36, 95% CI=[0.22, 0.16]). The homozygous carrier state for allele 2 was associated with greater likelihood of atherosclerosis (OR=6.93, 95% CI=[2.20, 21.86]). A gene dose effect was detected. The data strongly suggests an association between this polymorphism and the development of atherosclerosis. (submitted to the Annals of Neurology for publication) Two novel single nucleotide polymorphisms (SNPs) have been identified in the monocyte chemotactic protein-1 (MCP-1) promoter region by our laboratory. The SNP at the ?361 base site lies near the promoter region that is activated by shear forces such as elevated blood pressure. This region is a highly conserved region. The existence ?361 polymorphism in this region lying as the single base separating two ?Stat 01? binding sites suggests the potential for effect on gene function. The SNP, consisting of a cytosine for guanine in the ?928 position, created two new transcription binding sites. These were proto-oncogene c-myb in the sen