Sodium Azide in Commercially Available C-Reactive Protein Preparations Does Not Influence Matrix Metalloproteinase-2 Synthesis and Release in Cultured Human Aortic Vascular Smooth Muscle Cells

Detection of circulating concentrations of the acute-phase reactant C-reactive protein (CRP), which is synthesized in response to proinflammatory cytokines, is a relevant tool for identifying the involvement of low-grade inflammation in atherosclerosis and for predicting future atherothrombotic events (1). Whether CRP is only a marker or is also an active player in atherosclerotic injury is a matter of intense debate (2). CRP is present in atherosclerotic lesions (3) and can contribute directly to atherothrombosis (4). In particular, CRP induces expression of proatherogenetic molecules in endothelial cells and promotes LDL uptake by macrophages (4). We recently observed that CRP increases synthesis and secretion of matrix metalloproteinase 2 (MMP-2) from cultured human vascular smooth muscle cells (hVSMCs) (5), a mechanism potentially involved in plaque destabilization. Recently, however, the reliability of results concerning CRP obtained in vitro has been questioned because commercial CRP preparations contain the biologically active bacteriostatic preservative sodium azide (6)(7)(8)(8)(9). In particular, the contaminating presence of sodium azide has been considered responsible for the proapoptotic, antimigratory, antiproliferative, antiangiogenetic, and vasodilating effects previously attributed to CRP (5)(6)(7)(8). Thus, it is now necessary to exclude any sodium azide–mediated effect when a new biological action of CRP is described. Because our study of CRP effects on MMP in VSMCs included the use of a commercial CRP preparation (Sigma Chemical Co.) containing 1 g/L sodium azide, we carried out control experiments to rule out possible effects of sodium azide on MMP-2 synthesis and secretion in VSMCs from microarterioles (5). Our results for aortic hVSMCs were interesting. Aortic hVSMCs (Cambrex Bioscience Srl) cultured in minimum essential medium supplemented with 100 mL/L fetal calf serum, 100 units/L penicillin, 100 μg/L streptomycin, 10 mmol/L glutamine, and vitamins and buffered with 10 mmol/L TES and 10 mmol/L HEPES were incubated for 24 h with sodium azide at a final concentration of 76.9 μmol/L (concentration reached in culture medium after CRP addition). After incubation, MMP-2 expression (Western blot) and activity (gelatin zymography) were detected with previously described methods (5) in both supernatants and cell lysates. Contamination of the cell cultures by lipopolysaccharide was excluded by the Limulus assay (Sigma). Endotoxin was also removed from the CRP solution by use of a Detoxigel column (Pierce Biochemicals), as described previously (10). After purification, the endotoxin concentration in the CRP solution was <0.06 endotoxin units (EU)/mL as measured by the Limulus assay. The control experiment was performed by incubation of VSMCs with purified CRP solution. As evidenced in Fig. 11 , sodium azide did not influence MMP-2 expression and activity in either cell lysates or supernatants. CRP was used as a positive control. MMP-2 synthesis and secretion were not changed by cell exposure to the lipopolysaccharide antagonist Polymyxin B (0.1 and 0.5 mg/L). Purified CRP increased MMP-2 secretion, expression, and activity, with no difference compared with the nonpurified preparation. Recent studies underline the need to check the biological effects of a commercial protein preparation derived from a biological source by means of a broad range of control experiments. The results of our study demonstrate that CRP influences MMP-2 in VSMCs independently of sodium azide, which is present in commercial CRP preparations. To the best of our knowledge, we are the first authors taking into account the effects of sodium azide on MMP-2 synthesis and secretion, providing the first evidence that this agent does not influence MMP-2 synthesis and secretion from hVSMCs. Effects of sodium azide on expression, secretion, and activity of MMP-2 from hVSMCs. Densitometric analysis of gelatin zymographs (A and B) and Western blots (C and D) of cell supernatants and lysates indicated that CRP (10 mg/L), but not sodium azide (76.9 μmol/L), influences MMP-2 expression, secretion, and activity (∗, P <0.05 with CRP and not significant with sodium azide in both supernatants and cell lysates, Student–Newman–Keuls test). Each panel is representative of at least 5 experiments. This study was supported by a grant from the Italian Ministero dell’Istruzione, Università e Ricerca (MIUR), as part of the Inflammatory, Metabolic and Genetic Markers of Vascular Atherosclerotic Disease project (PRIN 2001).