Supplementary Material for: Overexpression of Toll-Like Receptors 2, 3, 4, and 8 Is Correlated to the Vascular Atherosclerotic Process in the Hyperlipidemic Rabbit Model: The Effect of Statin Treatment

<p><b><i>Background:</i></b> Atherosclerosis is the major cause of cardiovascular disease; hypercholesterolemia is a major risk factor. We hypothesized that specific TLR members (TLR2, TLR3, TLR4, TLR8) may play a role in atherosclerosis progression and its accompanying inflammatory response. We determined the association of atherosclerotic lesions and TLR mRNA expression in different aortic sites. We also assessed the effects of fluvastatin (Flu) treatment on TLR expression and plaque characteristics. <b><i>Methods:</i></b> Male rabbits, fed with an atherogenic diet for a duration of 3 months, were screened for advanced atherosclerotic lesions in the aorta. Additional animals received normal diet or normal diet plus Flu for 1 additional month. TLR mRNA expression in various thoracic and abdominal aortic segments was assessed, together with atherosclerotic changes. <b><i>Results:</i></b> After high lipid diet, the atherosclerotic burden increased more in the abdominal than in the thoracic aorta; TLR2, 3, 4, and 8 also increased significantly. Flu decreased atherosclerotic plaque, calcium deposition, lipid cores, intraplaque hemorrhage, erythrocyte membranes, endothelial cells, and macrophage infiltration, while increasing smooth muscle cells in plaques of both aortic segments; it also lowered TLR2, 3, 4, and 8 expression in all aortic segments to a stronger degree than resumption of normal diet. There was a strong association between blood and tissue parameters during experimental period and finally a strong correlation found between these parameters with mRNA of TLR2, 3, 4, and 8 in various stages. <b><i>Conclusion:</i></b> For the first time TLR2, 3, 4, and 8 mRNA expression is prospectively explored after hypercholesterolemic diet in the rabbit model. TLR2, 3, 4, and 8 mRNA expression is strongly upregulated and correlates with the progression of atherosclerosis in the aorta. Flu significantly inhibited this progress and reduced inflammation via TLR downregulation which was strongly associated with regression of plaque morphology and atherosclerosis promoting factors.</p>