| Backgrounds and ObjectivesAtherosclerosis (AS) is a chronic, progressive.and inflammatory artery disease, characterized by lipid accumulation, inflammatory cell infiltration, smooth muscle cell migration and extracellular matrix degradation. The precise pathological mechanisms of AS are remained to be clarified, clinical and experimental studies in the past two decades suggest that AS is initiated by endothelial dysfunction in response to various risk factors, orchestrated by a network of inflammatory cytokines(IL-1β, TNF-a, MCP-1, IL-8, ICAM-1, VCAM-1) and matrix proteinases (MMPs, Tryptase/Chymase, Serine proteinases plasminogen activator and plasminogen, Cathepsins, ADAMTSs), involving large and medium-sized elasticity artery of circulatory system.Acute coronary syndromes (ACS) are the end manifestation of atherosclerosis, leading to unstable angina pectoris (UAP), acute myocardial infarction (AMI), and cardiac sudden death. A number of studies have shown that development of a superimposed thrombus at site of the atherosclerotic plaque, which initiate abrupt arterial stenosis or even total occlusion is the proximate event responsible for the vast majority of ACS. Detailed histological assessment of vulnerable plaques has shown several distinctive features:1) a thin fibrous cap lack of smooth muscle cells and collagen;2):a large lipid core (≥30-40%plaque volume) composed of free cholesterol crystals, cholesterol esters, oxidized LDL-cholesterol impregnated with tissue factors;3):positive remodeling;4):inflammatory cells infiltration of fibrous caps and adventitia;5):enhanced adventitial and intraplaque neovascularizationIntimal macrophages contribute importantly to atherogenesis, and their accumulation progress during plaque growth and is associated with subsequent thrombotic complications. Four main processes regulated macrophage content in atherosclerotic plaques:chemotaxis and adhesion, migration, differentiation, and apoptosis. Multiple lines of evidence indicate that up-regulation of chemokines (MCP-1, IL-8, et al) and adhesion molecules (ICAM-1, VCAM-1, et al) are keys steps in the recruitment and activation of leukocytes into atherosclerotic lesions, resulting in plaque growth and instability.The evidence for matrix proteinases, such as MMPs, Cathepsins S, K, L, Tryptase/Chymase, Serine proteinases plasminogen activator and plasminogen, during atherogenesis and plaque destabilization is overwhelming. Lately, a newly identified family of secreted metalloproteinases, a disintegrin and metalloproteinase with thrombospodin motif (ADAMTS), for which diverse substrates have been reported. However, its specific importance and underlying molecular modulation mechanisms are remained to be clarifiedSeveral reports suggest that statins may confer cardiovascular benefits in addition to their well-known cholesterol-lowering effects, at least partly by modulating the inflammatory arm of atherosclerosis, improving endothelial function, down-regulating adhesion molecules and chemokines and reducing oxi dative stress. The atheroprotective and plaque-stabilizing role of statins, which are proved to be independent of their lip-lowering effect, render them top choice in treatment of patients with both ACS and chronic stable angina (CSA). We do believe that novel mechanisms underlying the atheroprotective effects of statins will be unveiled in the future studies.Unstable angina pectoris (UAP) represents the most common clinical type of plaque destabilization. According to statistics, about12-13%UAP patients experience AMI in1year, with a mortality rate at3-18%. Therefore, intensive monitoring and treatment should be mandatory for this patient cohort expecting to reduce AMI-related mortality. To search for an safe, easy, convenient, non-invasive and effective monitor methods or biological marker is the priority for all cardiologists at the moment.S100A4is a member of S100family that was originally isolated as a gene that was expressed in mouse adenocarcinoma cells. It is confirmed that the overexpression of S100A4was correlated with the lymph node metastasis, vascular invasion, malignancy grade and TNM stage and could be serve as an independent prognostic factor. It is found on various cell types and exerts both intra-and extracellular activities. In addition to various malignant pathological conditions, current data gained from experimental and clinical studies have been implicated S100A4in the pathogenesis of some inflammatory disorders, such as rheumatoid arthritis (RA), psoriasis, inflammatory bowel disease (IBS), idiopathic inflammatory myopathy. Recently, researchers reported that heightened expression of S100A4in restenotic and atheromatous lesions of human coronary artery. Furthermore, strong immunostaining of S100A4has also been detected in advanced atherosclerosis, especially plaques with a vulnerable phenotype, suggesting a relationship of S100A4with pathogenesis of plaque instability.Given the relationship of inflammation with plaque instability, it is tempting to hypothesize that S100A4may contribute to atherogenesis, and also to the triggering of acute coronary events, by representing a novel pathway of destabilization in human atherosclerotic lesions. In the current study, this hypothesis was tested by several approaches including both clinical and experimental studies, particularly focusing on its modulation effects on matrix metalloproteinases (MMP-9, ADAMTS4), chemokines and adhesion molecules. At the same time, we also examined the ability of statins to modulate the release of S100A4in vivo and vitro. Finally, we assessed the prognostic value of serum S100A4levels on short-term cardiovascular events in patients with unstable angina pectoris.Materials and Methods1. Serum levels of S100A4in patients with coronary artery disease A total of78consecutive patients who underwent elective CAG for suspected CAD at our coronary care unit were recruited in the present study. Arterial blood samples were collected just before the procedure for S100A4, hs-CRP and ADAMTS4measurements by ELISAs. All patients studies were categorized as CAD and non-CAD group according to the presence of a significant luminal stenosis at CAG. Blood levels of the S100A4, hs-CRP and ADAMTS4were compared between the two groups, respectively. And then a multiple logistic regression analysis were performed to identify clinical factors that independently associated with the presence of coronary artery disease. In another pilot study,91patients consecutively hospitalized in our department with a diagnosis of angina pectoris were included, of which59were diagnosed with unstable angina,32were stable angina. Control subjects comprised of30age-and sex-matched healthy volunteer blood donors. Venous blood samples were obtained from all patients upon admission, and the levels of serum S100A4and hs-CRP were measured by ELISAs. Receiver operating curves were plotted to evaluate and compare the accuracy of S100A4and hs-CRP as diagnostic biomarkers to predict UAP. All CAD patients were subjected to CAG according to the Judkins techiques in48hours after hospitalization. The coronary artery lesions is regarded as the'culprit lesion'on the basis of clinical symptoms and ECG foundings. Angiographic morphology of coronary artery stenosis was evaluated in all culprit lesions on the basis of Ambrose's classification. Levels of S100A4between the two types of angiography-detected morphology of the culprit lesions (complex or simple lesions in the culprit artery) were compared in patients with SAP and UAP, respectively. Multiple logistic regression analysis was carried out to determine the independent factors associated with angiographically-detected complex lesion in both CAD and UAP patients, respectively.2. Expression and release of S100A4in atherosclerotic plaquesA total10of consecutive patients underwent carotid endarterectomy were categorized as symptomatic and asymptomatic carotid plaque group depending on whether or not they had experienced ipsilateral stoke transient ischemic attack, amaurosis fugax in the prior6months. Plaque tissues were snap-frozen at-80℃until Trizol was added. Semi-quantitive RT-PCR was performed to assess the relative levels of S100A4and CD68mRNA with GADPH as an internal standard. At the same time,6patients with SAP undergoing PCI (balloon angioplasty followed by stent placement) for a de novo lesion of a native coronary artery were included. Serial venous blood samples from SAP patients were drawn before, and at2,4,8, and24hours after PCI for S100A4measurement by ELISA.3. Inflammatory regulation of S100A4The human monocytic cell line THP-1were seeded at5×105cells/ml in6-well trays and differentiated into macrophages by incubation for72hours with160nmol/ml phorbol myristate acetate (PMA). The changes of S100A4expression before and after differentiation of THP-1cells were compared at both mRNA and protein levels by RT-PCR and Western-blot, respectively. PMBCs isolated from UAP (n=9), SAP (n=9) and health subjects (n=9) were stimulated with oxLDL (20ng/ml) or IL-1β (10ng/ml) for24hours respectively, and then the cell-free supernatants were collected to determine the levels of S100A4by ELISA. Moreover, THP-1derived macrophages and HUEVCs were incubated for24hours in the absence (control) or presence of oxLDL (20ng/ml) or IL-1β(10ng/ml) alone or in combination. and then the conditioned medium were collected for S100A4measurements as described above.4. Biological effects of S100A4THP-1macrophages were incubated with different concentrations of rhS100A4(vehicle,100ng/ml,500ng/ml,1000ng/ml and2000ng/ml), at different time points (6hours and18hours), cell-free conditioned medium were collected and store at-80℃until further analysis. For inhibition studies, THP-1macrophages were pretreated with PTDC (10umol/l) for30minutes before incubating with rhS100A4at the concentration of1000ng/ml for24hours, supernatants were harvested at24hours after stimulation. Levels of ADAMTS4in conditioned medium were examined by ELISA. HUEVCs were were stimulated with different concentrations of rhS100A4(vehicle,100ng/ml,500ng/ml,1000ng/ml and2000ng/ml)5hours or24hours, respectively. Total cellular expression of ICAM-1and VCAM-1was assessed by cellular ELISA on adherent endothelial cells fixed by1%glutaraldehyde (5hours). Levels of MCP-1and IL-8in the supernatants were measured by using ELISAs (24hours). For chemotaxis assays, Increasing concentrations of rhS100A4(vehicle,100ng/ml,500ng/ml,1000ng/ml and2000ng/ml) was added to the lower well of the chambers. THP-1cells were seeded at2×105/ml into the upper well of the chambers and allowed to migrated through the membrane for30minutes in5%CO2at37℃. Then the upper chambers were sequentially washed, fixed with4%paraformaldehyde and stained with haematoxylin, Cells that had migrated through the membrane were counted using a microscope in five randomized high-power fields, and the results were given in mean number of cells per high-power field. In separated experiments,20ug/ml anti-S100A4were added into the lower chambers before addition of rhS100A4(1000ng/ml) in the lower chamber and THP-1cells in the upper chambers, respectively.5. Effects of statins on S100A4levelsA total of36angiographically confirmed CAD patients without previous statin treatment were included in the present study. Fasting venous blood were drawed before and at1month after medication intiation for monitoring the levels of lipid and S100A4. In vitro studies, HUEVCs and THP-1macrophages were stimulated with oxLDL (20ng/ml) firstly for24hours to mimic the in vivo situation in atherosclerosis, then the media were then discarded, and HUVECs were stimulated with IL-1β(10ng/ml). for24hours with or without preincubation with ortho-hydroxy atorvastatin (10μmol/1) for2hours before addition of IL-1β(10ng/ml). Levels of S100A4were measure by ELISA as previously described. In a separated set of experiments, both HUVECs and THP-1macrophages were pretreated with atorvastatin (10μmol/l) for2hours followed by stimulating by IL-1β(10ng/ml), and the cells were harvested at6hours and24hours to elucidate the changes of S100A4expression at both mRNA and protein levels by real-time PCR and Western-blot, respectively.6. The prognostic value of serum S100A4levels in patients with unstable angina pectorisA total of66patients with UAP were followed-up at6months by telephone contact for occurrence of any of the following events:(a) cardiac death,(b) non-fatal myocardial infarction,(c) recurrence of unstable angina requiring hospitalization. Given the cohort size, the UAP patients were further divided into4sub-groups based on their baseline serum S100A4levels that fell into4quartiles (1st<481.1ng/ml,2st481.1-576.1ng/ml,3st576.21-<676ng/ml,4st>676.9ng/ml), relative risk of6-month cardiac events according to quartiles of levels of S100A4were compared across the4groups. Multiple logistic regression analysis was carried out to identify factors that were independently associated with the occurrence MACE during6months follow-up in patients with UAP.Results1. Serum levels of S100A4were significantly higher in patients with chronic stable angina (CSA) as confirmed by CAG than those without CAD (436.5±151.0vs76.6±28.1, P<0.001). So did the levels of hs-CRP and ADAMTS4. Multiple logistic regression analysis showed that blood levels of S100A4and ADAMTS4were the independent factors associated with the presence of coronary artery disease. In another study, we found that both patient subgroups, particularly those with UAP, showed significant increase in serum S100A4over the healthy control group (346.2±106.30ng/ml and565.0±128.1ng/ml in SA and UA, respectively, vs.99.0±44.7ng/ml, P<0.01for both). In patients with UAP, serum levels of S100A4were significantly and positively correlated with that of hs-CRP (r=0.473, P=0.001). Serum levels of S100A4were significantly elevated in UAP patients with complex lesion than in those with simple lesions (598.55±117.67ng/ml vs445.02±91.06ng/ml, P<0.001). While there were no significant difference in serum concentrations of S100A4between those with simple and those with complex lesions in patients with SAP (334.03±110.37ng/ml vs389.07±68.78ng/ml, P=0.3). In the multivariate analysis, serum level of S100A4appeared to be the independent factor that associated with the presence of complex stenose in both patients with CAD and UAP (CAD:OR=1.009,95%CI=1.001-1.017. P=0.031; UAP:OR=1.010,95%CI=1.001-1.019,P=0.028).2. Plaque from symptomatic patients showed siginicanlty elevated mRNA levels of S100A4as compared with that from asymptomatic patients (P=0.047). In all carotid plaques, the mRNA expression of S100A4within the atherosclerotic plauqes was significantly correlated with mRNA levels of CD68(r=0.66, P=0.038). In SAP patients, the PCI procedures induced a significant increase in serum S100A4levels within2hours that gradually returned to baseline levels after24hours.3. After differentiation, mRNA expression of S100A4was significantly up-regulated, which was also observed at protein level using Western blot. Both oxLDL (20ug/ml) and IL-1β (lOng/ml) were able to induce marked S100A4release, particularly in PBMCs isolated from patients with UAP. In THP-1macrophges, both cytokines induced significant S100A4release in the condioned medium as determined by ELISA (35.39±3.02ng/ml vs65.70±3.35ng/ml vs55.36±4.99ng/ml, P<0.001). Furthermore, the cytokines in combination produced results that appeared to be additive with respect to the effects observed when administrated alone (91.24±6.42ng/ml, P=0.002). In contrast, in HUEVCs, stimulation by either oxLDL (20ug/ml) or IL-1β(10ng/ml) was not able to change the levels of S100A4in the conditioned medium (17.70±1.39ng/ml,19.75±1.12ng/ml,19.58±0.97ng/ml, P=0.137). However, when applied in combination, a remarkable increase in S100A4concentrations could be detected (36.15±3.77ng/ml, P<0.001).4. rhS100A4was able to significantly induce the release of MMP-9in THP-1macrophages in a dose-dependent manner, with the most marked effects after incubating for24hours. Concomitantly, a similar pattern was also observed in rhS100A4induced ADAMTS4release in THP-1macrophages. In THP-1macrophages pretreated with PTDC (10umol/l), the rhS100A4(1000ng/ml) enhancing effects on ADAMTS4and MMP-9release was markedly attenuated or even nearly abolished, which suggests the involvement of NF-κB signaling. rhS100A4markedly and dose-dependently enhanced the protein levels of ICAM-1and VCAM-1in HUVECs as assessed by cellular ELISA. The inductive effects of different concentrations of rhS100A4on MCP-1and IL-8release in HUVECs were also seen, demonstrating a dose-dependent pattern as determined by ELISAs in HUVECs supernatants. In chemotaxis assays, S100A4was shown to be a potent chemotactic factor for THP-1monocytes at concentrations varying from100ng/ml to2000ng/ml. THP-1monocytes chamotaxis was inhibited in the presence of anti-S100A4in the lower chambers, suggesting the migration detected was an induced events that followed by the activation of monocytes by extracellular S100A4proteins.5. Treatment with atorvastatin markedly reduced the blood levels of S100A4in CAD patients by30days (482.98±153.63ng/ml vs396.18±125.87ng/ml, P=0.011). In vitro studies, Ortho-hydroxy atorvastatin induced a modest, but significant decrease in S100A4levels from IL-1β-stimulated THP-1macrophages, however, ortho-hydroxy atorvastatin had no effect on release of S100A4in resting THP-1macrophages. A similar pattern was also observed in HUVECs. Ortho-hydroxy atorvastatin had no effect on mRNA expression of S100A4in both THP-1macrophages and HUVECs stimulated with IL-1β. In contrast, the results from Western-blot indicated that Ortho-hydroxy atorvastatin significantly increased S100A4expression at the protein levels (P<0.05).6. All patients in the UAP group were followed up for6-months. A total of21patients reached the end-piont (2cardiac deaths,3nonfatal acute myocardial infarction, and16recurrent unstable angina). All the patients with subsequent cardiovascular events showed elevated serum S100A4at admission compared with those without (656.9.5±97.8ng/ml vs522.3±124.9ng/ml, P=0.001). Furthermore, relative risk of6-month cardiac events increase as the quartiles of levels of S100A4increased (P=0.008). Multiple logistic regression analysis showed that blood levels of were independently associated with the occurrence of MACE in UAP patients (OR=1.010,95%CI:1.003-1.020, P=0.007).Conclusions1. Locally up-regulated and inflammatory regulated S100A4may involved in the pathogenesis of atherosclerosis and plaque destabilization at least partly by contributing to promote monocytes/macrophages and endothelial cells activation in the vessel wall.2. Statins (atorvastatin) were able to inhibit the release of S100A4from macrophages and endothelial cells induced by proinflammatory cytokines in vitro, which may serve as the cellular basis of its S100A4-lowering effect in patients with CAD that was shown to be independent of its lipid-lowering properties.3. S100A4may serve as a promising biological marker predictive of short-term outcome in patients with unstable angina.4. S100A4could represent a potential target for therapy in CAD.
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