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(Circulation. 2004;110:e542.)
© 2004 American Heart Association, Inc.

Correspondence

Letter Regarding Article by Li et al, "C-Reactive Protein Upregulates Complement-Inhibitory Factors in Endothelial Cells"

Carmen W. van den Berg, PhD; Karolina E. Taylor, BSc

Department of Pharmacology, Therapeutics, and Toxicology, Wales Heart Research Institute, University of Wales College of Medicine, Cardiff, United Kingdom

To the Editor:

Recently, Li et al reported an increase in decay accelerating factor (DAF) expression on human endothelial cells on incubation with C-reactive protein (CRP).¹ Using our in-house–generated recombinant CRP,² we were unable to reproduce these effects on human umbilical vein endothelial cells or the endothelial cell line EA.hy926. Commercial CRP (recombinant, Calbiochem), however, increased DAF expression on both cell types. Commercial CRP preparations contain 0.05% to 0.1% azide per milligram CRP. When azide was removed by dialysis, the commercial CRP lost its ability to induce DAF (Figure). When azide was added to our own CRP, this preparation gained the ability to increase DAF expression (see Figure). Azide on its own also increased DAF expression significantly (see Figure). We obtained similar results with EA.hy926 and human umbilical vein endothelial cells. The commercial CRP preparation (Trichem Resources) used by Li et al also contains azide (0.1%/mg CRP). These authors did not mention the removal of the azide before the addition of CRP to the cells. We show here convincingly that azide, present in commercial CRP preparations, is responsible for the increased expression of DAF, and therefore conclude that the results published by Li et al are artifacts and must be attributed to azide and not to CRP. Furthermore, CRP-induced endothelial cell activation studies with commercial preparations of CRP must be interpreted with care because some of the components in these preparations may induce artifacts. Indeed, we have found recently that azide and not CRP is responsible for the reported CRP-induced vasorelaxation.²

View larger version (13K): [in this window] [in a new window]   EA.hy926 cells were incubated for 24 h in medium without additional additives (–); with azide (Az; equivalent to 50 µg/mL CRP=0.0025% azide); Calbiochem CRP (C; 50 µg/mL), dialyzed Calbiochem CRP (C dial; 50 µg/mL); our recombinant azide-free CRP (O; 50 µg/mL); or our recombinant CRP with azide added (O + Az; 0.0025% azide). DAF expression was assessed by flow cytometry. Results are expressed as median fluorescent intensity (MFI)±SD of experiments carried out in triplicate. Statistical analysis was carried out by one-way ANOVA followed by the Tukey multiple comparison test with 95% CI.

Acknowledgments

The authors’ research on this topic received financial support from the British Heart Foundation.

References

1. Li SH, Szmitko PE, Weisel RD, Wang CH, Fedak PW, Li RK, Mickle DA, Verma S. C-reactive protein upregulates complement-inhibitory factors in endothelial cells. Circulation. 2004; 109: 833–836.[Abstract/Free Full Text]

2. van den Berg CW, Taylor KE, Lang D. C-reactive protein-induced in vitro vasorelaxation is an artefact caused by the presence of sodium azide in commercial preparations. Arterioscler Thromb Vasc Biol. 2004; 24: e168–e171.[Abstract/Free Full Text]

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This Article Extract Full Text (PDF) Alert me when this article is cited 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 van den Berg, C. W. Articles by Taylor, K. E. Search for Related Content PubMed PubMed Citation Articles by van den Berg, C. W. Articles by Taylor, K. E. Related Collections Risk Factors Endothelium/vascular type/nitric oxide Mechanism of atherosclerosis/growth factors