Biomarkers And Regulatory Mechanisms For Coronary Plaque Instability

Background:Acute coronary events are dangerous disease to shortened human lifespan, and about 75% of them are caused by unstable plaque rupture. So it’s important to develop screening method for early identification of the risk plaque and explore the regulation mechanism of the plaque instability for prevention and treatment of acute cardiovascular events. Detection serological of biomarkers is more economical and convenient relative to the imaging examination more suitable for large-scale population. Matrix metalloproteinases (MMPs) are zinc dependent enzymes for extracellular matrix (ECM) degradation, which is not only involved in the physiological processes, such as embryonic development, angiogenesis and wound healing, but also related closely to the pathological process of tissue remodeling, tumor metastasis, atherosclerosis and others. Our previous small case-control study found circulating MMP-9 levels increased in patients with non-calcified plaques contrast to those with calcified plaques and non-plaque. However, the value of MMP-9 and MMP-2 identifying non-calcified plaques during routine hospital practice remains to be explored. MMPs degrades ECM in the plaque to make fibrous cap thinner, and eventually leads to plaque rupture. Therefore, exploring the regulation mechanism of MMPs may provide new intervention targets for preventing unstable plaque rupture. Lysophosphatidic acid (LPA), as an bioactive and extracellular phospholipid signaling molecule, functions through the activation of at least six known G protein-coupled receptors (GPCRs), LPAR1-LPAR6, thereby controlling a broad range of biological effects, including cell proliferation, differentiation, migration, survival and apoptosis. LPA has been reported to induce expression and secretion of MMPs involving in the invasion process of human ovarian cancer cells, glioma cells and other tumor cells. But in the development of atherosclerosis, the relationship between LPA and MMPs is unclear. Therefore, our study explores biomarkers of atherosclerosis in the peripheral blood, which may provide new targets to stabilize the fibrous cap and prevent plaque rupture.Objectives:The present study aimed at investigating the feasibility of serum MMPs as biomarkers to identify unstable plaque, which provides new approaches and diagnostic tools for the prevention of cardiovascular events induced by plaque rupture; and determining that LPA induces the expression and secretion of MMP-9, as well as molecular mechanism to make plaque more rupture-prone.Methods:From July 2012 to February 2014,862 unselected consecutive outpatients with stable chest pain who underwent coronary computed tomographic angiography (CCTA) were divided into non-plaque (474 cases), calcified (159 cases), non-calcified (80 cases) and mixed plaque (149 cases) groups. Serum levels of MMP-9 and MMP-2 were detected by ELISA to clarify diagnostic capabilities for non-calcified plaque.15 cases in each group were selected randomly and serum levels of candidate inflammatory cytokines, including TNF-a, Eotaxin, PTX3, MPO, were measured as a preliminary exploration of new biomarkers for plaque instability.On this basis, colocalization of ATX, enzyme of producing LPA, and MMP-9 in coronary plaque tissue was detected by immunohistochemistry and immunofluorescence. The expression, secretion and activity of MMP-9 were detected by RT-PCR, western blot, ELISA and gelatin zymography. Inhibitor U0126 was used to block the ERK1/2 pathway, knockdown by using siRNA or miRNA, and then luciferase reporter assay and western blot was used to investigate the regulation mechanism of MMP-9 through multiple signaling pathways. All the results were analyzed by SPSS 17.0 and P<0.05 was considered statistically significant.Results:We found that serum MMP-9 level in the non-calcified plaque group was significantly higher than the control group, calcified plaque and mixed plaque group. The area under the ROC curve (AUC) was 0.70 of serum MMP-9 for predicting non-calcified plaques even no increase after combination with other indicators. Further analysis revealed that stratification with serum high-density lipoprotein cholesterol helped to improve the diagnostic value. When high-density lipoprotein cholesterol levels≥1.55 mmol/L in patients, the AUC of serum MMP-9 for identifying non-calcified plaques rose to 0.84. In addition, serum levels of MMP-9 and MMP-2 were different between the presence of plaque group and control group. And in patients of high-density lipoprotein cholesterol levels ≥1.55 mmol/L, the combination of serum MMP-9 and MMP-2 to predict the presence of plaques was 0.80. Unfortunately, the screening results showed that there were no differences of six kinds candidate inflammatory cytokines among non-plaque, calcified, non-calcified and mixed plaque groups.Our study also showed that peripheral levels of LPA and MMP-9 elevated in acute coronary syndrome patients compared with the control group. There is a positive correlation between the two. Also found in the human coronary plaque tissue, more expression and co-localization of ATX and MMP-9 with the increase of atherosclerotic plaque instability, suggesting that there may be a relationship between LPA and MMP-9. Therefore we demonstrated that LPA promoted activation of transcription factor NF-κB through ERK1/2 signaling pathway, which upregulated expression levels of MMP-9. In addition, miR-21 also invovled the expression of LPA-induced MMP-9. LPA2 mediated the regulation of LPA to MMP-9.Conclusions:MMP-9 have a high diagnostic efficacy in elevated high-density lipoprotein cholesterol populations as biomarkers, identifying non-calcified plaque singly, which could be used in early screening of unstable plaque and applied to routine clinical practice. We explained the mechanism that LPA induced the expression of MMP-9, promote its secretion and activity in macrophages. And LPA2 maybe an intervention target to prevent plaque rupture and acutecardiovascular events.