| Background: acute myocardial infarction(AMI)causes severe damage to the coronary microcirculation,and the dysfunction of coronary microcirculation will affect myocardial perfusion.In the pathological process of vascular obstruction,stenosis or injury,the change of mechanical force on the lumen causes endothelial cells to express mechanosensors.Rho protein and its downstream effector,Rho associated kinase(ROCK),are involved in regulating a variety of cellular behaviors.As a mechanical signal transducer from integrin to cytoskeleton,Rho protein can convert the sensed mechanical force into electrical or biological signals and trigger blood vessel sprouting.It mediates perceived shear stress and promotes its action on endothelial cells,and it also drives vascular endothelial factors to promote angiogenesis.In tumor diseases,ROCK activates the extracellular regulated kinase ERK1/2 to promote endothelial cell proliferation,reorganization,and capillary stabilization.Extracorporeal cardiac shock wave therapy(CSWT)is a low-energy pulse wave imported from the body surface,which can produce shear stress on tissues and cells,thereby up-regulating the expression of pro-angiogenic factors in the infarct border area and inducing local angiogenesis.To date,the role of Rho/ROCK/ERK1/2signaling pathway in CSWT-induced angiogenesis in the peri-infarct zone has not been reported.Our previous study used a rat myocardial infarction model to investigate the effect of CSWT on endothelial progenitor cell proliferation after AMI.It was found that CSWT could up-regulate stromal cell-derived factor-1(SDF-1)and down-regulate transforming growth factor-β1(TGF-β1).TGF-β1)together promote the proliferation,transformation and mobilization of endothelial progenitor cells into ischemic myocardium.On the basis of previous studies,the rat AMI model was established to study the mechanism of CSWT promoting the regeneration of myocardial infarction border zone,and to provide a new theoretical basis for the treatment of ischemic heart disease(IHD)with CSWT.Objectives: The rat model of AMI was established and CSWT intervention was performed to explore whether CSWT could induce the expression of pro-angiogenic proteins and angiogenesis in the infarct border zone,and to further study whether these effects of CSWT were related to the Rho/ROCK/ERK signaling pathway.Methods:(1)Male Sprague-Dawley rats aged 1.5-2 months were used to establish the AMI rat model by surgical thoracotomy and ligation of the left anterior descending branch of the coronary artery.Intraoperative electrocardiogram,postoperative echocardiography and pathological staining were used to evaluate the modeling effect and the degree of infarction.(2)The successfully established AMI model was randomly divided into five groups: SHAM operation group(SHAM group,N=6),myocardial infarction group(MI group,N=6),myocardial infarction + shock wave group(MI+CSWT group,N=6),myocardial infarction +ROCK inhibitor + shock wave group(MI+Y-27632+CSWT group,N=6),myocardial infarction +Y-27632 group(MI+Y-27632 group,N=6).The SHAM group only underwent thoracotomy without coronary artery ligation.(3)On the 16 th day after operation,echocardiography was used to evaluate the cardiac function of rats in each group.On the 18 th day after surgery,rats in MI+Y-27632+CSWT and MI+Y-27632 groups were intraperitoneally injected with Y-27632(5mg/kg)for three consecutive days,and the other three groups were intraperitoneally injected with the same amount of normal saline.The rats in MI+CSWT group and MI+Y-27632+CSWT group were treated with CSWT on the21 st day after surgery.The shock wave energy was 0.09 m J/mm2,200 pulses per point,1 shock per second,3 days a week,once a day for 3 weeks.(4)On the 72 nd day after operation,the cardiac function indexes of rats were evaluated again by echocardiography.After completion of the experiment,the heart tissues of rats in each group were collected,the morphology and edema of myocardial cells in each group were observed by H&E staining,the angiogenesis in each area of myocardial infarction was observed by factor VIII staining,and the degree of myocardial fibrosis was observed by MASSON staining.Western blot was used to observe the protein expression levels of pro-angiogenic cytokines in the border zone of myocardial infarction.Results:Part one:(1)The acute ST-segment elevation myocardial infarction model was successfully established in 56 rats,the success rate of modeling was 70%,and the72-day survival rate was 55.3%.(2)Echocardiography on the 16 th day after operation showed that the left ventricular diameter and left ventricular volume in the MI group were significantly increased,and the left ventricular systolic function was decreased.The abnormal ventricular wall motion was mainly in the interventricular septum and left ventricular anterior wall.Compared with the SHAM group,LVEDD:(7.93±1.25)vs(6.08±0.73)mm,LVESD:(5.83±1.30)vs(3.26±0.53)mm,LVEDV(176.90±32.93)vs(104.75±16.69)ml,LVESV(96.45±22.37)vs(35.27±9.82)ml,M-mode ultrasound:LVEF: 54.47(51.45,57.11)% vs 76.06(72.75,77.70)%,LVFS: 27.98(26.14,29.68)% vs 46.93(43.38,48.79)%,Simpson’s EF: 50.97(43.54,54.96)% vs 67.31(61.22,72.61)%,all p < 0.05,indicating that the AMI model was successfully established.Part two:In the CSWT efficacy and safety study,echocardiography showed that LV ESV in the MI+CSWT group was significantly lower than that in the MI grou p [(102.46±30.73)vs(168.63±43.56)ml] on day 72(30 days after the end of shock wave treatment).Left ventricular ejection fraction increased(Simpson’s L VEF:51.71(45.36,57.69)vs 34.98(30.97,37.13)%,p< 0.05;The results of H&E staining of paraffin sections in the border zone of myocardial infarction showed cell edema,necrosis,irregular arrangement of cells,and disordered arr angement of muscle fibers in MI group,and regular arrangement of myocardial fibers and uniform cytoplasm in MI+CSWT group.MASSON staining showed that myocardial collagen volume fraction at the border zone of myocardial inf arction was 14.35(12.83,15.05)% in MI+CSWT group and 19.69(18.12,21.61)in MI group(p < 0.01).The level of von willebrand VIII(v WF)in MI+CSWT group was significantly higher than that in MI group(0.34±0.03 vs 0.24±0.01,p < 0.05).No adverse events occurred during the intervention.The re sults suggest that CSWT can increase the density of neovascularization in the marginal zone of myocardial infarction,reduce the area of myocardial fibrosis,and improve cardiac function.Part three:(1)In the study of the pro-angiogenic mechanism of CSWT,Compared with the MI group,the expression levels of FGF2,PGF,VEGFA and VEGFR2 in the MI+CSWT group were significantly higher,while the expression levels of MI+Y-27632+CSWT group were significantly lower than those in the MI+CSWT group(p < 0.05).The results of VIII immunohistochemical staining showed that the vascular positive area ratio of MI+CSWT group was significantly higher than that of MI+Y-27632+CSWT group(0.34±0.04 vs 0.01±0.01,p< 0.001).Distal myocardial infarction: vascular positive area ratio 0.10±0.02 vs 0.03±0.03,p < 0.05.The results suggest that the effect of CSWT on up-regulating the levels of pro-angiogenic cytokines in the peri-infarct zone and the pro-angiogenic effect in the peri-infarct zone can be inhibited by ROCK inhibitor.(2)MASSON staining showed that the area of collagen in the MI+Y-27632 group was significantly lower than that in the MI group [10.44(9.67,12.36)vs 19.69(18.12,21.61)%,p< 0.01].The Simpson’s EF was significantly increased in MI+Y-27632 group,suggesting that Y-27632 may improve cardiac function in rats with MI,which may be related to the improvement of cardiac function.Conclusions(1)A stable rat model of AMI can be obtained by ligation of the anterior descending coronary artery through thoracotomy.(2)CSWT treatment is safe for the hearts of rats after myocardial infarction.The experimental results showed that CSWT partially improved cardiac systolic function and myocardial fibrosis in rats after myocardial infarction.(3)CSWT could up-regulate the expression of pro-angiogenic cytokines and increase the area of neovascularization in the peri-infarct zone of MI rats,which was inhibited by ROCK inhibitor,indicating that the pro-angiogenic ability of CSWT was achieved by activating the Rho/ROCK/ERK pathway.(4)Y-27632 may improve cardiac function in rats with myocardial infarction,which may be related to the improvement of myocardial fibrosis. |
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