Plasma Level Of Tissue Factor Pathway Inhibitor 2 In Patients With Acute Coronary Syndrome And Its Influences On The Proliferation And Migration Of Vascular Smooth Muscle Cells And Related Mechanisms

Tissue factor pathway inhibitor-2 (TFPI-2) is a Kunitz-type serine proteinase inhibitor synthesized by endothelial cells (ECs), smooth muscle cells (SMCs), and syncytiotrophoblasts, which associates through ionic interactions with the extracellular matrix (ECM) and inhibits trypsin, plasmin, and plasma kallikrein among others. The major role of TFPI-2 seems to be plasmin inhibition and thus of proMMP-1, proMMP-3, and proMMP-13 activation. As a direct inhibitor of MMP-2 and MMP-9 and potent inhibitor of both matrix-bound and cell-associated plasmin, TFPI-2 could regulate extracellular proteolysis and ECM remodeling, which are highly relevant both for normal development and for atherosclerosis.Its expression has been shown to be lower at the shoulder of atherosclerosis plaque, which suggests that downregulation of TFPI-2 contributes to atherosclerosis progression. Proliferation and migration of VSMCs from media to intima is pivotal to atherosclerotic progression. Oxidized low density lipoprotein (ox-LDL), which is an important detrimental factor of atherosclerosis, is well established as an important stimulus of VSMCs proliferation and a chemoattractant directing the migration of VSMCs toward the vessel intima during atherosclerosis and neointima hyperplasia.To test the role of TFPI-2 in atherosclerotic progression, we measured plasma levels of TFPI-2 and MMP-1,3,9 in patients with ACS and stable angina pectoris and healthy controls, and study the function of TFPI-2 on proliferation, apoptosis and migration of VSMCs under the treatment of ox-LDL.Part I Low plasma levels of tissue factor pathway inhibitor-2 in patients with acute coronary syndromeBackground:Recent evidence implied that tissue factor pathway inhibitor-2 (TFPI-2). a Kunitz-type protease inhibitor with the ability to inhibit matrix metalloproteinases (MMPs) activity, which can regulate extracellular proteolysis and extracellular matrix remodeling, may be relevant for stability of atherosclerotic plaque. However, plasma levels of TFPI-2 and in patients with acute coronary syndrome (ACS) have not yet been determined.Methods:Plasma concentrations of TFPI-2, MMP-1,3 and 9 were measured in 109 subjects with ACS,41 patients with stable angina pectoris (SA) and 30 controls, using commercial enzyme-linked immunoadsorbent assay kits. Results:Circulating TFPI-2 was notably lower and MMP-1 was higher in patients with ACS than in those with stable angina pectoris and controls. No differences have been observed in the plasma levels of MMP-3 and MMP-9 among the three groups. A negative correlation was observed between TFPI-2 and MMP-1 plasma concentrations. (r=-0.388, P<0.001). Low circulating TFPI-2 level was an independent predictor for ACS after adjustment for MMP-1, hsCRP, LVEF, serum creatinine and HDL-C (OR=0.549,95% CI:0.415-0.728, P<0.001).Conclusions:Patients with ACS showed decreased circulating level of TFPI-2 and low plasma levels of TFPI-2 is independently associated with ACS, providing new insights into ACS pathophysiology with appealing therapeutic implications.Part?Tissue factor pathway inhibitor-2 is downregulated by ox-LDL and inhibits ox-LDL induced vascular smooth muscle cells proliferation and migrationIntroduction:Tissue factor pathway inhibitor-2 (TFPI-2) is a member of the Kunitz-type family of serine protease inhibitors, which inhibits several matrix metalloproteinases activity involved in extracellular matrix degradation. Studies have shown low TFPI-2 expression in the shoulder regions of atherosclerotic plaques. But studies evaluated its role in the progression of atherosclerotic plaque are scarce. Vascular smooth muscle cells (VSMCs) are important component of atherosclerotic plaques and oxidized low density lipoprotein (ox-LDL) is an important detrimental factor of atherosclerosis. The aim of this study is to elucidate the effect of TFPI-2 on smooth muscle cell proliferation and migration induced by ox-LDL.Methods:Retroviruses expressing human TFPI-2 and lentivirus vector for TFPI-2 knockdown were constructed. Cell proliferation was determined by CCK-8 assay. Cell apoptosis was analyzed by double staining of FITC-Annexin?and propidium iodide. Cell migration was studied through a Transwell chamber and with a scratch-wound assay. Results:TFPI-2 over-expression and knockdown of mRNA and protein was confirmed in infected VSMCs. CCK-8 assay and flow cytometry analysis showed that TFPI-2 inhibit VSMCs proliferation induced by ox-LDL while without cytotoxicity to VSMCs. Transwell and scratch wound assay confirmed TFPI-2 over-expression can inhibit VSMC migration.Conclusion:TFPI-2 may strongly inhibit the proliferation and migration of VSMCs while do not influence cell apoptosis, making it a promising candidate for treatment of atherosclerotic process.Part?:Mechanisms of TFPI-2 influence on proliferation and migration of vascular smooth muscle cellsObjective:To investigate the potential mechanisms of TFPI-2 on proliferation and migration of vascular smooth muscle cells in vitro.Methods:After being infected with TFPI-2 overexpression virus vector and knockdown vector, expression of cyclin D1 and phosphorylation of ERK 1/2, Akt, FAK and STAT3 were detected by Western blot. The matrix metalloproteinase-2 and-9 activities were analyzed by gelatin zymography.Results:TFPI-2 could inhibit cycin D1 expression induced by ox-LDL. Ox-LDL treatment could induce expression of MMP-2,9 in VSMCs and the influence was inhibited by TFPI-2 overexpression. TFPI-2 overexpression inhibited the phosphorylation of Akt, FAK and STAT3, while no significant influence of TFPI-2 on phosphorylation of ERK 1/2 was detected.Conclusion:TFPI-2 may inhibit proliferation and migration of VSMCs through inhibition of cyclin D1 and MMP-2,9. Cell signal transduction pathway Akt, FAK and STAT3 may be involved.