| Coronary heart disease (CHD) is one of the leading causes of death worldwide. Accordingly, about3,800,000men and3,400,000women die of CHD ever year. It is suggested that timely thrombolytic therapy or open vascular PCI operation and emergency coronary revascularization after the occurrence of acute myocardial infarction is the most effective strategy to reduce the size of a myocardial infarct and improve the clinical outcomes. After an onset of acute myocardial infarction, early and timely methods to restore blood flow of the infarct-related artery, including the thrombolytic therapy, percutaneous coronary intervention (PCI) and emergency coronary artery bypass grafting (CABG).However, the re-open of the artery could increase the damage to the local tissue and cause the cardiomyoctytes death, leading to the enlargement of the infarct size and the reduction of the cardiac function. This paradoxical phenomenon is termed "myocardial ischemia/reperfusion (I/R) injury", which is first described by Jennings in1960.Inflammation is one of the major reasons for the myocardial I/R injury. Inflammatory response as well as calcium overload, ER stress response, and reactive oxygen species involve and mediate the cardiomycytes injury during the whole process of I/R.Evidence from the animal experiments showed that the ani-inflammation interventions could reduce the infarct size of up to50%. Chemokines triggered the initiation of the inflammation. CXCL16was a newly founded chemokine, which could bind to its unique receptor CXCR6, together formed a whole signal transduction axis. CXCL16/CXCR6axis played a pivotal role in the inflammation-associated diseases including atherosclerosis, rheumatoid arthritis and cancer. However, its role in the cardiac I/R injury has not been explored previously.Cardiac I/R injury could cause the loss of the cardiomyctyes via the process of the apoptosis and necrosis, which was called the Type I programmed cell death. While, recent a group of researches supported the idea that autophagy, Type II programmed cell death, could also account for the cell death, which was much more important than the Type I PCD in the process of the cell death.Therefore, in this study, we studied the role of chemokine CXCL16/CXCR6axis in cardiac autophagy and myocarcial function during the process of I/R injury, and explored their therapeutic penitential in the treatment of I/R injury.This paper is divided into five parts.Part1:The variation and clinic implication of the sCXCL16in the patients with acute ST-segment elevation myocardial infarction (STEMI)Background:The exact role of sCXCL16in CHD is not clear in the previous studies, and there is less information about its role in the patients with STEMI;Aim:To explore the variation and clinic implication of the sCXCL16in the patients with acute ST-segment elevation myocardial infarction (STEMI);Materials and Methods:A total of54patients with STEMI were recruited from the Department of Cardiology, affiliated Zhongshan Hospital, Fundan University, China. Among them,32patients received an emergency percutaneous coronary intervention (with E-PCI) due to the admission time of less than12h, and the other22patients didn’t receive an emergency PCI (without E-PCI) for the long admission time. Another18controls without CHD, diagnosed by CAG operation were also involved in. Venous blood from all the patients was collected at indicated time of7h,9h,12h,16h,24h,48h,72h (3d),168h (7d) after the onset of chest pain. The serum levels of sCXCL16were detected using ELISA kits.Results:(1) STEMI patients in the both group of "with E-PCI" and "without E-PCI" have a higher level of sCXCL16than control group at all the indicated time after the onset of symptoms.(2) Compared to "without E-PCI" group, the patients in the "with E-PCI" group have a much lower level of sCXCL16at the indicated time of48h,72h,168h (7d) after the onset of the symptoms.(3) Peak wave of the sCXCL16time curve is shifted from48-72h in the "without E-PCI" group to16-24h in the "with E-PCI" group.Conclusion:STEMI patients have a special time curve of sCXCL16after the onset of the symptoms. Soluble CXCL16is an important biological marker to reflect inflammatory levels in the STEMI patients.Part2:Blocking CXCL16/CXCR6axis could attenuate the myocardial ischemia/reperfusion injury and improve the cardiac functionBackground:sCXCL16is a bio-marker to reflect inflammatory level in the STEMI patients. However its role in myocardial I/R injury has not been explored;Aim:To explore the role of the CXCL16/CXCR6signal axis in myocardial I/R injury;Materials and Methods:Eight weeks old (weight20-24g) male CXCR6KO mice and their littermate wild-type (WT) mice were subjected to build a cardiac I/R injury model by ligating the left anterior descending (LAD) artery for45min followed by reperfusion for24h. At the end of the experiments, all the animals in the four groups (Sham-WT, Sham-KO, I/R-WT and I/R-KO)received AAR (the area at risk) and IS (infarct size) detection by TTC staining, LVEF(left ventricular ejection fraction) and LVFS (left ventricular fraction shortening) by UCG,+dp/dt (maximal velocity of left ventricular pressure development), LVEDP(left ventricular end-diastolic pressure), LVSP (left ventricular systolic pressure) by hemodynamics. These animals were also used to measure SUV (Standardized Uptake Value) by small animal PET/CT after the treatment of I45min/R48h.Results:Quantification of AAR was similar in all animals, but IS was significantly reduced in CXCR6KO mice24hours after reperfusion (31.86±1.808%vs.43.09±1.519%,≈26.06%reduction; P<0.001). Both LVEF and LVFS were improved in KO mice compared to WT mice (39.65±2.204%vs.32.98±1.525%,≈26.75%increase; P<0.05and20.81±1.001%vs.15.68±0.8257%,≈32.72%increase; P<0.05, respectively). In addition, I/R injury caused significant systolic dysfunction in WT mice (vs. CXCR6KO mice), measured by+dp/dt (9910±420.3vs.11366±381.5mmHg/s), as well as altered LVEDP (13.36±1.349vs.9.741±1.001mmHg) and LVSP (100.6±2.589vs.108.2±3.215mmHg). CXCR6KO mice also exhibited a much higher SUV than the WT mice due to the I45min/R48h (5.03±0.423vs.1.55±0.357P=0.003, n=4per group);Conclusion:Blocking CXCL16/CXCR6axis could attenuate the myocardial ischemia/reperfusion injury and improve the cardiac function. Part3:CXCR6deficiency attenuated the myocardial I/R injury by depressing the cardiac autophagy during the reperfusion periodBackground:Recent study suggested that autophagy played an important role in cardiac I/R injury, and autophagic response had cardioprotective effect during the ischemia period, while it was vise versa during the reperfusion time. This appeared the conflicts on the beneficial or detrimental role of autophagy in cardiac I/R injury;Aim:To determine whether or not cardiac autophagy was increased during the reperfusion period, and to explore the underlying roles of CXCL16/CXCR6axis in the process of cardiac autophagy in a mouse model of I/R;Materials and Methods:Eight weeks old (weight20-24g) male CXCR6KO mice and their wild-type (WT) littermates were used to build the model of ligating the left anterior descending (LAD) artery for45min followed by reperfusion for24h. At the end of the experiments, all the heart tissue samples in the four groups (Sham-WT, Sham-KO, I/R-WT and I/R-KO) were systematically detected for the autophagy markers by the techniques, including Laser Confocal Scanning Microscopy (LCSM), Transmission Electron Microscopy (TEM) and Western Blotting. Moreover, the serum levels of TNF-aα IFN-γ and IL-10were also measured by ELISA at the indicated times after reperfusion;Results:Qualitative analysis of LCSM showed that cytoplasmic distribution of LC3(autophagy maker) in the living cells were reduced in CXCR6KO mice when compared with WT mice. Quantitative analysis of autophagic vacuoles (double plasma membranes) and Beclin-1expression were both reduced in KO mice when compared with WT mice, respectively. Furthermore, I/R-induced a significant upregulation of autophagic flux that determined by LC3B-Ⅱ/LC3B-Ⅰ, P62and Cathepsin D, which could be attenuated by CXCR6knockout. In addition, serum levels of TNF-α and IL-10had no significant difference between groups, while the level of IFN-y was significantly lower in CXCR6KO mice than in WT mice during the period of reperfusion;Conclusion:Cardiac autophagy increased significantly and had a positive relationship with the cardiac injury during the reperfusion period. CXCR6deficiency attenuated the myocardial I/R injury by depressing the cardiac autophagy during the reperfusion phase, which might be associated with IFN-γ down-regulation. Part4:Exogenous IFN-γ reverse the protection of CXCR6deficiency on the myocardial I/R injury.Background:Data from the Part3implied that down-regulated secretion of IFN-γ might play a cardioprotective role in I/R-induced autophagy. But the underlying mechanism is not clear.Aim:To investigate whether attenuation of IFN-γ was crucial for suppressing of IFN-γ-dependent autophagy and improved cardiac function in CXCR6KO mice.Materials and Methods:Eight weeks old (weight20-24g) male CXCR6KO mice were subjected to IFN-γ (100U/g body weight) via intraperitoneal injection once a day for3consecutive days. All the CXCR6knockout mice in the four groups (Sham+PBS, Sham+IFN-γ,IR+PBS and I/R+IFN-γ) were received AAR and IS detection by TTC staining; LVEF and LVFS by UCG;+dp/dt, LVEDP and LVSP by hemodynamics after ischemia for45min and followed by reperfusion for24h.Results:The quantification of AAR was no difference in the four groups, but I/R-induced IS (39.94±1.544%,≈39.15%increase; P<0.05) was markedly increased in CXCR6KO mice after the treatment of IFN-γ compared with PBS treatment (28.71±1.746%). In addition, cardiac functions, such as LVEF (32.02±1.535%vs.41.96±1.831%,≈23.69%reduction; P,0.05), LVFS (14.74±0.7201%vs.20.41±1.079%,≈27.78%reduction; P<0.05),+dp/dt (9374±546.0mmHg/s vs.11158±458.9mmHg/s,≈15.99%reduction; P<0.05), LVEDP (12.97±1.836mmHg vs.8.81±1.274mmHg,≈47.22%increase; P<0.05), and LVSP (87.88±3.824mmHg vs.113.0±5.499mmHg,≈22.23%reduction; P<0.05), all deteriorated in CXCR6KO mice pretreated with IFN-γ in response to I/R injury. However, IFN-γ treatment did not induce cardiac dysfunction in sham-operated mice, indicating a critical role of I/R in activating the IFN-γ-dependent autophagy pathway.Conclusion:Exogenous IFN-γ abrogates the protection from myocardial I/R injury in CXCR6mice.Part5:CDllb+cells induce IFN-γ secretion in response to I/RBackground:IFN-γ triggers the activation of autophagy, and autophagy also recruits the inflammatory cells to local microenvironment to secret IFN-γ. Results from the Part3show that CXCR6-/-mouse has a down-regulated secretion of IFN-γ in response to I/R. The origin of IFN-γ, however, is not clear; and the relationship between IFN-γ and CXCL16/CXCR6axis has not been well documented.Aim:To explore which kind of cells contribute to the secretion of IFN-γ and to investigate the molecular mechanism of the interaction between the IFN-γ and chemokine CXCL16/CXCR6axis.Materials and Methods:CD11b+splenocytes were isolated and purified from the spleen of the WT or CXCR6KO mice by CD11b+MicroBeads, and were seeded into the24-well palates at a density of3×105/well; cardiomyocytes isolated from the hearts of the WT or CXCR6KO neonatal mice were cultured in the24-well palates for36-48h. Both kinds of the cells were stimulated with the CXCL16(100ng/ml, R&D Systems, Inc., USA). After cultured for another48h, the supernatant were collected for IFN-γ detection. CD11b+splenocytes isolated from the spleens of WT mice were co-cultured with neonatal cardiomyocytes from WT or CXCR6KO mice using transwell plates under the condition of anoxia for4h/reoxygenation for20h (A/R). Autophagy in cardiomyocytes was then determined by Western Blot. Meanwhile, immunohistochemistry and flow-cytometry analyses were used to evaluate the quantity of the CD11b+inflammatory cells infiltrating into the injured myocardium in all the four groups (Sham-WT, Sham-KO, I/R-WT and I/R-KO).Results:IFN-y production was far less in a supernatant of stimulated cardiomyocytes than in that of monocytes, indicating that cardiac IFN-y might not be secreted by the myocardium but by the infiltrated immune cells. Immunohistochemistry and Flow-cytometry analyses confirmed multiple infiltrations of CD11b+cells in ischemic myocardium, but it was significantly attenuated in CXCR6KO mice (4.922±0.698%vs.12.22±1.275%, P<0.05; n=6per group). To further confirm the function of these kind of cell subpopulations, the cells were isolated and purified from spleen in WT mice by CD11b+MicroBeads, and this kind of cell population (CD11b+splenocytes) were then co-cultured with cardiomyocytes from WT or from CXCR6KO mice using transwell plates under the condition of anoxia/reoxygenation (A/R). Beclin-1expression was significantly enhanced in cardiomyocytes from both WT and CXCR6KO mice. These data suggested that IFN-γ triggered cardiac autophagy after I/R injury in a paracrine secretion manner.Conclusion:Infiltration of CD11b+cells contribute to myocardial IFN-y secretion and heart autophagy injury in CXCR6-/-mouse in response to I/R. |
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