Modulation of Adhesion Molecules by Tumor Necrosis Factor-a and Salicylic Acid in Human Coronary Artery Endothelial Cells: Insights into the Pathogenesis of Kawasaki Syndrome
Pong Kian Chua¹, Richard Yanagihara¹, Marian E. Melish², and Vivek R. Nerurkar¹

Background: Kawasaki syndrome (KS), an acute febrile illness of unknown etiology affecting infants and young children, is characterized by inflammation of coronary arteries and other medium-sized muscular arteries, leading to coronary aneurysms and thromboses. Infiltration of immune cells into the intima and adventitia are observed in autopsy tissues. Treatment for KS includes high-dose aspirin and intravenous gamma globulin. Hypothesis: We hypothesized that elevated levels of tumor necrosis factor-alpha (TNF-a ) in KS increase the expression of adhesion molecules in vivo, allowing inflammatory and immune cells to adhere to the vascular endothelium. Subsequently, immune cells migrate into the extracellular matrix where they release matrix metalloproteinase 9 and assist in vascular remodeling, resulting in aneurysm formation. Objective: To determine if TNF-a is capable of modulating the transcriptional activities of adhesion molecules intercellular adhesion molecule (ICAM-1), E-selectin and monocyte chemoattractant protein-1 (MCP-1) in human coronary artery endothelial cells (HCAEC), as well as ascertain if salicylic acid (NaSal) can block gene and protein expression induced by TNF-a . Methods: HCAEC, cultured in 6- or 96-well plates, were treated with 1 ng/mL TNF-a or pre-treated with various concentrations of NaSal prior to stimulation with TNF-a . Reverse transcriptase-polymerase chain reaction (RT-PCR) and cell-based enzyme-linked immunosorbent assay (ELISA) were employed to determine gene and cell-surface expression of adhesion molecules, respectively. Results: Using semi-quantitative RT-PCR, TNF-a up-regulated the gene expression of ICAM-1, E-selectin and MCP-1 in HCAEC in a time- and dose-dependent manner. Cell-surface expression of ICAM-1 and E-selectin was similarly induced in a time- and dose-dependent manner. High levels of salicylic acid (>10 mM) were required to inhibit the transcriptional activities and cell-surface expression of these adhesion molecules induced by TNF-a . Conclusions: These results indicate that TNF-a may play an important role in mediating increased levels of adhesion molecules in endothelial cells. Furthermore, MCP-1 may provide a gradient to attract monocytes to sites of inflammation, thus providing a mechanism by which immune and inflammatory cells home in and enter the extracellular matrix, releasing enzymes that assist in vascular remodeling, thereby weakening the endothelium and hastening the process of aneurysm formation. NaSal, in addition to preventing thrombosis and lowering fever in KS, may also function in down-regulating the expression of adhesion molecules during the inflammatory stage. Further studies on the effects of TGF-b on modulating adhesion molecule expression and the underlying signal transduction mechanisms in HCAEC are underway. [Supported by US Public Health Service Grant G12RR/AI-03061 from the Research Centers in Minority Institutions Program, NCRR, NIH]