ABSTRACT
There are many controversies surrounding the biological activities of native C-reactive protein (nCRP) and its various modified forms such as monomerized and biotinylated CRP (mCRP and bCRP). No simple methods have been described to distinguish among these forms. By adapting established electrophoresis methods, we have developed a useful quality control method with which we have investigated the structural and functional characteristics of these forms of CRP. Under all electrophoresis conditions, biotinylation altered the electrophoretic mobility of CRP. nCRP was sensitive to sodium dodecyl sulphate (SDS)-induced monomerization, and only mCRP was susceptible to digestion by trypsin or neutrophil-derived serine proteases. bCRP and mCRP but not nCRP bound to cells, suggesting that chemical modification by biotin and denaturation had altered the structural integrity of CRP. Neither nCRP nor mCRP had the ability to induce secretion of chemokines, nor did they increase intracellular adhesion molecule 1 (ICAM-1) expression in endothelial cells.
Subject(s)
C-Reactive Protein/chemistry , Biotin , C-Reactive Protein/immunology , C-Reactive Protein/metabolism , Electrophoresis, Polyacrylamide Gel/methods , Endothelial Cells/metabolism , Endothelium, Vascular/cytology , Endothelium, Vascular/metabolism , Hot Temperature , Humans , Protein Denaturation , Serine Endopeptidases , Structure-Activity Relationship , TrypsinABSTRACT
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.
Subject(s)
Azides/pharmacology , C-Reactive Protein/pharmacology , Endothelium, Vascular/drug effects , Lipopolysaccharides/pharmacology , Artifacts , C-Reactive Protein/isolation & purification , Cell Division/drug effects , Cell Survival/drug effects , Cells, Cultured , Chemokine CCL2/metabolism , Dialysis , Endothelium, Vascular/cytology , Endothelium, Vascular/metabolism , Humans , In Vitro Techniques , Intercellular Adhesion Molecule-1/metabolism , Interleukin-8/metabolism , Nitric Oxide Synthase Type III/metabolism , Umbilical Veins/cytology , von Willebrand Factor/metabolismSubject(s)
Artifacts , Azides/pharmacology , C-Reactive Protein/pharmacology , CD55 Antigens/biosynthesis , Drug Contamination , Endothelial Cells/drug effects , Azides/analysis , C-Reactive Protein/isolation & purification , CD55 Antigens/genetics , Dialysis , Endothelial Cells/metabolism , Humans , Recombinant Proteins/pharmacology , Vasodilation/drug effectsABSTRACT
OBJECTIVE: Although C-reactive protein (CRP) is increasingly recognized as an independent risk factor for acute myocardial events, recent evidence suggests that it can directly induce vasorelaxation. This study aimed to investigate the mechanism of this CRP-induced response. METHODS AND RESULTS: Isometric tension recordings were used to measure endothelium-dependent and endothelium-independent vascular smooth muscle relaxation in isolated rabbit aortic rings. CRP generated in-house by genetic engineering and expressed in Chinese hamster ovary cells, CRP purified from ascites, and CRP obtained from commercial sources were assessed for vasorelaxing properties. Only the commercial CRP preparation induced vasorelaxation; more than half maximal relaxation was observed at 0.025 microg/mL and maximum relaxation attained at 0.25 microg/mL. Commercial CRP contains high levels of sodium azide, a well-known vasorelaxant. Removal of this agent by dialysis abolished the vasodilatory effect of commercial CRP. Sodium azide alone at concentrations equivalent to that present in the commercial CRP produced a near-identical relaxation pattern to the undialyzed commercial product. CONCLUSIONS: CRP has no vasorelaxant properties per se, and the reported vasorelaxant ability of CRP is an artifact caused by sodium azide present in commercial preparations of this agent.
