title: C-reactive protein-induced in vitro endothelial cell activation is an artefact caused by azide and lipopolysaccharide.
abstract: 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.
  
  
  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.
authors.affiliation: Department of Pharmacology, Therapeutics and Toxicology, Cardiff University, Wales College of Medicine, Cardiff, UK.
authors.forename: Karolina E
authors.initials: KE
authors.lastname: Taylor
authors.forename: John C
authors.initials: JC
authors.lastname: Giddings
authors.forename: Carmen W
authors.initials: CW
authors.lastname: van den Berg
mesh_terms: D016477:Artifacts; D001386:Azides / Q000494:pharmacology*; D002097:C-Reactive Protein / Q000302:isolation & purification / Q000494:pharmacology*; D002455:Cell Division / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D018932:Chemokine CCL2 / Q000378:metabolism; D003956:Dialysis; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism; D016209:Interleukin-8 / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology*; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D014471:Umbilical Veins / Q000166:cytology; D014841:von Willebrand Factor / Q000378:metabolism
chemical_list: D001386:Azides; C111835:CCL2 protein, human; D018932:Chemokine CCL2; D016209:Interleukin-8; D008070:Lipopolysaccharides; D014841:von Willebrand Factor; D018799:Intercellular Adhesion Molecule-1; D002097:C-Reactive Protein; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III