Experimental Study On The Relationship Between Matrix Metalloproteinases And Stability Of Atherosclerotic Plaques

Acute coronary syndromes (ACS), like myocardial infarction (MI) and unstable angina (UA), are primarily responsible for coronary atherosclerotic heart disease (CHD) mortality. These syndromes are a result of the rupture of unstable atherosclerotic plaques and thrombosis. A better knowledge of the risk factors that may determine the stabilities of the plaques would help to understand the mechanisms responsible for ACS formation and provide new clues to clinical diagnosis and therapy of ACS. The matrix metalloproteinases (MMPs), a family of Zinc-binding endopeptidases, are likely to be involved in CHD by degrading the extracellular matrix. Therefore, perturbed regulation of MMP activity would influence the risk of cardiovascular disease. To elucidate the relationship between matrix metalloproteinases and stability of atherosclerotic plaques, the present experimental study was performed in several parts as follows:1. Made an attempt to investigate the effects of oxidized low density lipoprotein (oxLDL), which was implicated in many proatherogenic events, on the expression and secretion of MMP-9 in an in vitro murine peritoneal macrophage system. The macrophages were incubated with various concentrations of LDL, copper-oxidized LDL. RT-PCR analysis was used to measure the mRNA expressions of MMP-9. The enzymatic activity of secreted MMP-9 was determined by gelatin zymography. The secreted protein levels of MMP-9 were also determined by Western blot analysis.The results showed that oxLDL (20 to 50 mg/L) stimulated the gelatinolytic activity of both MMP-9 and MMP-2, especially MMP-9. Western blot analysis showed that oxLDL increased the secreted MMP-9 protein levels in a dose-dependent manner. After oxLDL treatment for 6 h, the mRNA levels of MMP-9 were also increased in a dose-dependent manner. The signal intensity of MMP-9 mRNA induced by 50 mg/L oxLDL was 2.65-fold versus control. Native LDL (80mg/L) had no significant effect on MMP-9 expression and activity.2. Investigated the effects of oxLDL and Lysophosphatidylcholine (LysoPC), one ofthe major chemical components of oxLDL.on the expression and secretion of MMP-12 in an in vitro murine peritoneal macrophage system. In particular, we examined the potential involvement of ERK1/2 activation in oxLDL-induced MMP-12 expression.The results indicated that control macrophages demonstrate hardly detectable amounts of MMP-12 in p-casein zymography. Treatment with oxLDL can significantly increase gel lysis at both 22 and 29 Kda. The Murine MMP-12 proenzyme (54 kD) were not seen. The results demonstrated that oxLDL increased MMP-12 protein secretion and conversion of pro-MMP-12 to its active form. oxLDL also significantly increased the mRNA level of MMP-12 in a dose-dependent manner, with a peak (6.64-fold versus control) at 50 mg/L oxLDL. Native LDL(80mg/L) had no significant effect on MMP-12 expression and activity. Furthermore, our data showed that LysoPC at nontoxic concentrations (10~20 umol/L) had no significant effect on MMP-12 enzymatic activity and mRNA expression. It seemed that LysoPC did not contribute to oxLDL-induced upregulation of MMP-12 expression in macrophages.In an attempt to illustrate the cellular signaling mechanisms by which oxLDL induced MMP-12 expression, we next determined the phosphorylation of the extra-cellular signal -regulated kinase (ERK), p38 and c-jun NH2-terminal kinase (JNK) in macrophages after stimulation with oxLDL or LysoPC .Western blot analysis showed that the phospharylation levels of the three kinases were low in normal macrophages. LysoPC stimulated ERK1/2 , p38 and JNK phosphorylation in murine macrophages, especially p38 . After macrophages were stimulated by oxLDL, both p38 and ERK1/2 were significantly phosphorylated. But the phosphorylation level of JNK was not significant. oxLDL stimulates phosphorylation of ERK, mainly ERK-2, in a dose-dependent manner, with peak phosphorylation (5.74?.9, 10.60 ?1.07- fold versus the basal level) at 50 mg/L oxLDL. However, the phosphorylation levels of ERK 1 and ERK2 increased...