Association Of Acid Sphingomyelinase With The Stability Of Coronary Atherosclerosis Plaque

Background Acute coronary syndrome (ACS) has high morbidity and mortality. It is a series of clinical syndrome based on atherosclerotic plaque rupture or erosion, then leads to complete or incomplete occlusive thrombosis. Vulnerable plaque rupture is the initial stage of ACS. A number of studies have found that the degradation of extracellular matrix by activated matrix metalloproteinase (MMPs) is one of the main causes leading to vulnerable plaque rupture.Recently, many studies have confirmed that ASMase is closely related to the development of AS, and it could be a predictor of coronary artery disease (CAD). ASMase, which could hydrolyze sphingomyelin into ceramide, participates in many pathological changes in the process of AS, such as inducing apoptosis of smooth muscle cells, increasing aggregation of low density lipoprotein (LDL), promoting foam cells formation, et al. However, there was no study investigated whether ASMase play a critical role in coronary atherosclerosis plaque stability. Activation of MMPs was closely associated with coronary atherosclerosis plaque stability. Many studies have found that ASMase could regulate activation of MMPs induced by inflammatory factors. Thus, at first, we investigated the relationship between ASMase level and clinical ACS patients. Secondly, we further discussed whether ASMase affected stability of coronary atherosclerosis plaque via regulation of MMPs activity in macrophages.Chapter Ⅰ Relationship between plasma ASMase activity, ceramide levels and coronary atherosclerosis plaque stabilityObjectives:To investigate the relationship between plasma ASMase activity, ceramide level with coronary atherosclerosis plaque stability.Methods:214CAD patients and52healthy people were collected, all subjects were divided into three groups, ACS group (n=114), SAP group (n=98) and control group (n=52). Plasma Sphingomyelin level was measured by chemiluminescence assay; ASMase activity in plasma was detected by high performance liquid chromatography assay (HPLC); plasma ceramide level was measured by liquid spectrometry and mass spectrometry assay (LS-MS); plasma MMP-9and TNF-α levels were measured by enzyme-linked immunosorbent assay (ELISA).Results:(1) The plasma level of Sphingomyelin in patients with ACS and SAP was significantly higher than that of control group (P<0.01). However, there was no difference between ACS and SAP group (P>0.05).(2) The plasma ASMase activity, ceramide level in patients with ACS and SAP were significantly higher than control group (P<0.05). Importantly, the ASMase activity and ceramide level in ACS group was significantly higher than SAP group (P<0.05).(3) The plasma level of MMP-9in ACS group was significantly higher than SAP group and control group (P<0.05). However, there was no significant difference between SAP and control group (P>0.05).(4) The plasma level of TNF-a in patients with ACS and SAP was significantly higher than that of the control group (P<0.05). Meanwhile, the TNF-α level of ACS group was extremely higher that of SAP group(P<0.05).(5) The plasma ASMase activity in ACS patients was significantly correlated with plasma ceramide level, and both of them are correlated with MMP-9, while have no relationship with plasma Sphingomyelin level.Conclusion:Plasma ASMase activity, ceramide, MMP-9and TNF-α levels were associated with coronary atherosclerosis plaque stability. Measurement of plasma ASMase activity may be a helpful method for discovering the population with higher risk of ACS. Chapter II ASMase regulates TNF-α-induced expressions of MMPs in macrophagesObjective:To investigate whether ASMase regulates TNF-a-inducec expressions of MMPs in macrophages.Methods:(1) The effect of TNF-α on the ceramide level and MMP-9 expression in macrophages was observed. Macrophages were treated with different concentrations of TNF-a (10,30or100ng/ml) for24h. The expressions of ASMase and MMP-9were determined by Western-Blot and Real-Time PCR. The ceramide level was determined by LS-MS.(2) To study the effect of ASMase inhibitor desipramine on TNF-α induced MMP-9expression. Cultured macrophages were randomly divided into three groups:control group:cells were incubated in high glucose DMEM containing1%fetal bovine serum for24hours;100ng/ml TNF-α treatment group:cells were incubated in high glucose DMEM containing100ng/ml TNF-α for24hours;+desipramine group:cells were pretreated with5μM desipramine (ASMase inhibitor) for120min prior to TNF-α exposure.Results:(1) TNF-α treatment increased ASMase expression, ceramide level and MMP-9expression with a concentration-dependent manner.(2) Compared with the control group, TNF-α (100ng/ml) significantly increased ceramide level and the expression of MMP-9in macrophages and these effect were inhibited by the pretreatment of desipramine, the specific ASMase inhibitor (P<0.05).Conclusion:ASMase/cernide pathway was involeved in the TNF-α-induced MMP-9expression.