Published online before print March 31, 2005, doi: 10.1161/01.ATV.0000164623.41250.28
© 2005 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
C-Reactive Protein–Induced In Vitro Endothelial Cell Activation Is an Artefact Caused by Azide and Lipopolysaccharide
From the Departments of Pharmacology, Therapeutics and Toxicology (K.E.T., C.W.vdB.), and Haematology (J.C.G.), Cardiff University, Wales College of Medicine, Cardiff, UK.
Correspondence to Dr Carmen W. van den Berg, Department of Pharmacology, Therapeutics and Toxicology, Wales Heart Research Institute, Cardiff University, Wales College of Medicine, Heath Park, Cardiff, CF144XN, United Kingdom. E-mail vandenbergcw{at}cardiff.ac.uk
Objective— C-reactive protein (CRP) has been proposed to be an independent risk factor for cardiovascular disease. In vitro studies investigating the mechanism behind this have used purified commercial CRP (cCRP) and endothelial cells. We investigated the role of contaminants in cCRP preparations.
Methods and Results— Human umbilical vein endothelial cells and the human endothelial cell line EA.hy926 were incubated with Escherichia coli–derived cCRP, in-house–generated azide-free recombinant, and ascites-purified CRP, azide, or lipopolysaccharide (LPS) equivalent to the concentration present in cCRP preparations. Cells were investigated for change in cell proliferation, morphology, apoptosis, and expression of endothelial NO synthase and intercellular adhesion molecule-1. Cell supernatants were assessed for monocyte chemoattractant protein-1 (MCP-1), interleukin-8, von Willebrand factor secretion, and pH change. Only cCRP was able to induce all activation events analyzed; however, this ability was lost on extensive dialysis, suggesting that low molecular weight contaminants were responsible for these events. Indeed, the effects of cCRP were mirrored by azide or LPS.
Conclusions— We investigated a wide range of effects on endothelial cells ascribed to CRP; however, azide and LPS, but never CRP itself, were responsible for the cell activation events. We conclude that CRP, per se, does not activate endothelial cells.
We investigated the validity of the numerous responses of endothelial cells attributed to CRP and demonstrate here that the reported effects are caused by low molecular weight contaminants, like azide and LPS, which are present in commercial CRP preparations.
Key Words: C-reactive protein • endothelial cells • azide • LPS
Related Article:
Arterioscler Thromb Vasc Biol 2005 25: 1091-1094.
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