A Mechanism Research About ATRA Mediated KLF5 During The Prevention Of Post Transplant Vein Stenosis

Background and Purposes: CABG(Coronary artery bypass graft)is one of the most important therapy methods for atherosclerosis coronary disease.So far,great saphenous veins are still the commonest vessel graft material for CABG.Although surgical techniques and surgical instruments have been significantly improved,VGF(vein graft failure)occurs frequently in CABG patients and brings seriously bad impact to surgical operation effectiveness and CABG patients’ prognosis.Some studies investigated CABG patients as long as 10 years and showed that the incidence of VGF was more 50%.More than 90% acute VGF which occurred in one month after operation were due to acute thrombosis.Regular anticoagulation therapy could significantly reduce the incidence of acute VGF.But more than 60% chronic VGFs which occurred after 10 years since CABG operation were due to the proliferation of SMC(smooth muscle cell).Furthermore,the proliferation of SMC could mediate platelet adhesion and atheromatous plaque which could aggravate VGF.However,the mechanism of SMC proliferation in VGF is still unclear.There is no effective therapy method to prevent SMC proliferation.So the mechanism of SMC proliferation in VGF is the focus in cardiac surgery research field.ATRA(all—trans retinoic acid)is the metabolic intermediate of vitamin A in human body.ATRA has a wild physiological and pharmacological activity.ATRA can regulate vertebrate animal development and cell differentiation.Especially,ATRA plays an important role in embryonic development and regulates morphogenesis of vertebrate embryogenesis.With the continuous research of ATRA function,the tight relationship between ATRA and cardiovascular diseases has been proven.Lots of studies showed that ATRA could inhibit the proliferation of vessel endothelial cells and SMC.Inhibition of vessel SMC is an effective method for VGF prevention.According to a lot of basic studies,we speculate that ATRA could inhibit the proliferation of vessel graft SMC to prevent VGF.KLF5(Krüppel-like Factor 5)is an important member of Krüppel-like Factor family.KLF5 plays a role as transcriptional regulatory factor in embryonic development,cell proliferation and tumorigenesis.KLF5 can start the transcription of gene Myo D(Myogenic Differentiation Antigen)which can mediate myofibril and myotube proliferation in injured muscle cell and promote muscle tissue growth.Inhibition of KLF5 gene expression can reduce vessel muscle cell proliferation effectively.Lots of basic studies showed that there was a positive correlation between KLF5 gene expression and SMC proliferation.In our pre-experiment,we observed that the level of KLF5 gene expression in vessel graft was obviously lower in ATRA intervention group than control group.Furthermore,we obtain a big speculation: could ATRA inhibit SMC proliferation and prevent vessel graft angiostegnosis due to adjust the gene expression level of KLF5? This study is aimed to figure out the regulation role of ATRA in KLF5 gene expression which can reduce vessel graft SMC proliferation.Methods: I.Animal experiment Animal model establishment: we randomly chosen 42 new Zealand rabbits which were randomly separated into 7 groups(group A、B、C、D、E、F、G).Each group had 6 new Zealand rabbits.Group A、B、C were control groups and Group D、E、F were experiment groups.Group G was a blank control group.Right cleidomastoid incision was adapted in both experiment groups and control groups.We freed right jugular vein as long as possible and trimmed a section as long as 2cm as vessel graft.Then we replaced vessel graft to right carotid artery.After operation,rabbits in experiment groups were feed ATRA.Rabbits in control group were feed placebo.Rabbits in group G were sacrificed directly to harvest jugular vein specimens.Rabbits in group A and D were sacrificed to harvest vessel graft specimens after 2 weeks.Rabbits in group B and E were sacrificed to harvest vessel graft specimens after 4weeks.Rabbits in group C and F were sacrificed to harvest vessel graft specimens after 8 weeks.Immunohistochemical tests: compared of group A、B、C、G,we observed vessel graft lumen situation by HE stain.The situation of KLF5 and Ki67 distribution on vessel graft wall was showed by immunohistochemistry.The level of vessel graft SMC was also measured.Compared of group A、B、C、D、E、F,we analyzed the level of KLF5 gene expression influenced by ATRA and the vessel graft angiostegnosis situation.II.Experiment in vitro In vitro cell model establishment: we bought Huv SMCs(human umbilical vein smooth muscle cells)and cultured Huv SMCs.We identified Huv SMCs with α-SMA staining.Then we used human adenovirus which carried KLF5 gene to transfect Huv SMCs.⑴ Scratch test was executed to observe the inhibition of Huv SMCs migration.⑵ CCK-8 test was executed to observe the inhibition of Huv SMCs proliferation.⑶ RT-PCR was executed to test the expression levels of KLF5 downstream genes.⑷ Western-blot test was executed to analyze the correlation with ATRA and KLF5 downstream genes.⑸ Co-immunpprecipatation test was executed to find the RAR-α receptor competitive binding between ATRA and KLF5 protein.III.Statistics methods We used SPSS version 21.0 for statistics analysis.The measurement data was described as mean ± standard deviation.Two independent samples t-test was used to measure the data between two groups if they obey normal distribution and homogeneity of variance.Single factor analysis of variance was used to measure the data of multiple groups if they obey normal distribution and homogeneity of variance.Two groups were compared by LSD-t test.Image processing was achieved by software Graph Pad.P<0.05 was considered statistically significant.Results: ⑴ The proliferation of vessel graft SMC was observed obviously and the level of KLF5 gene expression was significantly higher.We established the venous arterial rabbit model by cuff cannulation.We observed that vessel intima-media thickness was significantly increased through HE stain.According to KLF5 and Ki67 immunohistochemical test results,KLF5 protein distribution on vessel graft wall was wider than control group and the level of Ki67 expression which was considered to be relative with SMC proliferation was also much higher.⑵ ATRA could be used to prevent vessel graft angiostegnosis,but had no influence to KLF5 gene expression.We observed that vessel intima-media thickness in experiment groups was significantly decreased than in control groups by HE stain.According to KLF5 and Ki67 immunohistochemical test results,KLF5 protein distribution on vessel graft wall in experiment groups had no difference with in control groups and the level of Ki67 expression which was considered to be relative with SMC proliferation was also much lower.⑶ KLF5 gene could promote vessel SMC proliferation and migration.In vitro cell CCK-8 test showed that proliferation of SMC was obviously enhanced after Huv SMCs were transfected by human adenovirus.In vitro cell scratch test showed that migration of SMC was obviously enhanced after Huv SMCs were transfected by human adenovirus.⑷ ATRA influenced the function of KLF5 gene Western-bolt showed that ATRA had no influence to the gene expression of KLF5.CCK-8 test result proved that ATRA could inhibit vessel SMC proliferation against KLF5.Scratch test result also proved that ATRA could inhibit vessel SMC migrant against KLF5.RT-PCR results showed that i NOS as a downstream gene of KLF5 was considered to be negative correlation with ATRA.Co-immunpprecipatation test results proved that ATRA could competitively bind RAR-α receptor with KLF5 protein.Conclusions: According to those experiments results above,we got conclusions that ATRA could inhibit Huv SMCs migration and proliferation.ATRA could not inhibit KLF5 gene expression directly.ATRA could competitively bind RAR-α receptor with KLF5 protein.i NOS as a downstream gene of KLF5 was considered to be negative correlation with ATRA.