| Cardiovascular disease is the leading cause of disease and death in the developed world. Acute coronary syndromes are most frequently caused by acute disruption of an atheromatous plaque. Plaque rupture is the most frequent cause of thrombosis. The excessive degradation of the extracellular matrix scaffold has been implicated as one of the major molecular mechanisms in this process. Apparently healthy subjects at risk for major cardiovascular events (acute coronary syndrome and/or sudden cardiac death) fuels demand for detection, interventions early.The unstable plaque are subject to excessive inflammation. Recent investigations have indicated that increases in biomarkers may provide an earlier assessment of overall patient risk and aid in identifying patients with higher risk of having an adverse event. Peripheral blood levels of biomarkers may be increased in patients with acute coronary syndrome, raising the interesting question of the possibility to develop noninvasive tests for detection of plaque vulnerability.There is growing interest in the search of a novel specific and sensitive circulating markers of vulnerable plaque. Recent studies suggest that MMP-2 and MMP9 may be of value in recognizing patients with ACS as an important biomarkers. MMPs expressed in human atherosclerotic lesions around the lipid core; they generally colocalize with macrophages/foam cells.Focal overexpression of activated MMPs may promote destabilization and complication of atherosclerotic plaques by degrading ECM collagen of overlying cap.ADAMTSs is a recently described family of proteinases. Structure of ADAMTS are compared with that of MMPs. The catalytic site consensus is present in ADAMTSs and MMPs, which is highly conserved Zn-dependent endopeptidases involved in proteolytic processes in the ECM. Different from MMPs,in the structure of ADAMTS,the disintegrin-like domain and TSP motif is followedReal time RT-PCR showed that ADAMTS-4 mRNA was induced in monocytes. Expression of ADAMTS-4 mRNA were enhanced 20-30-fold during monocyte to macrophage differentiation. In addition, ADAMTS-4 expression is upregulated during the development of atherosclerosis in mice.Because these vulnerable areas contain numerous macrophages, it is generally thought that the presence of these cells and their local release of matrix-degrading proteinases cause rupture of the plaque structure.The present work show that ADAMTS-4 were expressed in macrophage rich areas of human atherosclerotic carotid plaques and coronary unstable plaques. Whereas ADAMTS-4 expression was low in non-atherosclerotic aortas.We hypothesized that macrophage-derived ADAMTS4 play a role in initiating rupture of susceptible atherosclerotic lesions by degrading some structural ECM matrix component.ECM maintains the structural integrity of plaques. In addition to collagens, proteoglycans(versican, biglycan, and decorin), elastin, laminin,fibronectin, entactin are all ECM.Thinning of the fibrous cap has been shown to increase circumferential stress on the plaque and is generally regarded as a transitional step to plaque rupture.Versican, a high molecular weight chondroitin sulfate proteoglycan, is a component of normal blood vessels. Thus, cleavage of this structural proteoglycan at the periphery of the lipid cores could conceivably lead to cap destabilization and rupture.Collagen degradation within the fibrous cap is a critical factor in provoking rupture, the loss of versican in this region has received much attention. The degradation of versican in advanced human atheroma has been attributed to the expression of MMP7, MMP12 as well as serine protease plasminand the ADAMTSs family of metalloproteinases.Versican is one number of matrix component that maintains the structural integrity of plaques at the junction of the lipid core and cellular fibrous regions. Four isoforms of versican (V0, V1, V2, and V3), which vary by the presence or absence of two glycosaminoglycan (GAG) binding domains named aGAG and (3GAG. All forms of versican share the remaining domains that include the amino-terminal globular domain (G1; which contains the hyaluronan-binding link modules) and the carboxy-terminal G3 domain. In the blood vessel wall, in which V0 and V1 predominantly found.Not only do these molecules contribute to plaque burden, they also influence fundamental cellular and extracellular events associated with the pathogenesis of vascular lesions, such as thrombosis, lipid metabolism, and vascular proliferation and migration.Consequently, ADAMTS4 possesses substrate specificity and efficiently degrades extracellular matrix components aggrecan and versican. The aggrecan products generated by this enzyme are found in synovial fluid from patients with arthritis, suggesting that ADAMTS4 may be important in diseases involving cartilage destruction. ADAMTS4 is believed to play a key role in the destruction of articular cartilage through aggrecan degradation in human arthritides such as rheumatoid arthritis and osteoarthritis and. By analogy to its action on aggrecan in arthritis, it is likely that ADAMTS4 cleaves the core protein of versican or/and aggrecan in atherosclerotic lesions remains unclear. ADAMTS-4 has been reported to cleave either native versican or versican peptide substrates, ADAMTS-4 have been shown to cleave V1/V0 versican at the V1/V0 versican at the Glu441-Ala442/Glu1428-Ala1429 bond and the product of this cleavage was shown to be present in human atherosclerotic plaques and aneurysms by immunohistochemistry using neo-epitope antibodies. Such lower molecular weight species were also observed in extracts of normal human aorta..Recent work that suggests that cleavage of versican into discrete fragments may occur not only as normal physiologic turnover of the vascular extracellular matrix, but also be involved in vascular pathology.The G3 domain also increases migration of endothelial cells and the secretion of fibronectin and vascular endothelial growth factor, both of which are chemotactic for SMCs. The G3 domain has also been implicated in macrophage aggregation, as it has been shown to bind P-selectin glycoprotein ligand-1 (PSGL-1) a glycoprotein expressed on the cell surface of leukocytes, and this activity could turn out to be critically important in promoting leukocyte accumulation thrombus formation during the inflammatory phases in developing lesions of atherosclerosis.By virtue of their polyanionic nature, the chondroitin sulfate chains attached to the aGAG and PGAG domains of versican are involved in a range of functions. Binding of versican to CD44, L selectin, and P selectin is also mediated by the chondroitin sulfate chains. That changes in CS chains can alter factor Xa-anti-thrombin-CS interactions and influence the thrombotic balance in atherosclerotic tissue.There is accumulating evidence that disintegrin domain also displays potent proatherogenic properties. Cell adhesion and proteolytic matrix degradation are central processes in atherosclerosis. The disintegrin domain has been widely described as being able to interact with integrin molecules and therefore mediats cell-cell and cell-matrix interactions. The TSP type I motif of thrombospondin has been implicated in binding to matrix macromolecules and cell adhesion.TSP-1 motif of ADAMTS4 is critical for substrate recognition and cleavage.On the basis of the fact that ADAMTS4 was highly prominent at the advanced plaque lesions; versican is a high-risk substrate of ADAMTS4. Thus, it is tempting to speculate that ADAMTS4 provoke atherosclerotic plaque rupture by degrading the versican.On the other hand,ADAMTS4 potently and thoroughly degrades versican; the discrete fragments of versican were involved in vascular pathology;the coexistence of a zinc-protease and a disintegrin domain and the thrombospondin type I motifs, therefore, the different domains composing ADAMTS4 have independent but complementary functions. We speculate that ADAMTS4 is likely a key player in atherosclerosis development.Similar to the MMPs, the ADAMTSs can be inhibited by tissue inhibitors of metalloproteinases (TIMPs) because the C-terminal domain of ADAMTS participates in the binding of TIMPs. TIMP-3 is a potent inhibitor of ADAMTS4. So we suppose that the unbalance of circulating levels of ADAMTS4 and TIMP3 determine cap thinning, and sequently resulted in plaque stability.Early in atherogenesis, VSMCs from the media are thought to migrate into the intima and contribute to the development of atherosclerotic lesions. Although what initially triggers these events is not known, it is thought that proteases released by VSMCs degrade the matrix proteins in the intima, particularly the main proteoglycan of the arterial intima versican, making the intima more permissive for invasion by VSMCs. It is unclear wether ADAMTS-4 may modulate VSMC migration.Therefore, we undertook this study to:(i)assess ADAMTS4 may be of value in recognizing patients with ACS as an important biomarkers; (ii)investigat mechanisms of ADAMTS4 provoking atherosclerotic plaque rupture.The research contents and main results are as follows:1. ADAMTS4 level in patients with stable coronary artery disease and acute coronary syndromesA total number of 132 patients and 40 healthy subjects were involved in the study.25 patients with SAP,32 patients with UAP,35 patients with NSTEMI and 40patients with STEAMI were admitted to our institution within 3h-9h from the onset of symptom onset. Exclusion criteria were infection, tumor, liver or kidney diseases.Plasma levels of ADAMTS4 were measured in patients with stable effort angina pectoris (SAP),ACS and in controls. Venous blood was sampled upon admission before angiography and drug administration. In patients with ACS who underwent medical treatment, serial blood samples were also collected on days 1,2,3, 5 and 7 after admission. ADAMTS4 was measured using an enzyme immunoassay. Plasma ADAMTS4 level in cases (99.2(65.8; 149.2)ng/mL was significantly greater than in controls (51.2 (38.1; 64.8) ng/mL) (P<0.001). Higher levels of ADAMTS4 were found with progression of CAD from SAP (63.1 (45.2-90.3) ng/mL) to UAP (87.5(59.4-135.6)ng/mL) to NSTEMI (113.6(68.5-160.2) ng/mL) and to STEMI (127.3 (76.8-220.2) ng/mL) (P < 0.001). Elevated ADAMTS4 level was associated with ACS with an area under receiver operating characteristic (ROC) curve of 0.753 (95%CI 0.654-0.851; P< 0.001). The pattern of ADAMTS4 release observed was clearly different in various forms of ACS. In the UAP-M group, significant ADAMTS4 elevations was seen on day 2. The maximum value in both NSTEMI-M group and STEMI-M group was reached later than UAP group with a peak on day 3.There was a weak relationship between hs-CRP and ADAMTS4 in patients with ACS (r= 0.719, P=0.02;r=0.660,p=0.01; r=0.888,p=0.000 respectively)and Maximal ADAMTS4 levels were associated with maximal hsCRP levels (r= 0.700, P=0.03;r=0.632,p=0.02; r=0.769,p=0.03).however, no significant correlation was found between ADAMTS4 and TnT in ACS patients.Serial changes in plasma ADAMTS4 were documented in patients with ACS and may serve as a marker of plaque destabilization. 2. Elevated ADAMTS4 mRNA expression in peripheral monocytes and the correlation with the severity of coronary lesions in acute coronary syndrome patients150 subjects were divided into unstable angina pectoris (UAP) group (n= 50), acute myocardial infarction (AMI)group (n=30)stable angina pectoris (SAP) group (n =40), and controls (n= 30). coronary angiography was performed for all these patients. Total RNA was extracted from monocytes by use of TRIzol reagent. The expression of ADAMTS4 in monocytes was analyzed by RT-PCR. Coronary stenosis was assessed morphologically according to the Ambrose classification and was classified as either simple or complex. Gensini Score (GS)was used to evaluate the severity of coronary stenosis.ADAMTS4 mRNA level in cases was significantly greater than in controls (2.014±1.15 vs 1.201±0.402) P<0.001). Higher levels of ADAMTS4 mRNA were found in ACS group compared with SAP group (2.236±0.108 vs 1.333±0.432) (P<0.001).Correlation analysis showed that ADAMTS4 mRNA level was positively correlated with the complex coronary stenosis (r=0.58, p< 0.001). however, no significant correlation was found between ADAMTS4 mRNA level and the scores of Gensini in cases(r=0.118, P=0.201). Complex lesions were more frequent in the ACS group than in the SAP group (92%vs.13%,p<0.01).Patients with ACS show increased ADAMTS4 mRNA expression in peripheral monocytes which has positive relationship with the severity of coronary artery lesions, hence is an effective biomarker for ACS.3. Altered expression of ADAMTS4, TIMP3, Versican during the development of atherosclerosis in ApoE-/-mice.:results of quantitative tissue analysis using real-time RT-PCR method.Male apoE-/-mice on a primarily C57BL/6 genetic background acquired were 9 weeks old at the time of entry into the study and develops atherosclerosis on a normal diet. Mice were euthanized at 10 (n=8),20 (n=6),30 (n=7),40 (n=5),50 (n=5) and 40 (n=5) weeks and aortas were isolated in Trizol (Invitrogen) for RNA isolation.The amounts of mRNA for ADAMTS4, TIMP3, versican were determined by real-time RT-PCR method, and normalized with.betaactin.ADAMTS-4 mRNA was present in the mouse aortas before atherosclerotic lesions were visible (weeks 10). ADAMTS-4 mRNA was higher in mice with intermediate lesions (weeks 20 and 30) and almost 3-fold higher in late lesions (weeks 40 and 50) compared with the level in lesions (weeks 10 weeks). In contrast, aggrecan mRNA was undetectable in this model.TIMP3 and ADAMTS4 showed similar patterns of expression with age in apoE-/-mice. Although TIMP3 showed a steady increase with time (between 10 and 50 weeks, P<0.001). Interestingly, the indices of ADAMTS4/TIMP3 still significantly increased with time,.Before weeks30, there is no accociation between ADAMTS4 and versican (r=0.308,p=0.415; r=0.412,p=0.352; r=0.589,p=0.172); On weeks 40 and 50,there is strong accociation between ADAMTS4 and versican (r=0.688, p=0.02; r=0.712, p=0.000)Bofore weeks 30, TIMP-3 level was not positively correlated with the versican (r=0.411, p=0.395; r=0.282, p=0.572; r=0.379, p=0.220); On weeks 40 and 50, there is siginificant association between TIMP3 and versican (r=0.603, p=0.03; r=0.694, p=0.002).The upregulation of ADAMTS4 in development of atherosclerosis was disproportional to that of TIMP3 suggested that imbalanced degradation and synthesis of ECM versican.our study shows that the expression of versican is augmented during early atherogenesis suggesting that versican may contribute to the retention of lipoproteins at the earliest stages of murine atherosclerosis and at later stages decreacing possibly as a result of the degradation of ADAMTS4. Overall, versican accumulation reflected increased lesion development until such time as necrotic events deteriorated lesion architecture. 4. ADAMTS4 is expressed at sites of potential rupture in atherosclerotic lesions and localizes to areas of versican depositionAlthough previous studies have confirmed the involvement of versican and ADAMTS4 in atherosclerotic disease, it is not clear whether the degration of versican is associated with plaque rupture. There is little current knowledge of the relevance of versican and ADAMTS4 in the fibrous cap of advanced plaques. We performed immunohistochemical analysis to determine whether ADAMTS4 degrade versican in rupture-phone plaques lesion.Male apoE-/-mice were acquired were 10 weeks old at the time of entry into the study. Unless otherwise stated, the animals received a Western-type diet 2 weeks before surgery. Mice were anesthetized by subcutaneous injection of.barbitone.(0.04 mg/kg). Access to the anterior cervical triangles was gained through a sagittal anterior neck incision.20 weeks after collar placement, the animals were anesthetized and exsanguinated by femoral artery transection. Subsequently, both carotid bifurcations and common carotid arteries were removed, these were immediately snap-frozen in liquid nitrogen after having been embedded in OCT compound (Tissue-Tek; Sakura Finetek), the specimens were stored at-20℃until further use. Transverse 5-μm cryosections were prepared in a proximal direction from the carotid bifurcation and mounted in order on a parallel series of slides.Endogenous peroxidase activity was blocked by incubation in 0.3%H2O2 for 30 min at room temperature. Anti-ADAMTS4 antibody was diluted 1:200, and anti-versican antibody was diluted 1:250.Bound antibody was detected using a Vectastain ABC Elite kit following the manufacturer's instructions and 3,3'-diaminobenzidine tetrahydrochloride as the chromogenic substrate. Sections were counterstained with Harris hematoxylin.We found that ADAMTS4 was prominently expressed at sites susceptible to rupture, particularly where cellular and acellular areas are juxtaposed. We stained sections with Pentachrome to identify areas of elastin, collagen, proteoglycan, and fibrin deposition. Green-to-pale green staining, indicative of proteoglycan, was consistently seen in the areas where ADAMTS4-positive were detected. Immunostaining for versican, an was present in these same areas. The close spatial correlation of ADAMTS4 expression and versican in areas potentially vulnerable to rupture suggest that plaque destabilization may result upon degradation of versican by ADAMTS4.5. Effect of ADAMTS4 on the migration of Vascular smooth muscle cellsInvestigate ADAMTS4 effect on the migration in vitro of rat primary cultured thoracic aortic vascular smooth cells(VSMCs).Migration of VSMCs was assayed by a microchemotaxis chamber and a polycarbonate filter(Transwell's chamber)with pores of 8um in diameter. Real time polymerase chain reaction(RT-PCR)was used to detect the expression of ADAMTS4 mRNA.RT-PCR revealed that the expression of ADAMTS4 mRNA of migrating VSMCs(13.1±6.5) was significantly higher than that of resting VCMCs(4.3±1.5) (p <0.01).ADAMTS4 promotes the migration of VCMCs effectively in vitro.Our study showed that ADAMTS-4 may be involved in atherogenesis by modulating VSMC migration.
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