Effect Of Atorvastatin On Restenosis Post-PTA In Diabetic Lower Extremity Peripheral Arterial Disease

ObjectiveThe prevalence rate of Atherosclerosis(AS)was higher in diabetes mellitus.frequently-occurring in aorta,coronary artery and peripheral artery.AS of peripheral artery often occurred in lower extremity arteries especially in small arteries under the knees with a difficult treatment because of small diameter and low pressure,comparing with Endovascular Stent Implantation in coronary artery.Percutaneous transluminal angioplast(PTA)became an important therapeutic means for AS in lower extremity arteries especially in small arteries under the knees in diabetes,by means of advantages of good compliance of a new designed balloon catheter.However,a high restenosis rate of 30%after PTA was a big problem for its long term effect.As a result,the prevention and treatment of restenosis after PTA became the key to the question of treatment for lower limb arteriosclerosis in diabetes.The Statins is Hydroxymethyl glutaric acyl coenzyme A reductase inhibitors.Studies have shown that Statins have the effec of resistance to atherosclerosis and lower cholesterol levels including:inhibition of vascular endothelial inflammatory response,Stable atheromatous plaque,improvement endothelial function,inhibition of artery intimal hyperplasia.As a result.The Statins was used for the prevention of restenosis after PTA.However,the mechanism of restenosis was different from atherosis.The proliferation and migration of vascular smooth muscle cells played a key role to restenosis.Previous clinical trials found that the Statins had a poor Clinical curative effect for the prevention of restenosis,although the proliferation,migration and apoptosis of vascular smooth muscle cells could be inhibited by the Statins in some in vitro studies.Then could the restenosis after PTA be inhibited by the Statins?To solve this question,intuitive and accurate evidence from animal experiment was necessary as the limitations vascular specimens.A" single balloon injury" was used for the restenosis model at present.The disadvantage of this method is that this kind of restenosis model was build on the health of blood vessels instead of on the atherosclerotic vascular,which was different from the intravascular restenosis process.First of all,the model of Atherosclerosis of iliac artery in diabetic rabbit was build by once " single balloon injury",and then PAT was done.which could simulate the intravascular restenosis process after PAT in diabetics.Secondly,to clear the restenosis process and features after PAT by observingthe situation of Vascular restenosis at different time.Thirdly,to clear if restenosis after PAT could be restrained by the Statins.Methods1.Modeling and GroupingMale New Zealand big-eared white rabbit(1.7KG)was selected in group with 1-week adaptive feed and high fat feed till the end of the experiment.1.1 Building of restenosis model(1)Building of diabetes model:after alloxan(80mg/kg)was injected by ear marginal vein 1 week,blood glucose was monitored to verify if diabetes model was built successfully.(2)Building of AS model:" single balloon injury" was done on the iliac artery after 1 week later after the building of diabetes model,4 weeks later after "single balloon injury",AS model was verified by ultrasound.(3)Building of restenosis model:PTA was operated on the vascular area of AS.1.2 Group of Experiments:model rabbits of restenosis were divided into four groups random:7-days restenosis group,14-days restenosis group.28-days restenosis group,28-days atorvastatin group,and control group.Processing method:(1)7-days restenosis group:Same volumel physiologica saline lavage and model rabbits were put to death on the 7th day;(2)14-days restenosis group:Same volumel physiologica saline lavage and model rabbits were put to death on the 14th day;(3)28-days restenosis group:Same volumel physiologica saline lavage and model rabbits were put to death on the 28th day;(4)28-days atorvastatin group:atorvastatin(2.5mg/kg/d)lavage and model rabbits were put to death on the 28th day;(5)control group:The same operation was done on restenosis model rabbits but "Balloon dilatation".2.Subsistence of sample and Index detection2.1 According to different groups.blood sample and specimens of iliac artery were collected at different point-in-time(the 7th 14th,28th day after PTA).2.2 Blood index detection:serum total cholesterol,triglycerides,low density lipoprotein.high-density lipoprotein.2.3 Vascular morphology detection:Specimens of iliac artery were formaldehyde fixed,embedded in paraffin slice after dehydration and the detection was aa follows:(1)Vascular pattern was observed by HE staining;(2)The morphology of smooth muscle and collagen were observed by Masson staining;(3)Elastic fiber morphology was observed by EVG staining;(4)Vascular area with Vascular patches,intimal area and media area were measured by Image-Pro Plus 6.0 Image analysis software to calculate Vascular stenosis rate and The ratio of intima to media area.2.4 Immunohistochemical analysis:The number of proliferating cells of vascular plaque location was measured by immunohistochemical analysis of proliferating cell nuclear antigen(PCNA).PCNA index was calculated as Number of positive cells of PCNA/Total cell count× 100%.2.5 Immunofluorescence staining:The migration of vascular smooth muscle cell was identified by immunofluorescence staining of ?-SMA.3.Statistical analysisSPSS20.0 statistical software was used for data processing.Data was expressed as x ±s.One-way analysis of variance was used for comparison between the groups.P<0.05 was regarded as statistical differences.Results1.Animal model and Blood sugarDuring the process of diabetes model establishment,5 New Zealand big-eared white rabbit were dead and 10 were not qualified on the Blood sugar.Restenosis model was successfully built.Blood glucose level was above 300mg/dl in all the diabetes rabbit during the whole experimental process.2.Blood index detectionCompared with 28-days restenosis group and control group,triglycerideserum,total cholesterol and LDL-C of 28-days atorvastatin group declined(P<0.05).Triglycerideserum,serum total cholesterol and LDL-C were similar and no statistical differences between 7-days restenosis group,14-days restenosis group.28-days restenosis group and control group(P>0.05).HDL-C was no statistical differences among all the groups above(P>0.05).3.The progress of restenosis at different time point3.1 Vascular morphology detection:HE staining:Compared with control group.stenosis of vessel and Thickening of the intima happened in all the restenosis groups.Masson staining:The thicken inner membrance-was smooth muscle cells and substrate.EVG staining:The thicken inner membrance was elastic fibers,the elastic plate breaks out,mesolamella was scaling-down and lastic fiber fractured.With the passage of time,the changes above gradually deepened.3.2 Vascular stenosis rate and Inner membrance/Mesolamella:Compared with control group,the thickness of the inner membrance of all the restenosis groups increased obviously with statistical differences(P<0.05).From the 7th day to 14th day and to 28th day,the stenosis rate of all restenosis groups increased obviously with statistical differences(P<0.05),and from the 7th day to 14th day.increased more obviously.The increasing tendency of the thickness of the inner membrance and the ratio of inner membrance and mesolamella were similar with the stenosis rate with statistical differences(P<0.05).These results showed that the restenosis is a gradual progress with a fast speed at the beginning.3.3 The immunofluorescence of a-SMA:The positive expression of a-SMA in the arterial midbrain of the control group was strong while in the restenosis groups was not continuous.There was no a-SMA positive cells in the arterial intima of the control group.?-SMA positive cells were seen in the arterial intima of all the restenosis groups.Vascular smooth muscle cell appeared in the inner membrance of all the restenosis groups.From the 7th day to 14th day and to 28th day,the increasing tendency of the migration of vascular smooth muscle cell was consistent with the stenosis rate.3.4 The immunohistochemical of PCNA:Diffeerent from the index above,the PCNA index of was the maximum of 7-days restenosis group.Compared with 7-days restenosis group,the PCNA index decreased in 14-days restenosis group.There was no statistical differences between 14-days restenosis group and 28-days restenosis group(P>0.05).These results showed that the induce of endothelial cell proliferation was at the the greatest degree on the 7th day,then declined and became stable.Keeping the trend of proliferation,the speed of proliferation became reduced and stable 7days later.4.The effects of Atorvastatin to restenosis4.1 Vascular stenosis rate and Inner membrance/Mesolamella:The vascular stenosis rate and the ratio of inner membrance and mesolamella of 28-days atorvastatin group were similar with those of 28-days restenosis group(P>0.05).The restenosis was not inhibited by atorvastatin.4.2 The immunofluorescence of a-SMA:A large number of SMC could still be observed in the inner membrance of 28-days atorvastatin group,compared to 28-days restenosis group with no statistical differences(P>0.05).4.3 The immunohistochemical of PCNA:There was no statistical differences on PCNA index between 28-days atorvastatin group and 28-days restenosis group.All these results showed that.for animals experiment,the migration of vascular smooth muscle cell and the endothelial cell proliferation were not inhibited by atorvastatin,and further testified that the restenosis was not inhibited by atorvastatin.Conclusion1.In our study,diabetes AS model was built by " single balloon injury" method and then PTA was operated to build the restenosis model.This restenosis model could simulate the process of restenosis process after PTA of lower limb artherosclerosis of diabetes,and could be regarded as an ideal model.2.In animal models,the degree of restenosis post-PTA aggravated over time,with a fastest growing from the 7th day to 14th day and significant restenosis was observed on the 28th day.3.Although atherosclerosis could be prevented by the statins,and the proliferati on and migration could be inhibited by the statins in some In vitro and vivo s tudies,restenosis was not effected by the atorvastatin in our experiment.ObjectiveThe research results of our first part showed that:the migration,proliferation of VSMCs and the restenosis were not affected by the conventional dose atorvastatin in animal model.In the discussion part of first part,we summarized the probable cause of negative result including the insufficient blood drug concentration which including absolute and relative insufficience.The absolute insufficient blood drug concentration was caused by dose-dependent fashion of Statins which means improvement of therapy results could be done by dosage adjustment.Some results proved that Statins had dose-dependent fashion for the migration,proliferation of VSMCs,the inhibition of restenosis post coronary PCI,the inhibition of inflammatory reaction of restenosis and the protection of endothelial injury.The relative insufficient blood drug concentration was related to different animal model.A "single balloon injury" was used for the restenosis model at present,however,this kind of restenosis was caused by the injury of normal vessels which was not similar with the true restenosis.Our "double balloon injury" model was based on the AS vessels which was severe and similar with the true restenosis..And the blood drug concentration may be relative insufficient because of severe restenosis,as a result,intensive atorvastatin therapy was used for a probable relative ideal effect.So the research objective of the second part could be summarized as follows:if the migration,proliferation of VSMCs by intensive atorvastatin dose were different from that of conventional dose and if the influence on restenosis of atorvastatin was on dose-dependent fashion.Methods1.Modeling and GroupingMale New Zealand big-eared white rabbit(1.7KG)was selected in group with 1-week adaptive feed and high fat feed till the end of the experiment.1.1 Building of restenosis model(1)Building of diabetes model:after alloxan(80mg/kg)was injected by ear marginal vein 1 week,blood glucose was monitored to verify if diabetes model was built successfully.(2)Building of AS model:" single balloon injury" was done on the iliac artery after 1 week later after the building of diabetes model.4 weeks later after"single balloon injury",AS model was verified by ultrasound.(3)Building of restenosis model:PTA was operated on the vascular area of AS.1.2 Group of Experiments and Processing:model rabbits of restenosis were divided into 3 groups random:(1)Control group.Same volume]physiologica saline as Conventional group lavage from the the day of PTA to the 28th day:(2)Conventional atorvastatin dose group(Conventional group).Conventional dose atorvastatin(2.5mg/kg/d)lavage from the the day of PTA to the 28th day;(3)Intensive atorvastatin dose group(Intensive group).intensivedose atorvastatin(1 Omg/kg/d)lavage from the the day of PTA to the 28th day.2.Subsistence of sample and Index detection2.1 The animals were sacrificed at 28 day after operation.and Blood sample and Specimens of iliac artery were selected.2.2 Blood index detection:TC,TG,LDL-Cand HDL-C.2.3 Vascular morphology detection:Specimens of iliac artery were formaldehyde fixed-embedded in paraffin slice after dehydration and the detection was aa follows;(2)The morphology of smooth muscle and collagen were observed by Masson staining;(2)Elastic fiber morphology was observed by EVG staining;(3)Vascular area with Vascular patches,intimal area and media area were measured by Image-Pro Plus 6.0 Image analysis software to calculate Vascular stenosis rate and The ratio of intima to media area.2.4 PCNA Immunohistochemical analysis:The number of proliferating cells of vascular plaque location was measured by immunohistochemical analysis of proliferating cell nuclear antigen(PCNA).PCNA index was calculated as Number of positive cells of PCNA/Total cell count×100%.2.5 ?-SMA Immunohistochemical staining:The migration of vascular smooth muscle cell from tunica media vasorum to tunica intima was identified by ilmmunohistochemical staining of ?-SMA.3.Statistical analysisSPSS20.0 statistical software was used for data processing.Data was expressed as x ±s.One-way analysis of variance was used for comparison between the groups.P<0.05 was regarded as statistically significant.Results1.Animal model and Blood sugarDuring the process of diabetes model establishment,3 New Zealand big-eared white rabbit were dead and 9 were not qualified on the Blood sugar and 18 diabetes rabbits were successfully built.During the process of restenosis among 18 diabetes rabbits.3 were dead and 15 were successfully built as restenosis model Blood glucose level was above 300mg/dl(16.7mmol/L)in all the diabetes rabbit during the whole experimental process.2.Blood index detectionC'ompared with control group,TG,TCand LDL-C of Conventional group and Intensive group descend(P<0.05);There was no significant differences between Conventional group and Intensive group on HDL-C(P>0.05)and HDL-C of Conventional group was higher than control group and Conventional group(P<0.05).3 The effects of different dose of Atorvastatin to restenosis3.1 Vascular stenosis rate and Inner membrance/Mesolamella:The vascular stenosis rate and the ratio of inner membrance and mesolamella showed that:Compared with control group,both stenosis rate and the ratio of inner membrance and mesolamella of Conventional group and Intensive group showed no apparent decline and no significant differences(P>0.05).The restenosis was not inhibited by either Conventional dose nor Intensive dose of atorvastatin.3.2 The immunohistochemical of a-SMA:Compared with control group,the positive cell number of a-SMA of Conventional group and Intensive group showed no apparent decline and no significant differences(P>0.05).which showed that either Conventional dose nor Intensive dose of atorvastatin.could not reduce the the number of VSMCs and even the intensive dose of atorvastatin could not restrain the the migration,proliferation of VSMCsto the tunica intima.3.3 The immunohistochemical of PCNA:Compared with control group,the PCNA index of Conventional group and Intensive group showed no apparent decline and no significant differences(P>0.05),which showed that either conventional dose nor intensive dose of atorvastatin.could not reduce the the number of proliferating cells of restenosis part and even the intensive dose of atorvastatin could not restrain the proliferation and dedifferentiation.ConclusionSimilar with conventional dose of atorvastatin.the intensive dose of atorvastatin had no influence on migration,proliferation and dedifferentiation of VSMCsand the appearance,development of restenosis.Atorvastatin had no dose-dependent fashion on the restenosis in the post-PTA restenosis model of diabetic lower limb arteriosclerosis.