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Clinical Chemistry 51: 516-521, 2005. First published January 13, 2005; 10.1373/clinchem.2004.041277
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(Clinical Chemistry. 2005;51:516-521.)
© 2005 American Association for Clinical Chemistry, Inc.

Molecular Diagnostics and Genetics

Relationship between In Vitro Lipopolysaccharide-Induced Cytokine Response in Whole Blood, Angiographic In-Stent Restenosis, and Toll-Like Receptor 4 Gene Polymorphisms

Saskia Z.H. Rittersma1,a, Johanna A. Kremer Hovinga2, Karel T. Koch1, S. Matthijs Boekholdt1, Benien E. van Aken2, Arko Scheepmaker1, Matthijs Bax1, Carl E. Schotborgh1, Jan J. Piek1, Jan G.P. Tijssen1, Pieter H. Reitsma2 and Robbert J. de Winter1

Departments of1 Cardiology and 2 Experimental Internal Medicine, Academic Medical Center, Amsterdam, The Netherlands.

aAddress correspondence to this author at: Department of Cardiology, B2-115, Academic Medical Center, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. Fax 31-20-6962609; e-mail z.h.rittersma{at}amc.uva.nl.

Background: In coronary in-stent restenosis (ISR), a substantial contribution of inflammation is assumed. We evaluated the association between polymorphisms in the Toll-like receptor 4 (TLR4) gene and cytokine response after lipopolysaccharide (LPS) challenge and the development of ISR.

Methods: Patients were included after successful elective stent placement in a native coronary artery and were scheduled for follow-up angiography after 6 months. Quantitative coronary analysis was performed off-line. Patient whole blood was challenged with LPS for 24 h. Baseline and stimulated concentrations of interleukin (IL)-1ß, IL-6, tumor necrosis factor-{alpha}, and IL-10 were assessed by ELISA. Two cosegregating single-nucleotide polymorphisms in the TLR4 gene (Asp299Gly and Thr399Ile) were analyzed by allele-specific PCR amplification of genomic DNA.

Results: A total of 236 consecutive patients were included, and 40 (17%) developed ISR. Median baseline and stimulated cytokine concentrations did not differ between patients with and without ISR. In multivariate analysis, male sex, unstable angina, hypertension, and chronic total occlusion were predictors of ISR. TLR4 genotypes were not associated with baseline or stimulated cytokine concentrations or with angiographic variables at follow-up.

Conclusions: In vitro cytokine response to LPS challenge is not increased in patients with ISR. Functionality of the TLR4 Asp299Gly polymorphism could not be demonstrated in this setting, and this polymorphism was not associated with angiographic outcome, calling into question its role in the progression of neointimal tissue growth.




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