| Background: In recent years,the incidence of coronary heart disease(CHD)showed an increasing trend,and the tendency of younger cases has become a severe public health problem.Percutaneous Coronary Intervention(PCI)is an important clinical method for revascularization of CHD.In China,the number of patients underwent PCI has increased significantly year by year,with an average annual growth rate of 10%-15%.However,restenosis after PCI limits the long-term effect of revascularization severely,even with the restenosis rate of new generation DES up to 12%.So the research on restenosis has been a hot spot in recent years.The nature of in-stent restenosis after PCI is the excessive form of healing of localized injury.The major mechanism of restenosis is neointimal hyperplasia caused by vascular smooth muscle cells proliferation,migration and deposition of extracellular matrix.Endothelial injury is the initiating and key factor of restenosis,which can induce rapid re-endothelialization.Promoting the rapid endothelial repair can reduce the occurrence of restenosis.How is the endothelium repaired? The conventional viewed that the repair of endothelial injury depended on the migration and proliferation of local cells and mature endothelial cells.Until the 1990 s,endothelial progenitor cells(EPCs)were found as the important cell source of functional re-endothelializaiton.EPCs are derived from bone marrow stem cells.Under the stimulation of physiological or pathological factors,EPCs can mobilize from bone marrow to peripheral blood,home to injured vascularendothelium,and differentiate into mature endothelial cells,which can promote the repair of endothelial injury.EPCs colonize at the injured location after PCI,which can promote re-endothelializaiton,reduce smooth muscle proliferation and inhibit the endometrial hyperplasia.Studies have confirmed that the number,proliferation and migration of EPCs in the peripheral blood of patients with restenosis after PCI are significantly decreased,which means EPCs injury is a critical factor in the process of restenosis after PCI.So the quantities or/and functional decline are the critical factors of in-stent restenosis.Angiotensin II(Ang II)is the most active molecule of the renin-angiotensin system or the renin-angiotensin-aldosterone system(RAAS),growth and regulating vascular tone,blood volume.Many studies have shown Ang? can promote the apoptosis of EPCs,decline the quantity and function of EPCs.Blocking the receptors of Ang II can significantly reduce the apoptosis of EPCs and improve its function.The main mechanism of valsartan is blocking the bad effects of EPCs,such as generation of reactive oxygen species,oxidative stress,reduction activity of telomerase and acceleration the aging and apoptosis of EPCs.In recent years,some studies have found the presence of angiotensin II type 1 receptor autoantibodies(AT1-AA)in the serum of patients with preeclampsia,malignant hypertension or renal transplantation,which can be identified through specific identification(AT1R-ECII),activates the AT1 receptor,and produces a biological effect similar to that of Ang II-activated AT1 R.So far,the research of AT1-AA on in-stent restenosis after PCI is still at an early stage.It is unclear that whether AT1-AA can cause in-stent restenosis,effect the factors of restentosis and the underlying mechanisms.This paper will study these issues through the following aspects.Part 1 Analysis of relationship between AT1 receptor autoantibody and in-stent restenosisObjective: To investigate the relationship between in-stent restenosis and AT1-AA.Methods: Patients were divided into 4 groups according to whether occurred in-stent restenosis: restenosis group(n=50),non-restenosis group(n= 60),CHD group(coronary angiography without coronary stent,n=60)and control group(coronary angiography to exclude CHD,n=40).Patients(AMI)were excluded.At baseline,there was no significant difference of age,gender,risk factors(including hypertension,hyperlipidemia,diabetes mellitus and smoking),medication and stent implantation(restenosis group and no restenosis group)between groups.Vein blood 10 ml was drawn at decubitus.Ang II and ATI-AA were tested by ELISA.The difference of OD value and the positive rate of AT1-AA between the two groups were compared.In addition,fifteen cases of young healthy subjects were selected as the negative control group.Results: 62.0%(31/50)of patients in restenosis group with positive AT1-AA was significantly higher than 35%(21/60)in non-restenosis group,16.7%(10/60)in CHD group and 7.5%(3/40)in control group(p<0.05).The difference of OD value between restenosis group and the other three groups was statistically significant(p<0.05).The AT1-AA positive rate and OD value in CHD group were significantly higher than the control group(p<0.05).Ang II level had no significant difference of between restenosis group and non-restenosis group(P>0.05).Compared with CHD group,Ang II level of restenosis group and non-restenosis group were higher with no signicicnet differecnce(P> 0.05).Compare with control group,Ang II level of restenosis group andnon-restenosis group is significantly higher(p<0.05).Ang II level of CHD group was higher than control group with no significant difference(p>0.05).Conclusion: 1.AT1-AA in the stent restenosis group was higher than in the restenosis group,which suggested that AT1-AA may have an effect on in-stent restenosis.2.AT1-AA was higher in the stent group than in the non-implanted coronary stent group,which suggested that PCI process may lead to the elevation of AT1-AA.Part 2 Analysis of relationship between AT1 receptor autoantibody and restenosis after coronary stent implantationObjective: To observe the effects of AT1-AA on the repair of damaged blood vessels directly by using the immunized rat to produce AT1-AA and then underwent balloon injury in the arteria carotis communis.Methods: 55 rats were divided into four groups: group A(n=15,treated with AT1-AA and balloon injury),group B(n=15,treated with AT1-AA + valsartan + balloon injury),group C(n=20,treated with balloon injury only)and group D(n=5,control group).AT1 polypeptide conjugate was equilibrated with equal volume of Freund's complete adjuvant.Group A and B received active immunization by subcutaneous injection of 0.4 ?g/kg of AT1 polypeptide on the back of the rats.Group C and D received equivalent subcutaneous injection of Freund's complete adjuvant.After the initial immunization,the injections as above were repeated every 2 weeks for mass immunization campaign.Group B rats were given valsartan by gavage at 2mg/kg/d fromthe initial immunization to death.Rats in group A and group B had orbit blood draw at the beginning,2 weeks,4 weeks,6 weeks,8 weeks and 10 weeks of the experiment.AT1-AA titer was measured by ELISA.At 6 weeks of active immunization,the model of left common carotid artery balloon injury was established.Group A,group B and group C were model groups and group D was normal control group.In group A,B and C,anesthetized rats were surgically cut to separate the neck skin and tissues,exposed the left carotid artery,internal carotid artery and external carotid artery.From the external carotid artery,PTCA guidewire and 1.5 * 8mm PTCA balloon were sent to left common carotid artery.Pressurize the balloon and twitch back and forth,which can damage the vascular intima.In group D,left common carotid artery of rats were removed after anesthesia and divided into 3 parts for HE staining,electron microscopy and RT-PCR respectively.The nitric oxide synthase(e NOS)m RNA was detected by RT-PCT as baseline standard.Rats in group C were instantaneous injury group.The left common carotid artery was removed immediately after the balloon injury,tested as above.Rats in group A,group B and group C were sacrificed at 3 days,2 weeks and 4 weeks separately(5 rats each time).The left common carotid artery was removed and divided into 3 parts for HE staining,electron microscopy and real-time quantitative PCR(RT-PCR).The nitric oxide synthase(e NOS)m RNA was detected by real-time fluorescent quantitative PCR(RT-PCR).At the mean time,right common carotid artery was removed and treated for control.The mean intima thickness(MT),lumen area,calculated IT / MT,stenosis rate(left common carotid lumen area / right common carotid lumen area)were measured by HE staining.The specimens were cut along the longitudinal direction and observed by electron microscopy scanning.The rate of endothelial cell coverage(longitudinal profile)were measured.Nitric oxide synthase(e NOS)m RNA was tested by real-time quantitative PCR(RT-PCR).Results: In group A and B,the titers of AT1-AA were 1:(250±82.3)at 2 week,1:(2000±786.5)at 4 weeks,1:(3142±1124.3)at 6 weeks,1:(5428±1576.4)at 8 weeks and 1:(7000±2340.5)at 10 weeks.The titers at 6,8,10 weeks were significantly higher than the titer at 2 weeks.At the moment of balloon injury,endothelial cells completely stripped.Part of The elastic plate was broken.At 1 week of balloon injury,AT1-AA + balloon injury group,AT1-AA + valsartan + balloon injury group,simple balloon injury group were seen neointimal formation and lumen slightly narrowed.But between the three gourps,the membrane area(IA),medium area(MA),IA / MA,LA(lumen area)were not statistically significant(P> 0.05).At 2 weeks,the intima of the three groups were significantly thickened,with proliferation of the smooth muscle cells,disorganization of myocytes.The AT1-AA + valsartan + balloon injury group was the most severe followed by AT1-AA + balloon injury group and simple balloon injury group in sequence.IA and IA / MA were the highest and LA was the lowest in AT1-AA + balloon injury group,followed by AT1-AA + valsartan + balloon injury group.The simple balloon injury was the least affected(p<0.05).The difference between the two groups was significant(P <0.05).At 4 weeks,the differences were more pronounced.IA of IA1-AA + balloon injury groups reached to 0.914±0.024 mm2;IA/MA was 4.562±0.032 and LA was0.048±0.023 mm2.The injury was lowest in simple balloon injury group,IA was 0.336±0.027 mm2,IA/MA was 1.504±0.030 and LA was 0.138±0.026 mm2.The difference between the two groups was significant(P <0.01).There was no significant difference of the three groups(P> 0.05).At the same time,we collected the right common carotid artery samples from each rat at the time point by HE staining,and calculated the lumen area.The mean value was calculated at each time point,because the right common carotid artery was not undergoing balloon injury,no significant endometrial hyperplasia was observed.So we got the stenosis rate by the left/right of common carotid artery at the same time.At 1 week,the stenosis rate of AT1-AA + balloon injury group,AT1-AA + valsartan + balloon injury group and simple balloon injury group were 13.7%,12.4% and 11.2%,respectively,which were not statistically significant.The stenosis rates of the three groups were 48.6%,43.8% and 37.4% respectively at 2 week.The stenosis rates were 70.4%,61.8% and 52.3% at 4 weeks,respectively,and the difference was statistically significant(P <0.05).From the above,in AT1-AA + balloon injury group,AT1-AA + valsartan + balloon injury group and simple balloon injury group,neonatal intimal hyperplasia gradually repaired.At 2 weeks,the difference had shown.At 4 weeks,the difference had been more obvious.AT1-AA + balloon injury group had the most obvious neointimal hyperplasia,with the smallest lumen and the highest stenosis rate.The above situations were improved in the Valsartan group.The neointimal hyperplasia was the lowest in the balloon injury group,with the largest lumen area and the lowest rate of stenosis.These results suggested that AT1-AA can significantly induce neointima thicking,lumen smaller and lead to stenosis,whicn can be improved by valsartan.In the observation of electron microscopy,5 samples(1.0k)were randomly selected in each tissue specimen to count the number of endothelial cells.We clearly observed the change of endothelial cells number before and after balloon treatment in the field of view of 1.0k and 3.0k.The number of vascular endothelial cells changed obviously.At 1k and 3k Vision,we can clearly observe the vascular endothelial cells disappeared immediately after balloon treatment,which suggested balloon damages were more thorough.Under 1.0k view,the repair processes of endothelial cells were observed at the three group.Over time,the endothelial cells in each group were increased.At 1week and 2 weeks,there were no significantly differences of the three groups(p>0.05).At 4 weeks,the number of endothelial cells in was 46 in simple balloon injury group,36 in AT1-AA + valsartan + balloon injury group and 30 in AT1-AA + balloon injury group.Compared with AT1-AA + valsartan + balloon injury group and AT1-AA + balloon injury group,the number of endothelial cells had significant difference(p<0.05).There was also significant difference between AT1-AA + valsartan + balloon injury group and AT1-AA + balloon injury group(p<0.05).It was suggested that the endothelium of the balloon group was the fastest,and the AT1-AA + balloon injury group was the slowest.The endothelium could be improved by valsartan,which may be related to the effect of Valsartan antagonizing AT1 receptor and inhibiting the effect of AT1-AA.At the same time,the endothelium coverage rates(the number of endotracheal injury endothelium / the number of endothelium in the contralateral symmetrical site)were calculated at 1 week,2 weeks and 4 weeks.At 1week and 2 weeks,there were no significantly differences of the three groups(p>0.05).At 4 week,the endothelium coverage rate of AT-AA + balloon injury group was 64.2%,72.3% in AT-AA + valsartan + balloon injury group and 92.7% simple balloon group.The difference was statistically significant(P< 0.05).Endothelial cell coverage rates were consistent with the conclusion of endothelial cell counts.The 2-?CT value of e NOS m RNA was significantly reduced in the balloon injury compared with the normal control group(no balloon injury vessel).One week after balloon injury,the 2-?CT values of group A(AT1-AA group + balloon injury group),group B(AT1-AA + valsartan group + balloon injury group)and group C(simple balloon injury group)2-?CT value were still at a low level.With the passage of time,the e NOS m RNA gradually increased in each group.The value of C group increased rapidly and the value of group A recovered slowly.At 4 weeks,the differences among the three groups were significant(group C with highest value and group A with lowest value).The value of group C had significantly difference compared with the values of group A and B.The difference between group B and A was also significant.At 4 weeks,compared the e NOSm RNA of group A,B and C to normal control group,e NOSm RNA of group C was highest,0.547,which still didn't reach the baseline level.The e NOSm RNA of group A was lowest.These results had shown that AT1-AA had effects on the expression of e NOS m RNA,which could delay the recovery.Whereas the administration of valsartan could block AT1-AA and improve the repairmen.Conclusion: 1.AT1-AA acted on damaged blood vessels to make the neointima thickened,the lumen smaller and leaded to restenosis.Valsartan can significantly improve these conditions.2.Based on the scanning of electron microscope,we had found the differencs of the endothelial cell count,the endothelial cell coverage rates and delayed recover time between groups,which suggested AT1-AA can lead to delayed endothelial repair.3.It was confirmed that the delayed neointimal repair was significantly associated with the occurrence of restenosis.Part 3 Effects of AT1-AA on EPC in vitroObjective: To explore the effect of valsartan on AT1-AA-induced apoptosis in Endothelial progenitor cells.Methods: EPCs(8000 cells/well)were seeded in flat-bottomed 96-well plates.After 24 h,cells were exposed in AT1-AA(2.5,5 and 10?M)for 24 h or 10?M for 3,6,9,12 and 24 h.Then valsartan(0.5,1 and 2?M)for 24 or 1?M for 2,4,6,8,12 and 24 h were added to the wells to determine pre-treatment efficacy.The cells viability were detected by MTT.The apoptosis of EPCs was detected by DAPI staining.Briefly,EPCs were seeded at a density of 2×105 cells/well in flat-bottomed 12-well plates with glass slides.After 24 h,EPCs were treated with AT1-AA(2.5,5 and 10?M),valsartan(1?M)in medium alone or composite processing.EPCs were stained with DAPI and observed under laser confocal scanning microscope(LCSM).The apoptosis rate was measured by calculating the number of cells with nuclear phenotypic changes in 20 different microscopic fields.T1-AA-induced apoptosis in EPCs were measured by Flow cytometry using the Annexin V-FITC and propidium iodide(PI)staining method.Briefly,EPCs were palated at a density of 1×106 cells/dish into 60 mm dishes.After 24 h,EPCs were exposed in AT1-AA(2.5,5 and 10?M),valsartan(1?M)in medium alone or composite processing.AApoptosis was detected with a FAC Sort flow cytometer.Reactive oxygen species(ROS)was detected by cell permeant probe CM-H2 DCFDA.Briefly,EPCs were seeded in 6-well plates overnight and pre-treated with valsartan(1?M)and incubated with AT1-AA(10?M).Cells were incubated with fluorescent probe2',7'-dichlorofluorescein diacetate(DCF-DA)20?M.Then cells monitored at an excitation wavelength of 488 nm and emission of 530 nm by Fluostar Omega Spectrofluorimeter.The level of intracellular calcium in EPCs were determined by Fluo-4 AM staining.Briefly,cells were plated in 96-wells and incubated with Fluo-4 AM fluorescent.Fluorescence measurements were determined at 488 nm for excitation and 522 nm for emission by Fluostar Omega Spectrofluorimeter.EPCs were seeded in 24-well plates and pre-treated with BAPTA,a calcium chelator or calpeptin and an inhibitor of calpain for 1h.Then cells were loaded with Suc-Leu-Leu-Val-Tyr-AMC calpain protease substrate(40M).The fluorescent was detected by Fluostar Omega Spectrofluorimeter at 360 nm for excitation and 460 nm for emission.The express levels of p-ERK,p-e IF-2?,CHOP,Bcl-2 ? Caspase-3 were detected by western blot.Results: AT1-AA inhibited growth of EPCs in dose-and time-dependent.Meanwhile,effective AT1-AA(10?M for 12h)was chosen as optimum for further studies.Then valsartan(0.5,1 and 2?M)for 24 or 1?M for 2,4,6,8,12 and 24 h was added to the wells to determine the pre-treatment efficacy.The results indicated the decline of cell viability in EPCs,which was induced by AT1-AA,was elevated significantly by valsartan and the effective dose and time is 2?M for 4h.EPCs exhibited the apoptotic characteristics of chromatin condensation with typical apoptotic bodies after treated with AT1-AA and the percentage of apoptotic cells was significantly increased in a dose-dependent manner.After pre-treated with valsartan,the apoptosis rate of EPCs was significantly reverse compared with AT1-AA alone.The apoptosis rate of EPCs induced by AT1-AA(10?M)alone increased to 30.3% but reversed to 13.7% after pre-treated with valsartan.These results indicated valsartan inhibited AT1-AA-induced apoptosis in EPCs.AT1-AA increased ROS generation and intracellular calcium as early as 90 min and the effect of AT1-AA on intracellular calcium in EPCs in time-dependent manner.Meanwhile,after EPCs pre-treated with valsartan,the increasing of ROS andintracellular calcium was rescued.Calcium-activated neutral protease(Calpain)activity was also detected after exposed with AT1-AA and result suggested a marked increase.After pre-treated with valsartan,AT1-AA-induced increase of calpain activity was reversed.All these results indicated valsartan inhibited AT1-AA-induced cell apoptosis through decreasing ROS generation and intracellular calcium in EPCs.AT1-AA stimulated ERK1/2 phosphorylation in time-dependent manner and had a marked increase as early as 6h.AT1-AA effectively increased p-ERK,p-e IF-2?,CHOP,and Caspase-3 protein levels along with decreased Bcl-2 expression.After pre-treated with valsartan,the increase of p-ERK,p-e IF-2?,CHOP and Caspase-3 and decrease of Bcl-2 induced by AT1-AA were all reversed.These data indicated valsartan inhibits AT1-AA-induced apoptosis through inhibiting oxidative stress mediated ER stress in EPCs.Conclusions: 1.AT1-AA can regulate the expression levels of p-e IF2 ?,CHOP,Bcl-2 and caspase-3,and can escalate the ROS and Ca2+ Valsartan concentration in EPCs via the activation of ERK pathway,which indicates the potential mechanism that the EPC apoptosis might be induced by ERS-mediated patterm.2.Valsartan can down-regulate the AT1-AA-induced apoptosis in EPCs by suppressing ERS-mediated apoptosis. |
PDF Full Text Request