| BackgroundPercutaneous coronary intervention (PCI) has developed rapidly in China during the past30years, because the incidence of coronary artery disease(CAD),which has become the leading cause of death in China, has been increasing dramatically. According to the data from Health Statistic Yearbook of the China Ministry of Health, there were about3millions CAD patients performing PCI in2011.However, there were still too much patients who had recurrent cardiovascular events after PCI. A growing amount of clinical evidence has shown that clinical events after coronary stent deployment resulted from not only the restenosis of the target lesion, but also the progression of lesion remote from the site of stent deployment.With the introduction of drug-eluting stents, which were capable of reducing incidence of in-stent restenosis to5%～10%, targeting potentially unstable but non-target lesions has been postulated as an approach to reduce major adverse cardiac events(MACE). Although non-target-lesion events were less common than that of restenosis during the first year (12.4%versus18.3%), they dominated the events during years2through5(average annual hazard rate6.3%versus1.7%).Over the5-year follow-up, non-target-lesion events contributed46.4%to the overall events. This result was consistent with other studies of Stone and Nakachi,which suggested that most non-target lesions responsible for MACE during follow-up were angiographically mild but vulnerable at baseline.On multivariate analysis, nonculprit lesions associated with recurrent MACE were more likely to be characterized by:(1) a plaque burden of70%or greater;(2) a minimal luminal area of4.0mm2or less;(3) thin-cap fibroatheromas on the basis of intravascular ultrasonography(IVUS).And previous PCI has been one of the predictors of recurrent events.which means acceleration of atherosclerosis "natural" evolution after PCI. Clinical studies demonstrated that plasma CRP levels, regardless of pre-stent or post-stent,might be an independent predictor in evaluating the progression of non-target atherosclerotic lesion after stent implantment. The precise mechanisms underlying this process are still unknown.Vascular injury following cardiovascular intervention,including balloon angioplasty and stenting, is associated with inflammation and oxidative stress,moreover, inflammation and oxidative stress might be more significantly after stenting than that after balloon angioplasty. Stent implantation leads to mechanical injury that induces local and systemic inflammation, which stimulates macrophage infiltration,vascular smooth muscle cell proliferation and extracellular matrix deposition. Vascular inflammation after PCI involves complex interactions between multiple vascular cell types and the cellular and molecular processes that control vascular injury responses of repair and vascular healing. Aggarwal reported that increase in IL-6could be detected1hour after PCI, and thus IL-6may be an early initiator of the systemic inflammatory response to stent deployment.Oxidative stress, regardless of the source, induces cellular dysfunction in endothelial and smooth muscle cells after arterial injury.As an membrane protein, CD36belongs to the class B scavenger receptor family. It is expressed on the surface of many cells of the cardiovascular system, including platelets, monocytes,smooth and skeletal muscle cells, microvascular endothelial cells and cardiomyocytes.The CD36protein has a ’hairpin-like’ configuration, containing two transmembrane domains, one near the N-terminus and the other near the C-terminus, which are separated by a large, glycosylated extracellular loop. However, with the new knowledge of CD36ligands and the following signaling consequences, it becomes clear that the CD36protein acts in a receptor-type of manner and might be a signalling protein,which mediates signaling events involve activation of the pathways of mitogen-activated protein kinase (MAPK), nuclear factor κB (NFκB) and Rho kinase. In many cases, CD36ligandation causes a stress-like effect, enhancing defensive responses such as thrombus formation (platelets), production of inflammatory markers (endothelial cells, monocytes) and development of foam cells (macrophages)Studies confirmed that CD36is upregulated by cytokines IL-6、ox-LDL and stress hyperglycemia by signaling pathway involving protein kinase C and PPAR-y, mitogen-activated protein (MAP)kinase.However, whether inflammation and oxidative stress after stent deployment could affect the progression of non-target lesions through CD36signaling pathway is still unkown. The aim of the study was to investigate the effect of inflammation and oxidative stress after stent deployment on CD36and non-target lesions vulnerability in animal models.Methods1. Establishment animal models of the rabbit with vulnerable non-target plaques and stent deploymentTwenty male New Zealand white rabbits were randomly grouped into two groups at2th week:group A (n=15), group B (n=5). Rabbits in group B were fed cholesterol-rich diet (a normal chow supplemented with1%cholesterol), while rabbits in group A were underwent larger balloon-induced abdominal aortic wall injury and then fed the cholesterol-rich diet. At10th week, according to the abdomimal aortic angiography,11rabbits with multiple plaques in upper and lower segments of abdominal aorta were selected and randomly divided into two subgroups:group A1was performed stent deployment according to clinical methods (n=6), A2was still fed the cholesterol-rich diet (n=5). One rabbit in group A1was dead before stenting due to anesthetic overdose.At the end of14th weeks, the rabbits in both group A, B were killed.2. Examining the features of vulnerable plaques:The rabbits’ atheroslcerosis plaques in abdominal aorta were detected with intravascular ultrasound (IVUS). Plaque characteristic, plaque area and plaque distribution were examinated. The rabbits in group A1were taken the examination pre-and post-stenting at10th week, before being killed at14th week,while others were underwent the examination before being killed at14th week.3. Laboratory examinations:Blood samples were collected in different period to measure lipid profile and glucose. Using sandwich ELISA to quantify the amount of different inflammation mediators such as hs-CRP, IL-6,oxLDL and CD36.4. Histologic evaluation:The upper abdominal aorta was excised and examined by immunohistochemical stain of RAM-11(anti-rabbit macraphage), MMP-9, CD36, IL-6and TNF-α in local lesions,respectively.5. Western-blot:To examine the protein expression of MMP-9, CD36, IL-6and TNF-α in the upper abdominal aorta.6. Statistic analysis:Continuous variables are reported as mean±deviazione standard (SD) and have been compared using t test and ANOVA analysis, categorical variables by x2test. The relationship between variables was estimated by spearman analysis. For all analysis, a p value less than0.05was considered significant, using a statistical programme (spss version17.0).Results1. Compared with group B and group A2, cytokines IL-6、elevated oxidized LDL and blood glucose could be detected one hour after the rabbits abdominal aortic stenting. IL-6levels reached an earlier peak at24hours postprocedure, and then declined gradually at7days and4weeks, but still higher than that in group B and group A2; Hyperglycemia reached an earlier peak at24hours postprocedure, and then returned to baseline at7days, with no differences compared with group B and group A2; OxLDL level gradually increased, continuing to4weeks after stenting, and was significantly higher than that in group B and group A2(P<0.05)2. Intravascular ultrasound:External elastic membrance area (EEMA), plaque area (PA) and plaque burden of upper abdominal arterial lesions in group A1were significantly higher than those of group A2and group B (P<0.05).3. Histologic evaluation:Immunohistochemistry showed that the expression of RAM-11(anti-rabbit macraphage), MMP-9, IL-6, TNF-α, CD36in group A1were significantly higher than that in group A2and group B.4. Western blot:The expression of MMP-9, IL-6, TNF-a and CD36in group A1were significantly higher than that in group A2and group B (P<0.05).5. Serum CD36began to increase after24hours, and CD36level was positively correlated with elevated levels of inflammation and oxidative stress at24hours after stenting; CD36continued increasing until the rabbits were sacrificed (4weeks after stenting) and CD36levels and plaque burden before death was significantly positively correlated.Conclusions1. This study successfully constructed AS animal models stented with non-target lesions for further research.2. Inflammation and oxidative stress was more significant after stent deployment than that after balloon angioplasty.3. The results of our study demonstrated that the evolution of vulnerable non-target lesions acceleratd after PCI,which was in consistent with clinical studies.3. Preliminary results of this study found that inflammation, oxidative stress and CD36expression upregulating mediated non-target lesion plaque vulnerability and progression. BackgroundAtherosclerosis is a chronic inflammatory disease in the artery wall leading to the development and progression of atherosclerotic lesions, which may obstruct the arterial lumen and/or eventually rupture and thrombose, causing acute coronary syndrome (ACS) of unstable angina, myocardial infarction, and sudden death. The risk of plaque disruption depends more on plaque composition and vulnerability (plaque type) than on degree of stenosis (plaque size). Thus, coronary occlusion and myocardial infarction most frequently evolve from mild to moderate stenoses (<70%stenosis at baseline).CD36, is a multi-ligand class B scavenger receptor expressed by monocytes/macrophages. It is a key player in atherosclerosis development through oxLDL binding and internalization, thereby leading to macrophage-derived foam cell formation and accumulation in atherosclerotic lesions. Stimulation by oxLDL induces the activation of the transcription factor NF-kB in macrophages through a mechanism that is dependent on CD36and PKC.Fractalkine (CX3CL1) is the only known member of the CX3C chemokine subfamily, and is expressed as both a soluble protein and as a membrane-bound protein on the surface of inflamed endothelium. Fractalkine has been associated with atherogenesis, and two specific mutations of the fractalkine receptor (CX3CR1) gene reduce the risk of future coronary events. A previous study comparing patients with unstable angina pectoris (UAP) and those with stable angina pectoris (SAP) showed that fractalkine independently enhanced the vulnerability of coronary atherosclerotic plaques.Oxidized LDL also regulates monocyte trafficking by upregulating chemokines such as CCL2within the intima and/or endothelial lining of arteries. A reduced chemotactic response of CD36-deficient macrophages towards the CCL2chemokine has been observed in vitro. This suggests that crosstalk between CD36and chemokines regulates mononuclear phagocyte trafficking to atherosclerotic lesions and influences plaque progression. However, few additional studies have looked more closely at the interaction of CD36and chemokines. The combined effect of CD36and chemokines at different stages of atherogenesis is still unknown.This study assessed atherosclerotic lesions in patients with UAP by intravascular ultrasound (IVUS) and then compared serum CD36and fractalkine levels in patients with intermediate and severe lesions. The objective was to determine if there are correlations between CD36and fractalkine levels, and the severity of coronary artery atherosclerosis assessed by IVUS.Methods and resultsStudy populationOne hundred and twenty patients admitted for confirmed UAP and selective coronary angiography (CAG) were enrolled. A total of83men and37women, with a mean age of63.3±9.6years and range of49to82years, were divided into two groups according to the CAG results. Group A comprised80patients with intermediate lesions (lumen diameter stenosis50-70%) and group B included40patients with severe lesions (at least one lesion having lumen diameter stenosis>70%). The control group consisted of40healthy, age-and sex-matched subjects who visited the hospital for a routine physical check-up Blood Biomarker MeasurementsBlood samples were collected from every patient and control on admission to the hospital. Blood was collected by venipuncture into two foil-wrapped tubes containing5mL EDTA. The tubes were centrifuged for10minutes at3000rpm at4℃. Sera were transferred to2mL cryovials and stored at-80℃. Serum levels of total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), fasting glucose, and troponin I were measured by enzymatic assays. Leucocytes counts were carried out. The levels of high-sensitivity C-reactive protein level (hs-CRP) were measured by an immunonephelometric assay on a BN ProSpec nephelometer (Dade Behring, Siemens Healthcare Diagnostics, Germany). Serum CD36and fractalkine levels were measured by a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer’s protocol.Coronary AngiographyAll patients underwent CAG. The CAG images were analyzed using a Philips3000system. A coronary stenosis was defined as more than a50%decrease in vessel diameter compared with an adjacent normal segment. The number of stenotic lesions in each coronary artery was recorded.Intravascular Ultrasound StudiesThree coronary IVUS assessments were carried out in all patients, as described previously. The following parameters were measured:external elastic membrane area (EEMA), lumen area (LA), plaque area (PA), plaque burden (PB), plaque eccentricity index (El), and remodeling index (RI). RI>1.05was regarded as positive remodeling,0.95-1.05as intermediate remodeling, and<0.95as negative remodeling. Coronary plaque composition was assessed visually according to plaque echogenicity. Two independent observers reviewed the IVUS images, and the final consensus values were used for data analysis. End PointsThe primary end points targeted major adverse cardiovascular events (MACE) that occurred in the two years after admission, including non-fatal myocardial re-infarction, severe angina pectoris (Canadian Cardiovascular Society, CCS grades≥Ⅲ), revascularization, and death. Follow-up was performed by office visits and/or telephone interviews.Statistical AnalysisAll statistical analyses were performed with the SPSS software package (version16.0; SPSS Inc., Chicago, IL, USA), and data are presented as mean±SEM. Comparison of continuous variables among multiple groups was performed by analysis of variance (ANOVA), and Chi-square analysis was used to compare categorical data. The correlations between two variables were assessed by Pearson or Spearman correlation analysis. Kaplan-Meier curves were calculated for the cumulative incidence of the primary end points in the two groups. The hazard ratios of end-point incidence were also calculated. A two-tailed P<0.05was considered statistically significant.ResultsBaseline Characteristics and Blood Biomarker MeasurementsThere were no significant differences in age or gender among three groups. More patients with UAP had a history of hypertension, increased levels of LDL-C, troponin I, leucocytes, and hsCRP compared with controls (P<0.05). There were no significant differences in any of those parameters between group A and B. Serum CD36and fractalkine levels were significantly higher in UAP patients than controls. The differences in CD36and fractalkine levels in the two patient groups were also significant (P<0.05).CAG and IVUS MeasurementsThe CAG studies identified a total of97coronary stenotic lesions with a lumen diameter stenosis of50-70%. Among these group A patients,80%had one-vessel coronary artery disease and18.75%had two-vessel disease. The remaining (group B) patients had a total of96coronary lesions. Of those lesions,5.2%had total occlusion and85.4%had a lumen diameter stenosis>70%. Each group B patient had at least one lesion with a lumen diameter stenosis>70%;10%had one-vessel disease,50%had two-vessel disease and40%had three-vessel disease.IVUS found that most atherosclerotic plaques in both groups were eccentric plaques with positive remodeling. Patients in the intermediate lesion group had softer lipid plaques (73%vs.48%, P<0.01), while patients in the severe lesion group had more calcified plaques and mixed plaques (P<0.05). Patients with severe lesions also had larger PA (P<0.01) and PB (P<0.05) than patients with intermediate lesions (Table2, Figure1). Spearman correlation analysis showed that both serum CD36(r=0.4068,95%CI:0.24-0.55; P<0.01) and fractalkine (r=0.43,95%CI:0.27-0.57; P<0.01) levels had a significant positive correlation with the PB of atherosclerotic lesions as measured by IVUS. Importantly, there was also a significant positive correlation between CD36and fractalkine levels (r=0.183,95%Cl:-0.001-0.355; P=0.046)(Figure2).More patients with severe lesions than with intermediate lesions underwent percutaneous coronary intervention (PCI)(95%vs.73.75%, P<0.05), and also received more stents per patient (2.3stents vs.1.3stents, P<0.05) in the severe lesion group. However, there were no significant differences in the diameter and length of stents that were placed in patients in the two groups (Table2).End Point EventsDuring the two years of follow-up, primary end point events occurred in six patients (7.5%) in the intermediate lesions group and eight patients (20%) in the severe lesions group (P<0.05). A lower incidence of MACE was observed in the intermediate lesions group (HR:0.3118,95%CI:0.1013-0.9601; P=0.0424), mainly because of a lower risk of severe angina pectoris (6.25%vs.17.5%, P<0.05). There were no significant differences in risk of death, non-fatal myocardial infarction (MI), or revascularization between two groups (P>0.05). Three patients who returned for a clinically-driven re-PCI received a second CAG. The results showed two patients had plaque rupture, thrombosis of non-target vessels, and one experienced progression of non-target plaques.ConclusionsIn conclusion, CD36and fractalkine both promote, and might synergistically enhance, the progression of coronary atherosclerotic plaques. This study also showed that a higher incidence of MACE was observed in the severe lesion group during two years of follow-up. |
PDF Full Text Request