The Effect And Mechanism Of Rho Kinase On Apoptosis Of Smooth Muscle Cells After Arterial Intimal Injury In Rats

Background:With the developing of the economy and improving of the living standard,the incidence of coronary heart disease(CHD)is increasing these years,and the trend of onset age becoming younger.Nowadays CHD has become the biggest threat to human health.In recent 10 years,the death rate of CHD in China has increased rapidly.The mortality rate of CHD is 1.1091‰ and expecting the rate will keep soaring in the next 20 years in China,furthermore,it may become leading cause of death for our residents.With the advent of percutaneous coronary angioplasty(PTCA),CHD has entered a new era of interventional cardiology.In resent 40 years,PCI therapy which from the simple balloon dilation to the coronary stent implantation,from bare metal stents(BMS)to drug coated stents(DES)and bio-absorbable stents grows fast.PCI rapidly improves blood supply by opening narrow or occluded blood vessels.It has been widely used in patients with stable CHD or acute coronary syndromes and has successfully saved many lives.With the progress of stent technique,the rate of coronary restenosis after interventional surgery gradually decreased.But even with the new type method(DES),coronary stent restenosis(in-stent,restenosis,ISR)is still a problem that need to solve.ISR continues to be a difficult problem for clinical interventional physicians,for that the incidence in PCI patients is still up 3% to 20%.Therefore,further understanding of the mechanism of restenosis and finding new intervention targets for restenosis are of great significance for improving the prognosis of patients after PCI.The essence of ISR is healing reaction after arterial intimal injury,is the excessive damage of endothelial healing and adverse reactions of trauma caused blood vessel revascularization,is a series of vascular active substances and growth factors mediated complex biological processes.Endothelial cell injury is the key factor of restenosis,the damaged endothelial cells detached and exposed the basal membrane,resulting in platelet aggregation and co-secreting multiple cytokines with damaged endothelial cells,all the above mentioned situations lead to excessive proliferation and migration of vascular smooth muscle cells(VSMC)which is believed to be the leading cause of neo-intimal thickening and luminal loss.Neo-intimal proliferation is the important part of restenosis,except for the proliferation and migration of VSMC,the cause of neo-intimal proliferation is accompanied by the lack of VSMC apoptosis.After the injury of vascular endothelial,VSMC apoptosis throughout the entire process of vascular repair,and in the postoperative ISR occupies an important position.The imbalance between two ways of apoptosis and anti-apoptosis is the cytological basis of neo-intima formation.Promoting cell apoptosis can reduce the total number of cells and alleviate endometrial hyperplasia.Therefore,promoting VSMC apoptosis is an important treatment of PCI.Research shows that promoting apoptosis can reduce the excessive proliferation of VSMC,which will protect the vessels from restenosis after injury effectively.Hence,to fully understand the factors and mechanisms of promoting VSMC apoptosis,we can find an effective target to reduce the incidence of ISR after operation.Although there are many of the targets for promoting VSMC apoptosis drug therapy,gene therapy applied in preclinical and clinical,looking for more safe,effective,strong specificity of drugs is still seems particularly important.Rho is a subgroup of the Ras family,belonging to the small molecule G protein and widely distributed in mammalian tissues.Rho kinase(ROCK)acts as a signal transducer or a molecular switch in the cell signaling pathway and acts on the cytoskeleton or its target proteins,producing a wide range of biological effects.Many cells express ROCK.There may be different effects for different tissue cell ROCK activation.ROCK's high expression and the excessive activation can lead to disease,which has not yet to be found in ROCK low expression or lack of activity.The Rho/ROCK pathway interacts with a variety of vasoactive substances and is involved in the development of various cardiovascular diseases,including coronary heart disease,acute myocardial infarction,coronary artery spasm,hypertension,heart failure,etc..Previous animal experiments in our research group have also been confirmed that myocardial ROCK mRNA increased in acute myocardial infarction,myocardial ischemia/reperfusion injury and heart failure.In addition,after the use of ROCK specific inhibitor-fasudil,ROCK mRNA could be reduced to protect ischemic myocardium and improve cardiac function.Further studies also found that the myocardial protective effect of fasudil is related to the inhibition of ROCK mRNA and the decrease of myocardial apoptosis.In recent years,the study of the relationship between ROCK and restenosis is particularly attractive.ROCK can participate in the process of stent restenosis after a variety of approaches.Scholars have been attempt to find ways to prevent restenosis from the ROCK.Matsumoto first published ROCK's role in ISR,by implantation coronary artery stent in pig,the study shows that ROCK can regulate the process by acting on the contraction of vascular smooth muscle,endothelial function and inflammatory processes involved in stent neo-intimal proliferation and restenosis.Domestic studies have also confirmed that ROCK participates through restenosis,and most of the mechanisms are focused on endothelial injury,inflammatory cell infiltration,migration,proliferation,and so on.At present,there is a little research on the effect of ROCK on VSMC apoptosis.The mechanism is unknown and controversial.It is clear that ROCK can promote cardio myocyte apoptosis,but whether it can promote apoptosis in all cells? Is it different for different cells,or even different states of the same cell? And what is the mechanism of this action? In this study,we observed the apoptosis of VSMC and the dynamic changes of ROCK mRNA after vascular intimal injury,and further explored the relationship between ROCK and apoptosis of VSMC and the mechanism involved in the development of restenosis.Objective:This paper firstly,we observed the changes of ROCK mRNA expression and the condition of apoptosis on VSMCs in rats after arterial balloon injury;Secondly,we analyzed the effect of ROCK inhibitor-fasudil on vascular intima and intima media thickness;Finally we discussed the relationship between the apoptosis of the ROCK signaling pathway and the apoptosis of smooth muscle cells and the effect and mechanism of the fasudil on the prevention of arterial injury.And the relationship between ROCK and VSMC autophagy was preliminarily discussed.Methods:1.Establish animal models and groups: we selected male Wistar rats which were inserted into the self-made 2F balloon catheter from left common carotid artery and injured the Thoracoabdominal aorta.The rats were divided into 3 groups randomly: A.Model Group,B.Treatment Group,C.Control Group.In the control group,except for unthreading the balloon catheter without damaging the vascular endothelium,the rest of the operation was the same as the model group and the treatment group.Intraperitoneal injection of fasudil 5 mg/kg 2 times a day for 4 weeks on treatment group.The model group and control group injected the same amount of physiological saline on abdomen.2.To observe the changes of intimal and media film thickness by HE staining: taken the aorta of rats 4 weeks after surgery of Intra arterial lesions,fixed with formaldehyde and did paraffin-cut section method to HE staining.The condition of hyperplasia and changes of thickness in intimal and media film were observed in each group,at the meantime,we compared the hyperplasia and thickness in control group and treatment group which used the fasudil.3.The expression of apoptosis related proteins was observed by immunohistochemical staining: Using the aortic vessels of rats which 4 weeks after aortic endothelial injury,performed immunohistochemical staining and observed the expression of apoptosis related proteins Bcl-2 and Bax in aortic intima and tunica media smooth muscle cells of each group,we compared the expression levels of apoptosis related proteins Bcl-2 and Bax in model group and treatment group which used the fasudil.4.Using the TUNEL staining to observe the apoptotic quantity of VSMC: We taken the aortic vascular tissues of rats 4 weeks after operation to observe the number of apoptotic smooth muscle cells in the intima and media of aorta in each group,besides we compared the apoptotic rate of the smooth muscle cells and the value of IOD in model group and treatment group.5.Using RT-PCR method to detect the changes of ROCK and m RNA expression in VSMC: 4 weeks after operation,the aortic vascular tissue of rats was collected and cleaned,to extract the RNA in VSMC.The expression levels of ROCK mRNA of VSMC in each group were observed.We compared the expression levels of ROCK mRNA in model group and treatment group.6.Detecting the expression of autophagy related proteins by Western blot: 4 weeks after operation,we collected aortic vascular tissue of rats,homogenated and extracted protein to observe the expression of autophagy in VSMC related protein Beclin-1 and LC3-? and compared that in control group and treatment group.Results:1.52 rats survived in 24 hours after injury operation of aortic intima.20 rats in model group,20 rats in treatment group,12 in control group.A total of 3 deaths were found in the following 4 weeks' intervention,including 2 rats in the model group,1 in the treatment group and no rat died in control group.The death rate was 10% and 5% in the model group and the treatment group,respectively.The difference was statistically significant,P<0.05.Finally we choose 18 rats in model group,19 rats in treatment group and 12 rats in control group in this experiment.2.After 4 weeks balloon injury of aortic intima in rats,smooth muscle cells proliferated obviously which thickened the intima and tunica intima,comparing with the control group(intima: 21.8 ± 2.93,media: 194.3 ± 9.09),the intima and media intima of the vessels in the rats were obviously thickened after endothelial injury,the difference was statistically significant,P<0.05,comparing with the model group(intima: 99.6 ± 5.42,media: 311.6 ± 17.00),treatment group(intima: 91.3 ± 9.11,media: 291.6 ± 19.74)which after the usage of fasudil treatment,intima and media hyperplasia were significantly reduced,with a statistically significant difference(P<0.05).3.Compared with the control group(0.43 ± 0.041),the expression of ROCK mRNA in VSMC was significantly increased after the endometrial injury in rats.After 4 weeks using fasudil,the ROCK mRNA of the treatment group(0.31 ± 0.041)was significantly lower than that in the model group(0.51 ± 0.036),with a statistically significant difference(P<0.05).4.Compared with the mentioned control group(Bcl-2: 0.93 ± 0.08,Bax: 0.82 ± 0.54),after endothelial injury in rats,apoptosis related protein Bcl-2 and Bax were increased,the difference was statistically significant,P<0.05,after using fasudil the treatment group(Bcl-2: 1.15 ± 0.09,Bax: 2.39 ± 0.16)compared with model group(Bcl-2: 2.12 ± 0.12,Bax: 1.64 ± 0.13),the expression of Bcl-2 deceased but Bax expression increased significantly,the difference was in statistically significant,P<0.05.5.TUNEL staining indicated that the apoptosis of VSMC increased significantly after arterial intimal injury in rats.After 4 weeks of treatment with fasudil,compared with the mentioned model group(intima: p-rate 6.28±2.69,IOD 0.10±0.03;media: p-rate 7.32±2.41,IOD 0.11±0.04),the number of apoptotic VSMC increased,the rate of apoptosis(intima:9.64±2.76,media:10.51±2.71)and IOD(intima:9.64±2.76,media:10.51±2.71)increased with a statistically significant difference(P<0.05).6.Compared with the control group(Beclin-1: 9.80±2.09,LC3-?: 13.81±2.66),the rat's artery after the balloon injury,expression of autophagy related protein Beclin-1 and LC3-? that increased,and after 4 weeks using fasudil(Beclin-1: 26.04±4.83,LC3-?: 33.00±4.46),comparing with model group(Beclin-1: 21.38±3.24,LC3-?: 25.59±3.61),Beclin-1 and LC3-? expression increased,the difference was statistically significant,P<0.05.Conclusions:1.The significant proliferation of vascular smooth muscle cells leads to luminal stenosis after intimal injury in rats.However,the relative insufficiency of apoptosis leads to the occurrence of vascular stenosis.2.After endometrial injury in rats,the expression of ROCK mRNA in vascular smooth muscle cells was significantly elevated,cell proliferation was obvious.However,the apoptosis of smooth muscle cells increased obviously when ROCK and mRNA were inhibited by Fasudil.These results suggest that fasudil can reduce intimal hyperplasia and reduce the degree of vascular stenosis by promoting apoptosis of smooth muscle cells.3.Fasudil could promote the apoptosis of smooth muscle cells which may be associated with the autophagy of VSMC.