The Influence Of Cytokine On PAPP-A, MMP-3, TIMP-1, CD40L Gene Expression In Human Coronary Artery Smooth Muscle Cells

Background: Acute coronary syndrome is including a series of clinic emergency cases from instability angina pectoris to cardiac death, and its clinical manifestation occurrence suddenly and seriously. Evidence indicates that inflammatory plays an important contributory role in the development of acute coronary syndrome. Acute coronary syndrome was associated with rupture of instability plaque, and pregnancy-associated plasma protein-A, matrix metalloproteinase-3, tissue inhibitor of metalloproteinase-1, CD40L play important role inthe proceeding of plaque destabilization. Vascular smooth muscle cells are important cell components of atherosclerotic plaque, play important role in the process of atherosclerosis. They not only promote the atherosclerosis and stenosis in structure, also have crucial endocrine function, to participate the pathophysiology proceeding of plaque development. These plasma makers were regulated by inflammation cytokine, but at present the result about human coronary artery smooth muscle cells is unknown, we think that it maybe have the similar results.Objectives: To study the effect of interleukin-1β, interleukin-6 and interferon-αon pregnancy-associated plasma protein-A, matrix metalloproteinase-3, tissue inhibitor of metalloproteinase-1, CD40L mRNA expression in human coronary artery smooth muscle cells.Methods: Human coronary artery smooth muscle cells were cultured in a 37℃, 5% CO₂/95% air, humidified cell culture incubator. Cells of the fifth to seventh passage were used for experiments. Human coronary artery smooth muscle cells cultures seeded at 1×10⁴ cells/cm² each 25 cm² culture flask, change the culture medium after 24 hours. Once the culture reaches 80% confluence, add cytokine to stimulate human coronary artery smooth muscle cells. Human coronary artery smooth muscle cells were stimulated with interleukin-1β(20 ug/L), interleukin-6(10 ug/L) or interferon-a(20 ug/L), collect cells and culture media after 0, 2, 4, 8, 24 and 36h. Then stimulate human coronary artery smooth muscle cells with interleukin-1β(0, 5, 20, 40 ug/L), interleukin-6 (0, 5, 10, 50 ug/L) or interferon-α(0, 5, 20, 40 ug/L)respectively, collect cells and culture media after 6h. Culture media were stored at -70℃, cells were washed twice with normal saline. Total mRNA was isolated with TRIzol reagent. Real-time quantitation PCR was performed on RNA to detect pregnancy-associated plasma protein-A, matrix metalloproteinase-3, tissue inhibitor of metalloproteinase-1, CD40L mRNA expression. All data were analyzed with SPSS 10.0 software. Data were presented as means+SE. Statistical analyses were performed using one-way ANOVA and spearman correlation, followed by multiple comparisons (SNK). Results were considered statistically significant at P＜0.05, and were replicated in independent experiments.Results: All dissociation curves of products only had one peak, which manifested that the products were pure and the primer is specific. In the same concentration of interleukin-1β, matrix metalloproteinase-3, pregnancy-associated plasma protein-A mRNA expression was up-regulation at 2 hour, at 8 hour the expression got to the peak, then begin to descent; tissue inhibitor of metalloproteinase-1 mRNA expression will be down-regulation at 2 hour, the mRNA expression got to the lowest at 8 hour, then begin to go up. In different concentration of interleukin-1β, matrix metalloproteinase-3, pregnancy-associated plasma protein-A mRNA expression is up-regulation with the dose(MMP-3: r=0.907, p=0.000; PAPP-A: r=0.972, p=0.000), andtissueinhibitorofmetalloproteinase-1 mRNA expression is down-regulation with the dose(r=-0.768, p=0.004). There were significant difference in mRNA expression among different groups (MMP-3.. F=24.047, P=0.000; TIMP-1 : F=33.737, P=0.000; PAPP-A: F=264.699, P=0.000). There are no significant differences of matrix metalloproteinase-3 between 20 and 40μg/L groups (p=0.154). But there are significant difference of tissue inhibitor of metalloproteinase- 1 mRNA expression only between 40μg/L and other groups.In the same concentration of interleukin-6, matrix metalloproteinase-3, pregnancy-associated plasma protein-A mRNA expression was up-regulation at 2 hour, at 8 hour the expression got to the peak, then begin to descent; tissue inhibitor of metalloproteinase-1 mRNA expression will be down-regulation at 2 hour, the mRNA expression got to the lowest at 4 hour, then begin to go up. In different concentration of interleukin-6, matrix metalloproteinase-3,pregnancy-associated plasma protein-A mRNA expression is up-regulation with the dose(MMP-3: r=0.919, p=0.000; PAPP-A: r=0.941, p=0.000), and tissue inhibitor ofmetalloproteinase-1mRNA expression is down-regulation with the dose(r=-0.799, p=0.002). There were significant difference in mRNA expression among different groups (MMP-3: F=14.081, P=0.001; TIMP-I: F=5.727, P=0.022; PAPP-A: F=25.128, P=0.000).There are no significant differences of matrix metalloproteinase-3, pregnancy-associated plasma protein-A mRNA between 5 and 10 ug/L groups (MMP-3: p=0.292; PAPP-A: p=0.065). There are significant difference of tissue inhibitor of metalloproteinase-1 mRNA expression between control and 10, 50 ug/L groups.In the same concentration of interferon-a, matrix metalloproteinase-3, pregnancy-associated plasma protein-A mRNA expression was down-regulation at 2 hour, at 8 hour the expression got to the lowest, then begin to go up; tissue inhibitor of metalloproteinase-1 mRNA expression will be up-regulation at 2 hour, the mRNA expression got to the peak at 8 hour, then begin to descent. In different concentration of interferon-a, matrix metalloproteinase-3,pregnancy-associated plasma protein-A mRNA expression is down-regulation with the dose (MMP-3.. r=-0.928, p=0.000; PAPP-A: r=-0.919, p=0.000) , and tissue inhibitor of metalloproteinase-1 mRNA expression is up-regulation with the dose(r=0.864, p=0.000). There were significant difference in mRNA expression among different groups (MMP-3: F=23.849, P=0.000; TIMP-1: F=49.526, P=0.000; PAPP-A: F=70.822, P=0.000). There are no significant differences of matrix metalloproteinase-3 between control and 5μg/L groups (p=0.154), pregnancy-associated plasma protein-A between 20 and 40μg/L groups (p=0.783) and tissue inhibitor of metalloproteinase-1 between 5 and 20μg/L groups (p=0.597).There were no significant differences of CD40L mRNA expression.Conclusions: Inflammatory factors may influence the mRNA expression of markers of plaque destabilization, such as pregnancy- associated plasma protein-A, matrix metalloproteinase-3, tissue inhibitor ofmetalloproteinase-1, and participate in the pathophysiology of coronary plaque instability and subsequent rupture. It manifested that inflammatory reaction and inflammatory factors were important factor of plaque unstabilization, and matrix metalloproteinase-3, tissue inhibitor of metalloproteinase-1, pregnancy-associated plasma protein-A may be one of the important mechanisms and pathways of inflammation effect in plaque unstabilization. Therefore, inflammatory reaction and inflammatory factors play an important role in the development of atherosclerosis and acute coronary syndrome.