The Function And Mechanism Of Tiam1 In Intimal Hyperplasia

Intima hyperplasia is the general pathobiology phenomenon after the vascular injury. It is an important plot in the process of arterior atherosclerosis. Intima hyperplasia is also a significant reason for re-stenosis of bypass vessels and cornary stents. In detail, intima hyperplasia is the thicking of vascular intima layer. Normally, the intima layer is only composed of endothelial cells and subendothelial layer. The latter includes little elastic fiber、collagenous fiber and sometimes few vascular smooth muscles. Intima hyperplasia is a complicated process. Several mechanisms participate in it, including endothelial injury、the proliferation and migration of vascular smooth muscle cells、the degradation and reconstruction of extracellular matrix、lipid storage and imflammation reaction. Currently, there is no effective inhibition and curement for intima hyperpalasia. To investigate the mechanism and pathbiology of intima hyperplasia is beneficial to reveal effective methods.The guanine nucleotide exchange factor Tiam1 is mainly the regular protein for Rac1, one of small G proteins. By activating Rac1, Tiam1 regulates the cytoskeletal activities, cell migration, endocytosis and cell growth. What’more, it was reported that mutant mice lacking ephrin-B2 expression in vascular smooth muscle developed vessel wall defects and aortic aneurysms, which were associated with impaired Tiam1 expression. However, the direct relationship among Tiam1、vascular smooth muscle cell and intima hyperplasia has not been reported. Therefore, our experiment reveals that mutant mice without Tiam1 are less suspectible to intima hyperplasia and it also reveals the mechanism in relatively depth.In this study, five parts are included as follow:1. the expression of Tiam1 in vascular smooth muscle cells(i) objectiveTo detect the expression level of Tiam1 in vascular smooth muscle cells(ii) methodRat primary cultured VSMCs were assigned to four groups:siCtr group, siCtr+PDGF group、siTiam1 group、siTiam1 +PDGF group;Examining the expression of Tiam1 in this four groups by Western blotting;(iii) resultsiCtrl group expresses Tiam1 and it expression level increases with PDGF. The siTiam1 group espresses less than the siCtrl group even without expression. When applied with PDGF for 12 min, the expression level increased apparently.(iv) conclusionThe VSMC expresses Tiam1.2. the function of Tiam1 on VSMCs migration(i) objectiveTo detect the function of Tiam1 on VSMCs migration(ii) methodRat primary cultured VSMCs were assigned to four groups after transfection: siCtrl group、siCtrl+PDGF(20ng/ml)、si Tiam1 group、siTiam1+PDGF(20ng/ml) group. They are scratched seperately and observed by taking images.(iii) resultThe scratched areas in siTiam1+PDGF group after 12 h is larger than the ones in siCtrl+PDGF group.(iv) conclusionTiam1 promote the VSMC migration in a certain extent.3. the function of Tiam1 on VSMCs proliferation and apoptosis(i) objectiveTo detect the function of Tiam1 on VSMCs proliferation and apoptosis(ii) methodRat primary cultured VSMCs were assigned to five groups: the negative group、siCtrl+complete medium(20%FBS) group 、 siCtrl+starvation(0.4%FBS) group 、siTiam1+complete medium(20%FBS) group、siTiam1+starvation(0.4%FBS) group. The groups are stained with Annexin-FITC/PI for flow cytometry analysis for investgating apoptosis. The groups are stained with EdU for detacting cell proliferation.(iii) result(a) the flow cytometry analysis shows that both siCtrl group and siTiam1 group indicate late phase apoptosis indifferently.(b) compared with siCtrl group, less cells in the siTiam1 group are both passive.(iv) conclusionTiam1 promote the VSMC proliferation and have little function on apoptosis.4. the function of Tiam1 in VSMC through PI3K/Akt、MAPK/P38 signaling(i) objectiveTo detect the function of Tiam1 in VSMC through PI3K/Akt、MAPK/P38 signaling(ii) methodRat primary cultured VSMCs were assigned to two groups after transfection: siCtrl group、siTiam1 group. Then each one is divided into six subgroup according to the time after adding PDGF(20ng/ml): 0、5、15、30、60、90min. The cells are collected for Western Blot to detect the expression level of p-Akt、p-P38.(iii) resultAfter added with PDGF for 5min and 15 min, the expression level ofp-P38 in siTiam1 group is significantly less than siCtrl group. The expression level of p-Akt in siTiam1 group is significantly less than siCtrl groupAfter added with PDGF for 30min、60min and 90 min.(iv) conclusionTiam1 possibly regulate the intima hyperplasia in p-Akt、p-P38 signalings.5. the function of Tiam1 in intima hyperplasia after vasular injury(i) objectiveTo detect the function of Tiam1 in intima hyperplasia after vasular injury(ii) method(a) Identified the genetype of KO mice by PCR and gene sequencing(b) Performing carotid artery wire injury surgery on knockout(Tiam1-/-) mice and wild-type(Tiam1+/+)mice;Hematoxylin-eosin staining was performed on arteries(24 days after ligation);Examining the intima,the media and adventitia area,counting the intima-to-media(I/M) ratio in both the Tiam1-KO arteries and WT controls.(iii) result(a) according to gene sequencing diagram, the double mutation type is knockout, the single mutation type is hetre, the one without mutation is wild-type.(b) The neointima in the Tiam1 KO mice was significantly thinner compared with the WT mice(P<0.0.5). I/M ratio in KO mice is less than that in WT mice.The area of media and adventitia were similar between KO mice and WT mice.(iv) conclusionThe mutant mice without Tiam1 undergo less intima hyperplasia.