Inflammatory Cytokines Stimulated C-Reactive Protein Production by Human Coronary Artery Smooth Muscle Cells

Abstract

Background-Serum C-reactive protein (CRP) levels are good predictors of the development of cardiovascular events in apparently healthy men and women. CRP has been believed to be produced exclusively by hepatocytes during the acute-phase response. Several lines of evidence have suggested that atherosclerotic arteries can also produce CRP. However, the cell types that produce CRP locally in the atherosclerotic arterial wall have not been clearly identified. Methods and Results-Human coronary artery smooth muscle cells (HCASMCs) and human umbilical vein endothelial cells (HUVECs) were incubated with interleukin-1␤ (IL-1␤), IL-6, their combination, tumor necrosis factor-␣ (TNF-␣), or lipopolysaccharide (LPS) at different concentrations. The supernatants were concentrated and analyzed by a high-sensitivity enzyme-linked immunosorbent assay specific for human CRP. RNA was extracted from the HCASMCs for reverse transcriptase-polymerase chain reaction (RT-PCR) using specific primers for the CRP. Maximal CRP production was observed in HCASMCs after 48 hours of incubation with the combination of 25 ng/mL of IL-1␤ and 10 ng/mL of IL-6, whereas incubation with IL-1␤ or IL-6 alone only modestly induced CRP. Incubation with TNF-␣ (50 ng/mL) or LPS (1000 EU/mL) resulted in an increase in CRP production comparable to the IL-1␤ and IL-6 combination. [1][2][3][4] and could directly participate in the pathogenesis of atherosclerosis through activation of endothelial cells. 5-9 CRP, named for its capacity to bind to the C-polysaccharide of Streptococcus pneumoniae, was the first acute-phase protein to be described. 10 CRP, like other acute-phase proteins, is synthesized by the liver in response to microbial infection, tissue injury, and autoimmune disorders. It had been shown that interleukin-1␤ (IL-1␤) and IL-6 strongly induced the expression of CRP in human hepatocytes 11 and hepatoma cells. 12 Recently, human neuronal cells were found to produce CRP in Alzheimer's disease. 13 In addition, renal cortical tubular epithelial cells were shown to produce CRP after inflammatory stimuli. 14 Interestingly, CRP has also been found in human atherosclerotic plaques, 15 which could be the result of indirect deposit from circulating cells or direct production by cells in the arterial wall. We show that human coronary artery smooth muscle cells (HCASMCs), but not human umbilical vein endothelial cells (HUVECs), can synthesize CRP after stimulation by inflammatory cytokines. Methods Cell CultureHCASMCs, HUVECs, and endothelial cell supplements were purchased from Cascade Biologics; penicillin, streptomycin, medium 231, medium 199, and smooth muscle cell growth supplement were from Gibco BRL; and fetal bovine serum, human serum, heparin, and gelatin were obtained from Sigma. HCASMCs were plated onto 0.1% gelatin-coated culture dishes from Corning, Inc, and grown in 231 medium with growth supplement and 1% penicillin/streptomycin; HUVECs were plated onto 0.1% gelatin-coated culture dishes and grown in 199 medium with endothelial growth suppleme...

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