The Role Of Chemokine Receptor5in Myocardial Ischemia-reperfusion Injury

Coronary heart disease (CHD) is a common and high prevalent medical disorder. Acute myocardial infarction (AMI), as the most severe complication of CHD, is not only a common emergency in intensive care unit (ICS), but also has a high mortality rate. According to a recent survey, China currently has nearly40million patients with coronary heart disease, of which there are nearly100million people a year died from AMI or CHD-related complications. The main mechanism for the pathogenesis of CHD is myocardial ischemia. Different degrees of damage would occur on myocardial structure and function when myocardial oxygen provision is in deficient. Therefore, the most effective treatment is to improve coronary blood supply and restore myocardial blood reperfusion. Treatments used include thrombolytic therapy, surgical bypass surgery, and cardiac catheterization interventional procedures, in order to save the "dying" myocardium, reduce myocardial infarct size, improve patient prognosis. However, some studies found that the ischemia induced cardiac injury represented by myocardial metabolism, cardiac function, myocardial structure and electrophysiological injury, becomes aggravated after recanalization. This is called myocardial ischemia-reperfusion injury. The clinical manifestations include malignant arrhythmia, shock, heart failure, and even sudden death etc during a period time after ischemia-reperfusion. Therefore, the main focus on CHD and AMI treatment is to restore and improve the myocardial blood supply to the ischemia area but minimize myocardial ischemia-reperfusion injury as much as possible at the same time.Many theories have been proposed to be responsible for myocardial ischemia-reperfusion injury:oxygen free radical, calcium overload, apoptosis etc. Recent studies have shown that inflammation has played a more and more important role in the occurrence and development of CHD. It has been reported that the size of myocardial necrosis, the damage extent of myocardial function are high associated with the level of various inflammatory factors. The critical role of inflammation regulation factors and inflammatory responses in ischemia-reperfusion injury becomes more and more being recognized. Chemotactic factors (chemokines) is a group of small molecules which have various biological functions other than chemotactic effect on leukocytes. It mainly achieves its functions by binding to its receptors on specific cell surface. Ischemia-reperfusion injury is believed to be a stress reaction caused by inflammation. The whole process is characterized by accumulation of inflammatory cells in ischemic area and adhesion and migration of inflammatory cells through vascular walls via the binding of chemokines and their receptors. Therefore, the myocardial protection and prevention of ischemia-reperfusion injury can be achieved through the intervention on the binding of chemotactic factors and their receptors.Objective:In this study, we set up an in vivo rat model of myocardial ischemia-reperfusion. With this model, we have observed pathophysiological changes associated with ischemia reperfusion injury after AMI. Chemokine receptor5(CCR5) antibody and CCR5ligand RANTES were used to pre-treat the rat model to study their effects on myocardial structure, function and cardiac hemodynamics. This study would provide a new strategy and ideology to prevent myocardial ischemia-reperfusion injury via inhibition of inflammatory reaction. The study is divided into two parts:1) Set up ischemia preconditioning rat model and myocardial ischemia-reperfusion rat model, examine cardiac hemodynamics, myocardial tissue structure, markers of myocardial injury as well as CCR5changes in those models, and determine whether there was an increase of CCR5in ischemia-reperfusion rat.2) Examine the cardiac hemodynamics, myocardial tissue structure, markers of myocardial injury, inflammatory factors (TNF-a, NF-κB, ICAM-1) as well as CCR5changes after the interventions with CCR5antibody or CCR5receptor antagonist and determine whether CCR5antibody can protect heart function and prevent ischemia-reperfusion injury via suppression of chemokines expression and inflammatory cytokines.Methods:Part1:Ischemia-reperfusion model was achieved via three cycles of occlussion and reperfusion. In the first cycle, left anterior descending artery was ligated for5min and then released for5min and repeat; the second cycle, the ligation lasted10min and release for10min; the third cycle, ligation lasted30min followed by2hours reperfusion.36male Wistar rats were randomly divided into three groups respectively: sham group (12), ischemia-reperfusion group (12), ischemic preconditioning group (12). Multi-channel physiological recording system was used to observe rat heart hemodynamic parameters:left ventricular systolic pressure (LVSP); left ventricular end-diastolic pressure (LVEDP); heart rate (HR),+dP/dtmax and-dP/dtmax during the2h of reperfusion. Blood samples collection:In the end of reperfusion,4ml of blood was drawn from the carotid artery. After standing for30minutes and centrifugation for10min, the upper layer of serum was collected and stored in the-70℃refrigerator for the detection of CK, AST, LDH. Myocardial specimen collection:In the end of the experiment the heart was isolated. The left atrium and right atrium as removed and the blood was washed away with ice saline rinse. It was then dried with wipes and fixed with4%paraformaldehyde, dehydrated with ethanol gradient dehydration, embedded with paraffin, use hematoxylin-eosin (HE) staining myocardial tissue pathological structures. Another part is rinsed with0.9%ice cold sodium chloride solution and cryopreserved for malondialdehyde (MDA), superoxide dismutase (SOD) determination, as well as to use WesternBlot Determination of chemokine receptor5(CCR5) protein expressionPart2:60male Wistar rats were randomly divided into five groups, sham-operated group (SHAM,12), ischemia-reperfusion group (I/R,12), ischemic preconditioning group (I/R+Pre,12), the CCR5antibody group (I/R+CCR5Ab,12), of CCR5agonist group (I/R+CCR5Ago,12). CCR5antibody group before reperfusion from the external jugular vein injection CCR5antibody and agonist group were injected with RANTES for processing.Left ventricular systolic pressure (LVSP); left ventricular end-diastolic pressure (LVEDP); heart rate (HR),+dP/dtmax and-dP/dtmax) were measured through a short segment of saline-filled PE50tubing which was advanced to left ventricle through right carotid artery and connected to a multi-channel physiological monitoring system (LEAD2000, Sichuan, China) before LAD ligation or sham operation. Hemodynamic parameters were obtained immediately after2hours reperfusion. At the end of reperfusion, the LAD was ligated and normal myocardial tissue was marked with1%Evans blue but ischemic myocardial tissue was marked with1%TTC. Myocardial ischemic area (AR), left ventricular wall (LV) and infarcted myocardium (IS) were weighed with the help of staining and MPO activities in different areas were also measured. The collection of blood samples and myocardial samples were the same as part1. Detection method of the indicators are as follows:enzyme-linked immunosorbent assay (ELISA) method for measuring serum TNF-alpha levels, using immunehistochemical method determination of ICAM1expression in myocardial tissue, the electrophoretic mobility of variation analysis (EMSA)method for determining the activity of NF-kB and use the WesternBlot detection CCR5expression in the cellsStatistical methods:Quantitative data were expressed as mean±standard deviation (SD). One-way ANOVA was used for the analysisof normal distribution. Non-normal distribution of variables was analyzed with non-parametric "Mann-Whitney U" test. P<0.05was considered statistically significant. SPSS (version13.0) was used for all statistical analysis.ResultsPart Ⅰ:The hemodynamic results showed that LVSP,+dP/dtmax and-dP/dtmax were significantly decreased in I/R group than SHAM and I/R-Pre groups. Meanwhile, LVEDP was significantly higher in I/R group than SHAM and I/R-pre groups but no difference was observed between SHAM and I/R-Pre group. It suggested that cardiac hemodynamics were significantly damaged after ischemia-reperfusion, but were improved with pre-conditioning. Myocardial injury markers CK, LDH, AST activity were highest in ischemia-reperfusion group and not significant difference between other two groups suggesting that I/R would lead to myocardial injury. However, Myocardial tissue pathology examination showed no significant pathological changes in the myocardial tissue of the SHAM group. In ischemic preconditioning group, there were only mild cardiomyocytes swelling, mild myocardial fibrosis and interstitial edema in comparison, visible myocardial cell derangement, obvious swelling, nuclear condensation and irregular swelling of the myocardial fibers, interstitial edema, and inflammatory cells (mainly neutrophils) infiltration were observed in I/R group. It is therefore concluded that Ischemia-reperfusion injury will lead to changes in the structure of the myocardial tissue. Again, CCR5protein levels were highest in I/R group measured by western blot suggesting that CCR5expression was upregulated in the myocardial tissue after I/R injury. Part2:The level of hemodynamic parameters, LVSP and±dP/dtmax descended from group SHAM, I/R+CCR5Ab, I/R-Pre, I/R and I/R+CCR5Ago. It suggested that CCR5antibody can improve rat heart hemodynamic level. AR measurements showed ischemic preconditioning and CCR5antibody treatment significantly reduced myocardial infarct size so as to confer myocardial protection. Rat myocardial tissue MPO activity descended from I/R+CCR5Ago, I/R, I/R+Pre, I/R+CCR5Ab, SHAM suggesting that CCR5antibody can protect myocardial via suppression of MPO activity. The pathology assay showed that the severity of myocardial injury descended from I/R, I/R+CCR5Ago, I/R+Pre, I/R+CCR5Ab, SHAM showing that CCR5antibody can reduce the swelling of myocardial cells and degeneration and inflammatory response. The expression level of ICAM-1detected by immunohistochemical descended from I/R+CCR5Ago, I/R, I/R+Pre, I/R+CCR5Ab, SHAM suggesting CCR5antibody can reduce ICAM-1expression in the myocardial cells during I/R. The NF-kB activity detected by EMSA descended from descending I/R+CCR5Ago, I/R, I/R+Pre, I/R+CCR5Ab, SHAM demonstrating that NF-kB activity is much higher in I/R+CCR5Ago, I/R than other groups. The CCR5protein levels measured by Western blot was significantly reduced in I/R+CCR5Ab groop suggesting that CCR5antibody can inhibit CCR5protein expression to protect myocardial cells.Conclusions1. We have successfully established an I/R rat model through which we confirmed previous reports that I/R would induce myocardial structural damage, hemodynamic deterioration, myocardial injury and heart function impairment.2. I/R preconditioning or pretreatment can improve the I/R damage to myocardial to a certain degree.3. CCR5antibody can reduce I/R induced cardiac tissue structure damage, heart function impairment and blood flow dynamics deterioration.4. CCR5antibody protect heart and attenuate myocardial injury via the following mechanism/route:Inhibit the expression of cytokines to reduce the activation of NF-κB in myocardial cells; consequently the adhesion molecule ICAM-1expression was inhibited in the myocardial tissue and finally inflammatory cells (mainly neutrophils) aggregation was reduced.