C-reactive protein-induced in vitro endothelial cell activation is an artefact caused by azide and lipopolysaccharide

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.