| BackgroundAcute coronary syndrome (ACS) is a group of myocardium ischemia charactized syndrome caused by atherosclerotic coronary artery plaque rupture, hemorrhage and local thrombosis, or vasospasm, with subsequent total or subtotal occlusion of coronary arteries. ACS is a common emergency and warrants urgent, proper and effective treatment once diagnosed to minimize the harmful effects of myocardial ischemia and areas of myocardial infarction and save patients’ lives. The risks of recurrent angina, acute cardiac infarction and cardiac death during hospitalization and follow-up are relatively high, and how to improve the prognosis of recurrent cardiac events remains as a great challenge for the clinical physicians.The Global Registry of Acute Coronary Events (GRACE) is a widely used scoring system evaluating risks of ACS. The GRACE research is the first prospective study of ACS patients worldwide, with22645subjects from about100hospitals of14countries enrolled. The inclusion criteria were all types of ACS. The variables required for calculation of GRACE score include age, heart rate, systolic blood pressure, baseline creatinine level, history of congestive heart failure, in-hospital percutaneous coronary intervention, history of MI, ST-segment depression on admission electrocardiography (ECG) and elevated cardiac enzyme or marker levels. The variables for GRACE score calculation were based on risk factors from large scale objective clinical trials and data of the variables are easy to obtain, which make GRACE score rational and effective.Atherosclerosis, a multifocal lipidstatic disease characterized by lipid accumulation and inflammation in aortas and median arteries, is the most common known cause of cardiovascular diseases. Pathological studies showed that atherosclerotic plaques are filled with inflammatory cells, which alter morphology and characters of the plaques. However, atherosclerosis is more than a localized inflammatory disease. Various clinical trials claimed that secretary proinflammatory biochemical markers, such as IL-6, MPO, MCP-1and so on, are effective for cardiovascular events prediction and prognosis evaluation. C Reactive Protein (CRP) is one of the most widely studied and convincible clinical biochemical markers. Resent researches clarified that the Wnt signaling family plays an important role in inflammation modulation. Several secretory antagonists were confirmed to suppress the Wnt pathway, including FIZZLE and dickkopf family, and the mostly studied of the latter is DKK-1. Serous DKK-1level was showed effective to predict prognosis of osteoarthritis and some malignant cancers such as multiple myeloma. DKK-1was found upregulated in atherosclerotic plaques and playing a proinflammatory role to promote atherosclerosis and plaque vulnerability.Objectives1. To investigate the expression of DKK-1in ACS patients;2. To investigate the relevance between DKK-1and CRP or GRACE score;3. To investigate the role of peripheral concentration of DKK-1in predicting recurrent cardiovascular events of ACS patients and the feasibility of taking DKK-1as one supplementary variable for GRACE score predicting long-term adverse cardiac events.Methods1. Study populationWe included consecutive patients hospitalized in the Department of Cardiology of Qilu Hospital, Shandong University, from March2008to January2010. Inclusion criteria were diagnosis of ACS, including ST-segment elevation myocardial infarction (STEMI) and non-ST elevated ACS (NSTE-ACS); all patients underwent coronary angiography. The diagnosis of STEMI was typical chest pain with serum cardiac enzyme levels twice that of the upper level of normal or cardiac troponin I (cTnl) level≥0.1ng/ml, both with persistent electrocardiographic ST segment elevation>1mm in2or more contiguous leads or newly occurred left bundle branch block. NSTE-ACS included non-STEMI (NSTEMI) and unstable angina (UA). The diagnosis of NSTEMI was angina or discomfort at rest with ST segment depression or transient elevation and/or prominent T-wave inversion, with cardiac enzyme levels twice that of the upper level of normal or cTnI≥0.1ng/ml. Patients with clinical features and/or electrocardiographic expression of NSTEMI but normal cardiac biomarker levels were diagnosed as having UA. Exclusion criteria were valvular heart disease, severe arrhythmias, severe heart failure, severe or refractory hypertension, active hepatosis, malignant diseases, anemia and acute or chronic inflammatory diseases.2. Coronary angiogram (CAG) and percutaneous conronary intervention (PCI)The patients enrolled in the study all received coronary angiography (CAG) examination. Imaging of coronary arteries was taken and target arteries were diagnosed. Type, location, morphology and degree of stenosis of the target coronary artery lesions were recorded.3. Laboratory analysisAll laboratory data, including total cholesterol (TC), triglycerides (TG), high-density lipoprotein-cholesterol (HDL-C), low-density lipoprotein-cholesterol (LDL-C), blood glucose, uric acid level, creatinine level, creatinine kinase activity, and cTnl and hs-CRP levels were measured in the biochemical department of Qilu Hospital.4. Measurement of DKK-1in serumBlood samples of5ml each were collected in EDTA-containing tubes and then centrifuged at4℃.The collected plasma was stored in aliquots at-80℃. DKK-1concentration was measured by use of an ELISA kit (R&D Systems, Minneapolis, USA).5. Calculation of GRACE risk scoresThe variables required for calculation of the score include age, heart rate, systolic blood pressure, baseline creatinine level, history of congestive heart failure, in-hospital percutaneous coronary intervention, history of MI, ST-segment depression on admission electrocardiography (ECG) and elevated cardiac enzyme or marker levels. The risk categories of GRACE score were divided into low, medium and high.6. Hospital and post-discharge medicationsNitrates, beta receptor inhibitors, angiotensin enzyme inhibitors (ACEI), angiotensin II receptor blockers (ARB), calcium channel blockers and vasodilators were applied according to the pharmacopeia.7. Follow-up Endpoints after discharge were MACEs, including sudden cardiac death, MI, percutaneous coronary intervention, coronary artery bypass grafting and recurrent unstable angina pectoris. Patients were followed up by researchers from Qilu Hospital, Shandong University after discharge. Follow-up examinations included fasting blood glucose, TC, TG, LDL-C, HDL-C and resting electrocardiogram.8. Statistical analysisAll data were analyzed by use of SPSS v16.0(SPSS Inc., Chicago, IL, USA). Numeric variables are expressed as mean±SD. Categorical variables are expressed as frequencies and percentages. Kolmogorov-Smirnov test was used to assess normal distribution of quantitative variables, with log transformation for non-normal distribution. Categorical data were compared by chi-square test or Fisher’s exact test as appropriate. Bivariate correlation was used for correlation analysis. One-way ANOVA was used for comparison of multiple groups. Binary logistic regression was used to assess the independent association of DKK-1level with MACE. Differences in the predictive values were estimated by comparing the area under the receiver-operating characteristic curve (ROC). The level of statistical significance was set at P<0.05.Results1. Baseline characteristics of study subjectsA total of331patients with ACS met the inclusion criteria, and we had complete data for322. At the end of the study, data for291patients (193males,66.3%) with complete follow-up data were analyzed, including46with STEMI and245with NSTE-ACS (63with NSTEMI,182with UA), and68%of our patients underwent percutaneous coronary intervention. CAG was performed for all the subjects and among which198patients (68%) were qualified and treated with PCI. During the follow-up,44MACEs took place.2. Plasma levels of DKK-1in patients with ACSMedian plasma DKK-1level was713pg/ml (range129-2139pg/ml). DKK-1levels were correlated with hs-CRP level(r=0.308, P<0.001). DKK-1was significantly higher in patients with STEMI than those with NSTE-ACS at baseline. DKK-1level did not differ between patients with NSTEMI and those with UA. Treatment with PCI did not alter the DKK-1level in patients.3. Risk stratification by GRACE scoreThe median GRACE risk score was88(range38-149) for the whole population, 108(63-149) for patients with STEMI,84(38-148) for patients with NSTE-ACS. The GRACE scores were significantly higher for patients with STEMI than NSTE-ACS at baseline.4. Clinical index among groups divided by concentrations of DKK-1in plasmaThe patients were divided into three groups (T1, T2and T3) according to the concentrations of DKK-1, with97patients in each group. The patients in T3were much older and the levels of blood sugar and hs-CRP were higher than the other two groups (P<0.05). Till the end of follow-up, the rate of MACEs was2.1%in T1,13.4%in T2and29.9%in T3. There was significant difference in the rate of MACEs among the three groups (P<0.05).5. Clinical index between event and non-event groupsThe levels of TC, TG, LDL-C, BS, hs-CRP and DKK-1are significantly higer in event group than in non-event group (P<0.05); the level of HDL-C decreased in the event group (P<0.05). The rates of hypertension and diabetes were higher in the event group than in non-event group (P<0.05). There was no difference in the GRACE score between the two groups (P>0.05).6. The relationship of plasma levels of DKK-1and GRACE scoresDKK-1levels were correlated with GRACE scores (r=0.259, P<0.05). The medium concentrations of DKK-1were642,718and959pg/ml for low, intermediate and high GRACE category, respectively. The concentrations of DKK-1were elevated with high-risk than intermediate-or low-risk GRACE score (P=0.002and P<0.001). DKK-1levels were higher but not significantly with intermediate than low risk (P=0.100).7. DKK-1is an independent predictor of long-term MACE for patients with ACSAfter adjustment for cardiovascular risk factors (age, sex, BMI, smoking, blood glucose and blood lipids), binary logistic regression revealed a significant association of DKK-1and hs-CRP levels and MACE for ACS patients.8. DKK-1has Better prognostic value for patients with ACSReceiver-operating characteristic curve (ROC) analysis showed that the GRACE score alone was a poor predictor of MACE, the area under the ROC (AUC) was0.524(P=0.605). With the addition of DKK-1level, the AUC was increased to0.775(P<0.001) and to0.791with hs-CRP level (P<0.001). With both biomarkers added, the AUC was significantly increased to0.847(P<0.001), and the sensitivity of this model in evaluating prognosis was81.8%, with specificity71.7%. Conclusions1. Traditional CHD risk factors age, hypertension and diabetes are predictive for long-term MACE for patients with ACS.2. After adjustment for cardiovascular risk factors (age, sex, BMI, smoking, blood glucose and blood lipids), peripheral plasma DKK-1and hs-CRP levels are independent predictors of long-term MACE for patients with ACS.3. The long-term predictive ability of post-discharge GRACE score combined with hs-CRP may be enhanced by adding DKK-1level. BackgroundThe complete or part occlusion of coronary artery, generated by sudden rupture of atherosclerotic plaque and the following thrombosis, is the main cause of acute coronary syndrome (ACS). Atherosclerosis is the dominant pathological etiology of ACS. Canonical pathological research revealed that amount of inflammatory cells and cytokines existed in the atherosclerotic plaque, the activities of these factors correlated closely with stability of the plaque. The theory of inflammation provided us with a deeper recognition of atherogenesis. However, the complexity of pathogenesis and diversity of factors involved in atherosclerosis signify the difficulty in totally elucidating the role of inflammation in atherogenesis. The researches of relationship between inflammatory factors and vascular cells are still carried on all over the world.Wnt pathway is a conserved signaing transduction pathway. It participates in many aspects during embryonic development and plays a great role in maintaining the dynamic balance in adult tissue. The role of wnt pathway in cardiovascular system has become more and more important during recent years, it takes part in adipogenesis, angiogenesis, vascular calcification, myocardial infarction and ischemic adaption. In1998, researchers found that a secretory glycoprotein, DKK-1, can block wnt pathway by competitively bind to the receptors (LRP5/6, Kremen) on the cellular member. DKK-1takes part in the formation of cardiovascular system including endocardium, myocardial cells and cardiac valve, etc. Ueland et al. found that DKK-1participated in the activation of endothelial cells deduced by platelet. DKK-1derived from endothelial cells and platelet can enhance the inflammation between them, indicating that DKK-1promotes the inflammatory reaction in atherosclerotic plaque and DKK-1is an atherogenic factor. In the clinical study of ACS patients, we found that the expression of DKK-1in plasma was not only correlated with the severity of disease, but also predicted the prognosis of disease. The expression of DKK-1implies its ability in reflecting the severity and stability of coronary atherosclerosis.There is no report about the role and mechanism of DKK-1in atherogenesis. According to the results discovered in the clinical research, we tried to study the role of DKK-1in atherosclerotic animal model. The pathological changes of atherosclerosis in mice carotid are similar to those changes in human, including stenos is, formation of fibrous cap and plaque rupture, etc. By placement of a constrictive perivascular collar around the common carotid artery in ApoE-/-mice and high fat diet for about8weeks, von der Thusen et al. successfully induced vulnerable plaque similar to human lesion in the proximal part of the collar. In the animal study, we used this model to implement the formation of atherosclerotic plaque, and interfered the expression of DKK-1by RNAi mediated with lentiviral vector. Histopathological and immunohistochemical measurement were used to observe the formation and stability of atherosclerotic plaque. The research about mechanism of DKK-1in the study of atherosclerosis might provide a new target for the prevention and treatment of atherosclerosis.Objectives1. To study the effects of DKK-1interference on the development and stability of atherosclerotic plaque in ApoE-/-mice;2. To study the mechanisms of DKK-1interference on stability of atherosclerotic plaque;3. To observe the expression of DKK-1in HUVECs under stimulation of ox-LDL in vitro;4. To study the influence and mechanism about DKK-1on functions of HUVECs under stimulation of ox-LDL.Methods1. Construction of lentiviral vectorThe DKKli lentiviruses (KD sites:1,2,3) were transfected into RAW264.7cells. Three days after the transfection, cells were collected for real-time PCR analysis to select the most effective targeting site for the DKK-1gene to amplify. The siRNA NS vector containing lentivirus only was used as a control. Finally, two lentiviral vectors were produced:Lenti-DKKli and Lenti-GFP.2. Atherosclerosis animal experimental protocol80ApoE-/-mice (male,8-week old) were fed on an atherogenic chow after two weeks’adaptive feed. Atherosclerotic plaques were induced by placement of a perivascular collar around the right common carotid artery. All mice were randomly divided into3groups8weeks after the placement of collar:NS group (n=27) injected with normal saline; Lenti-GFP group (n=27) transfected with Lenti-GFP; Lenti-DKKli group (n=26) transfected with Lenti-DKKli. A total suspension of200ul was injected into the mice through tail vein. Four mice from NS and Lenti-GFP group were selected at week12for detection of lentiviral transfection efficiency. All mice were euthanized at the end of week14.3. Measurement of serum lipid and blood sugarBlood samples were collected and centrifuged. The serum levels of total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C) and blood sugar were measured.4. Histopathological and immunohistochemical measurementThe mice were perfused through the left ventricle with saline, followed by4%formaldehyde. The whole aorta, aortic root and right common carotid artery were excised and immersed in4%formaldehyde overnight at4°C. The vessel tissue was rinsed before embedded in OCT, and stored at-40℃until use. OCT-embedded aortic root and carotid artery were cross-sectioned into slices about5μm thick. The whole aorta was stained with oil red O. Sections were stained with hematoxylin and eosin for plaque morphology, and also stained with oil red O for lipids and picrosirius red for collagen. Smooth muscle cells, macrophages, inflammatory cytokines (MCP-1, IL-6, IL-1β, TNF-a), adhesive factors (ICAM-1, VCAM-1), MMP-2, MMP-9, P4Hal and RIP3were immuno-stained with corresponding antibodies. TUNEL test was used to detect cellular apoptosis in the plaque. Vulnerability index was calculated with the following formula:(macrophage staining%+lipid staining%)/(SMC staining%+collagen staining%). The content of each index in the plaque was quantified with the Image-Pro Plus6.0software.5. Molecular biological analysisThe expressions of DKK-1in each group were detected by Western Blot with specific antibodies.6. The role of DKK-1in HUVECsHUVECs were stimulated by ox-LDL. In order to elucidate the role of DKK-1in regulating oxidative stress, secretion of adhesive factors and apoptosis of HUVECs under stimulation of ox-LDL, siRNA for DKK-1and recombinant DKK-1were used according to the procedure.7. Statistical analysisQuantitative values are expressed as mean±SEM. And the data were analyzed by independent sample t-test or one-way ANOVA as appropriate. The level of P<0.05was considered significant.Results1. Screening the most effective targeting sites for DKK-1genes silencing by real-time PCRRAW264.7cell line was transfected with lentivirus-based vectors expressing three different DKK-1siRNAs and gene silencing analysis showed that the DKKli-KD1lentivirus was the most effective vector in blocking DKK-1expression. The DKK1-RNAi KD1, KD2, and KD3exhibited72.1%,68.1and36.5%reduction, respectively. The terminal virus titer was5x108IFU/mL.2. General condition of the miceThere were no significant differences in body weight, levels of blood sugar and serum lipids among the three groups at the end of experiment.3. The efficiency of lentivirus transfectionSignificant amount of GFP was expressed in carotid plaques2weeks after the injection of lentivirus. Western blot analysis of DKK-1expression in tissue protein from carotid artery exhibited that level of DKK-1in Lenti-DKKli group was significantly down-regulated.4. The influence of DKK-1interference on plaque burden of ApoE-/-miceThe gross oil red O staining revealed that atherosclerotic area of the whole aorta in Lenti-DKKli group significantly decreased (P<0.05). H&E staining revealed that cross-section of the aortic root was also reduced in the Lenti-DKKli group (P<0.05).5. Vulnerability index in different groupsSpecific staining revealed that the content of macrophage in carotid plaque in the Lenti-DKKli group significantly decreased compared with NS and Lenti-GFP groups (P<0.05), yet the contents of smooth muscle cells and collagen fibers increased (P<0.05). Though the content of lipids in the interference group slightly reduced, no significant differences existed among the three groups (P>0.05). Vulnerability index in the Lenti-DKKli group significantly diminished compared with the other two groups (P<0.05). The thickness of fibrous cap increased in the interference group (P<0.05).6. The effect of DKK-1interference on expression of inflammatory factors and matrix metalloproteinase in carotid plaquesThe content of inflammatory factors (MCP-1, IL-6, IL-1β, ICAM-1and VCAM-1) in Lenti-DKKli group decreased compared with NS and Lenti-GFP groups (P<0.05). The content of MMP-2and MMP-9decreased in the Lenti-DKKi group, yet the content of P4Hα1increased compared with NS and Lenti-GFP groups (P<0.05).7. The effect of DKK-1interference on cellular apoptosis in carotid plaquesThe content of apoptotic factor RIP3decreased in Lenti-DKKli group compared with NS and Lenti-GFP groups (P<0.05). The percentage of TUNEL positive cells in Lenti-DKKli group also decreased compared with NS and Lenti-GFP groups (P<0.05). Western Blot results showed the Lenti-DKKli group had less cleaved caspase-3expression in the carotid plaque tissue (P<0.05).8. The roles of DKK-1in HUVECs stimulated by ox-LDLThe expression of DKK-1in HUVECs was elevated after stimulation of ox-LDL. In the ox-LDL+siRNA group, the expression of adhesive factors (ICAM-1, VCAM-1) and apoptotic factor cleave caspase-3declined, yet the level of p-eNOS increased compared with the ox-LDL group and ox-LDL+NC group(P<0.05). The changes of the above factors weren’t different between recombinate DKK-1group and ox-LDL group. Though the levels of adhesive and apoptotic facters declined and p-eNOS increased in the ox-LDL+siRNA group, they were still higher or lower than those in the empty group (P<0.05). Gain and loss of function tests showed DKK-1participated in the stimulation of HUVECs by ox-LDL.Conclusions1. DKK-1interference can effectively decrease the formation of atherosclerotic plaque in ApoE-/-mice;2. By decreasing the content of macrophage and increasing the content of smooth muscle cells and collagen, DKK-1interference can effectively improve the stability of atherosclerotic plaque; 3. DKK-1interference can decrease the extent of inflammation and apoptosis in the atherosclerotic plaque in ApoE-/-mice;4. DKK-1participates in the influences of expression of adhesive factors, eNOS and apoptosis of HUVECs under stimulation of ox-LDL. |
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