of Work: Components of the inflammatory pathways have been theorized to play a significant role in the development 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 resulting in heart attack and stroke. Despite the presence of these prerequisite vascular changes, the pathophysiologic mechanisms that result in a symptomatic or prothrombotic state are not well defined. Hypothesis: Increased expression of inflammatory mediators on the vessel luminal endothelial surface and within 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 pro-inflammatory mediators within atherosclerotic plaque will be highest in patients with a prior history of stroke or TIA; 3) differential inflammatory expression within carotid plaque among patients is influenced by: a) clonal T-lymphocyte proliferation within the plaque activated by endogenous and exogenous antigens, and b) a differential profile of gene expression influenced by polymorphisms of key inflammatory mediators. Review of Results: Patients with symptomatic atherosclerotic plaque were found to have a pro-inflammatory profile both in the plaque and peripheral blood.(Stroke 1998;29(7):1405-1410). In addition, increased serum IgA levels for Chlamydia pneumonia was associated with symptomatic carotid atherosclerosis, suggesting infection plays a pivotal roll in some cases of stroke.(Stroke 2001;32:855-860). Further, presence of C. pneumoniae was associated with increased levels of T-lymphocytes including MHC class I restricted CD8+ T-cells in symptomatic versus asymptomatic plaques, suggesting that thromboembolic carotid disease is associated with an antigen specific activation of inflammatory cells. (Stroke 2001;32(9):1966-72). Using immunohistochemical staining on 28 plaques, we studied CD40-CD154 receptor ligand interaction. 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;56(3):A462 [abstract]) To characterize T-cell subtype, pro- and anti- inflammatory cytokine secretory patterns of T cell cultures isolated from carotid plaque at endarterectomy were stimulated with phytohemaglutinin (PHA) in the presence of irradiated autologous peripheral blood mononuclear cells (PBMC) and interleukin-2 (IL-2). Supernatants were collected after 24 and 48 hours and analyzed for IL-1, IL-2, IL-6, IL-8, IL-10, TNF;,IFNgamma;,IL-4 and IL-10. A pro-inflammatory cytokine profile revealed Th-0 T-lymphocyte predominence. (Stroke 2002;33(1):377. To examine if activation is a result of clonal expansion, T cell receptor (TCR) Vbeta region profile from T-lymphocytes harvested from 8 plaque were compared to T-cells in peripheral blood. TCR analysis for the 24 known Vbeta families, measured by flow cytometry, revealed an average of 7 Vbeta family expansions in the plaque compared to PBMC. These data suggests expansion of specific T cell clones occur within the carotid atherosclerotic plaque. To examine gene expression in symptomatic and asymptomatic plaque, preliminary study utilizing a 1152 cDNA microarray revealed a distinct pattern of gene expression in symptomatic patients, including CCR-5, CD4, integrin alpha-9, and caspase-1. Genes found to be increased predominantly in asymptomatic patients included IL-10r and FGF-5. Numerous genes were found in common supporting the feasibility of using cDNA microarray in atherosclerotic plaque tissue.(Neurology 2001;56(3);A368 [abstract]). A 9K human array is currently being employed to continue the study. Further, in a effort to study the similarities of a pig of model of atherosclerosis with human athero, a 15K mouse and 9K human micro-array are being used on carotid and iliac arteries from diabetic, hypercholesterolemic and normal pigs. (Poster accepted for the AHA Stroke meeting Feb 2002). IL-1ra, allele 2, VNTR polymorphism: A study involving 197 patients with atherosclerosis and 113 subjects with no atherosclerosis revealed 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. (Stroke, in press) We identified two novel single nucleotide polymorphisms (SNPs) in monocyte chemotactic protein-1 (MCP-1) promoter region at the -361 base site, a region activated by shear forces such as elevated blood pressure. The - 361 polymorphism is a single base separating two Stat 01 binding sites, one in the sense direction; one in the anti-sense direction, suggests the potential for effect on gene function. The second 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 sense direction and AH receptor nuclear translocator protein (ARNT) in the anti-sense. Work is ongoing to characterize the frequency of the c allele in populations with and without atherosclerosis, as well as their effects of these polymorphisms on MCP-1 transcription. (Ann Neurol 2001;50(3)Suppl1).
