This Article Full Text Full Text (PDF) All Versions of this Article: 25/6/1225    most recent 01.ATV.0000164623.41250.28v1 Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Taylor, K. E. Articles by van den Berg, C. W. Search for Related Content PubMed PubMed Citation Articles by Taylor, K. E. Articles by van den Berg, C. W. Pubmed/NCBI databases Substance via MeSH Related Collections Risk Factors Endothelium/vascular type/nitric oxide Mechanism of atherosclerosis/growth factors Related Article

(Arteriosclerosis, Thrombosis, and Vascular Biology. 2005;25:1225.)
© 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

Karolina E. Taylor; John C. Giddings; Carmen W. van den Berg

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:

CRP or not CRP? That Is the Question

Mark B. Pepys
Arterioscler Thromb Vasc Biol 2005 25: 1091-1094. [Extract] [Full Text] [PDF]

This article has been cited by other articles:

				C. Erridge and N. J. Samani Saturated Fatty Acids Do Not Directly Stimulate Toll-Like Receptor Signaling Arterioscler Thromb Vasc Biol, November 1, 2009; 29(11): 1944 - 1949. [Abstract] [Full Text] [PDF]

				M. Megyeri, V. Mako, L. Beinrohr, Z. Doleschall, Z. Prohaszka, L. Cervenak, P. Zavodszky, and P. Gal Complement Protease MASP-1 Activates Human Endothelial Cells: PAR4 Activation Is a Link between Complement and Endothelial Function J. Immunol., September 1, 2009; 183(5): 3409 - 3416. [Abstract] [Full Text] [PDF]

				M. A. Ortiz, G. L. Campana, J. R. Woods, G. Boguslawski, M. J. Sosa, C. L. Walker, and C. A. Labarrere Continuously-Infused Human C-Reactive Protein Is Neither Proatherosclerotic Nor Proinflammatory in Apolipoprotein E-Deficient Mice Experimental Biology and Medicine, June 1, 2009; 234(6): 624 - 631. [Abstract] [Full Text] [PDF]

				H. H. Shih, S. Zhang, W. Cao, A. Hahn, J. Wang, J. E. Paulsen, and D. C. Harnish CRP is a novel ligand for the oxidized LDL receptor LOX-1 Am J Physiol Heart Circ Physiol, May 1, 2009; 296(5): H1643 - H1650. [Abstract] [Full Text] [PDF]

				T. B. Grammer, W. Marz, W. Renner, B. O. Bohm, and M. M. Hoffmann C-reactive protein genotypes associated with circulating C-reactive protein but not with angiographic coronary artery disease: the LURIC study Eur. Heart J., January 2, 2009; 30(2): 170 - 182. [Abstract] [Full Text] [PDF]

				B. Molins, E. Pena, G. Vilahur, C. Mendieta, M. Slevin, and L. Badimon C-Reactive Protein Isoforms Differ in Their Effects on Thrombus Growth Arterioscler Thromb Vasc Biol, December 1, 2008; 28(12): 2239 - 2246. [Abstract] [Full Text] [PDF]

				F. Montecucco, S. Steffens, F. Burger, G. Pelli, C. Monaco, and F. Mach C-reactive protein (CRP) induces chemokine secretion via CD11b/ICAM-1 interaction in human adherent monocytes J. Leukoc. Biol., October 1, 2008; 84(4): 1109 - 1119. [Abstract] [Full Text] [PDF]

				F. Maingrette, L. Li, and G. Renier C-reactive protein enhances macrophage lipoprotein lipase expression J. Lipid Res., September 1, 2008; 49(9): 1926 - 1935. [Abstract] [Full Text] [PDF]

				M. A. Sardo, S. Campo, G. Mandraffino, C. Saitta, A. Bonaiuto, M. Castaldo, M. Cinquegrani, G. Pizzimenti, and A. Saitta Tissue Factor and Monocyte Chemoattractant Protein-1 Expression in Hypertensive Individuals with Normal or Increased Carotid Intima-Media Wall Thickness Clin. Chem., May 1, 2008; 54(5): 814 - 823. [Abstract] [Full Text] [PDF]

				J. Wu, M. J. Stevenson, J. M. Brown, E. A. Grunz, T. L. Strawn, and W. P. Fay C-Reactive Protein Enhances Tissue Factor Expression by Vascular Smooth Muscle Cells: Mechanisms and In Vivo Significance Arterioscler Thromb Vasc Biol, April 1, 2008; 28(4): 698 - 704. [Abstract] [Full Text] [PDF]

				S. M. Nelson, N. Sattar, D. J. Freeman, J. D. Walker, and R. S. Lindsay Inflammation and Endothelial Activation Is Evident at Birth in Offspring of Mothers With Type 1 Diabetes Diabetes, November 1, 2007; 56(11): 2697 - 2704. [Abstract] [Full Text] [PDF]

				E. Grad, M. Golomb, I. Mor-Yosef, N. Koroukhov, C. Lotan, E. R. Edelman, and H. D. Danenberg Transgenic expression of human C-reactive protein suppresses endothelial nitric oxide synthase expression and bioactivity after vascular injury Am J Physiol Heart Circ Physiol, July 1, 2007; 293(1): H489 - H495. [Abstract] [Full Text] [PDF]

				A. P. Sjoberg, L. A. Trouw, S. J. Clark, J. Sjolander, D. Heinegard, R. B. Sim, A. J. Day, and A. M. Blom The Factor H Variant Associated with Age-related Macular Degeneration (His-384) and the Non-disease-associated Form Bind Differentially to C-reactive Protein, Fibromodulin, DNA, and Necrotic Cells J. Biol. Chem., April 13, 2007; 282(15): 10894 - 10900. [Abstract] [Full Text] [PDF]

				C. Rocker, D. E. Manolov, E. V. Kuzmenkina, K. Tron, H. Slatosch, J. Torzewski, and G. U. Nienhaus Affinity of C-Reactive Protein toward Fc{gamma}RI Is Strongly Enhanced by the {gamma}-Chain Am. J. Pathol., February 1, 2007; 170(2): 755 - 763. [Abstract] [Full Text] [PDF]

				J. P Casas, T. Shah, J. Cooper, E. Hawe, A. D McMahon, D. Gaffney, C. J Packard, D. S O'Reilly, I. Juhan-Vague, J. S Yudkin, et al. Insight into the nature of the CRP-coronary event association using Mendelian randomization Int. J. Epidemiol., August 1, 2006; 35(4): 922 - 931. [Abstract] [Full Text] [PDF]

				M. Juonala, J. S.A. Viikari, T. Ronnemaa, L. Taittonen, J. Marniemi, and O. T. Raitakari Childhood C-Reactive Protein in Predicting CRP and Carotid Intima-Media Thickness in Adulthood: The Cardiovascular Risk in Young Finns Study Arterioscler Thromb Vasc Biol, August 1, 2006; 26(8): 1883 - 1888. [Abstract] [Full Text] [PDF]

				E. Paffen and M. P.M. deMaat C-reactive protein in atherosclerosis: A causal factor? Cardiovasc Res, July 1, 2006; 71(1): 30 - 39. [Abstract] [Full Text] [PDF]

				M. Oroszlan, E. Herczenik, S. Rugonfalvi-Kiss, A. Roos, A. J Nauta, M. R Daha, I. Gombos, I. Karadi, L. Romics, Z. Prohaszka, et al. Proinflammatory changes in human umbilical cord vein endothelial cells can be induced neither by native nor by modified CRP Int. Immunol., June 1, 2006; 18(6): 871 - 878. [Abstract] [Full Text] [PDF]

				B. M. Scirica, D. A. Morrow, S. Verma, S. Devaraj, I. Jialal, B. M. Scirica, D. A. Morrow, S. Verma, S. Devaraj, and I. Jialal The Verdict Is Still Out Circulation, May 2, 2006; 113(17): 2128 - 2151. [Full Text] [PDF]

				E. Ruiz, A. Gordillo-Moscoso, E. Padilla, S. Redondo, E. Rodriguez, F. Reguillo, A. M. Briones, C. van Breemen, E. Okon, and T. Tejerina Human vascular smooth muscle cells from diabetic patients are resistant to induced apoptosis due to high bcl-2 expression. Diabetes, May 1, 2006; 55(5): 1243 - 1251. [Abstract] [Full Text] [PDF]

				I. Montero, J. Orbe, N. Varo, O. Beloqui, J. I. Monreal, J. A. Rodriguez, J. Diez, P. Libby, and J. A. Paramo C-Reactive Protein Induces Matrix Metalloproteinase-1 and -10 in Human Endothelial Cells: Implications for Clinical and Subclinical Atherosclerosis J. Am. Coll. Cardiol., April 4, 2006; 47(7): 1369 - 1378. [Abstract] [Full Text] [PDF]

				N. Sattar High sensitivity C-reactive protein in cardiovascular disease and type 2 diabetes: evidence for a clinical role? The British Journal of Diabetes & Vascular Disease, January 1, 2006; 6(1): 5 - 8. [PDF]

				M. B. Pepys, P. N. Hawkins, M. C. Kahan, G. A. Tennent, J. R. Gallimore, D. Graham, C. A. Sabin, A. Zychlinsky, and J. de Diego Proinflammatory Effects of Bacterial Recombinant Human C-Reactive Protein Are Caused by Contamination With Bacterial Products, Not by C-Reactive Protein Itself Circ. Res., November 25, 2005; 97(11): e97 - e103. [Abstract] [Full Text] [PDF]

				J. Torzewski C-Reactive Protein and Atherogenesis: New Insights from Established Animal Models Am. J. Pathol., October 1, 2005; 167(4): 923 - 925. [Full Text] [PDF]

				U. Singh, S. Devaraj, and I. Jialal C-Reactive Protein Decreases Tissue Plasminogen Activator Activity in Human Aortic Endothelial Cells: Evidence that C-Reactive Protein Is a Procoagulant Arterioscler Thromb Vasc Biol, October 1, 2005; 25(10): 2216 - 2221. [Abstract] [Full Text] [PDF]

				E. T.H. Yeh A New Perspective on the Biology of C-Reactive Protein Circ. Res., September 30, 2005; 97(7): 609 - 611. [Full Text] [PDF]

				S. S. Nerurkar, P. J. McDevitt, G. F. Scott, K. O. Johanson, R. N. Willette, and T.-L. Yue Lipopolysaccharide (LPS) Contamination Plays the Real Role in C-Reactive Protein-Induced IL-6 Secretion From Human Endothelial Cells In Vitro Arterioscler Thromb Vasc Biol, September 1, 2005; 25(9): e136 - e136. [Full Text] [PDF]

				J. Nilsson CRP--Marker or Maker of Cardiovascular Disease? Arterioscler Thromb Vasc Biol, August 1, 2005; 25(8): 1527 - 1528. [Full Text] [PDF]

				C. W. van den Berg and K. E. Taylor Letter to the Editor: Letter in Response to Bisoendial et al: "Activation of Inflammation and Coagulation After Infusion of C-Reactive Protein in Humans" Circ. Res., July 8, 2005; 97(1): e2 - e2. [Full Text] [PDF]

				M. B. Pepys CRP or not CRP? That Is the Question Arterioscler Thromb Vasc Biol, June 1, 2005; 25(6): 1091 - 1094. [Full Text] [PDF]