The Effect And Mechanism Of Vildagliptin On Improving Carotid Artery Stenosis In Type 2 Diabetic Mice

PART Ⅰ: Effect and mechanism of vildagliptin on carotid stenosis in T2 DM mice OBJECTIVE: In stent restenosis(ISR)is one of the most serious complications next to percutaneous coronary intervention(PCI).Type 2 Diabetes mellitus(T2DM)is an independent risk factor for ISR and is closely related to the occurrence and development of ISR.In recent years,a new type of hypoglycemic drug,known as Dipeptidyl Peptidase-4 Inhibitor(DPP-4i)has been widely used in the treatment of T2 DM.An increasing number of studies have proved that DPP-4i not only lower blood glucose levels effectively but also suggest therapeutic potential for a variety of cardiovascular diseases.This section aims to investigate the effects of DPP-4i vildagliptin on carotid stenosis in T2 MD mice induced by injury and its related mechanisms.METHODS: Twenty male T2 MD mice were enrolled in this study.The carotid stenosis model was successfully constructed through ligation injury.The mice were randomly divided into 2 groups and treated with vildagliptin and placebo separately for 4 weeks.The carotid artery was taken for pathological section after that.HE staining was used to evaluate the neointimal thickening of the carotid artery and to measure the stenosis.Immunohistochemistry and immunofluorescence staining were performed to detect the density of α-SMA,PCNA and CD31 positive cells,and to assess the proliferation activity of the endothelial cells and vascular smooth muscle cells(VSMCs).Immunoblotting analysis of α-SMA,PCNA,CD31,Cyclin D1,CDK2,phospho-p65,phospho-IKKα/β,CHOP,GRP78,ATF-6,phospho-eIF2α,Phospho-IER1 expression level was used to evaluate the effect and mechanism of vildagliptin on ER-Stress and NF-kB signaling pathways.RESULTS: After vildagliptin treatment,the lumen area was increased while the neointimal area was decreased significantly.The numbers of abnormally proliferating VSMCs,as well as the number of α-SMA and PCNA double positive cells,were decreased in the carotid artery,while there was no significant change in the number of CD31 positive cells.Immunoblot results showed that the expression of Cyclin D1,CDK2,phospho-p65,phospho-IKKα/β,CHOP,GRP78,and phospho-IER1 were significantly decreased compare to those vildagliptin-free mice.Conclusion: Neointimal hyperplasia due to abnormal proliferation of VSMCs is the main cause of carotid stenosis.Vildagliptin inhibits the activation of ER-Stress and NF-kB pathways in carotid smooth muscle cells,and affects the proliferation of neoplastic intima by regulating the cell cycle of VSMCs to reduce its proliferative activity and to improve its hypertrophy,thus to reduce the abnormal proliferation of neointimal and to reverse the carotid stenosis.PART Ⅱ: Mechanism of proliferation and hypertrophy of VSMCs induced by high glucose regulated through ER-Stress and NF-κB signalingOBJECTIVE: Previous studies have shown that hyperglycemia and injury stimulation promote ER-Stress and NF-κB activation in arterial VSMCs and cause abnormal proliferation and hypertrophy of VSMCs.The interaction between ER-Stress and NF-κB pathway constitutes a positive feedback loop,which is involved in regulating cell proliferation and apoptosis.In the first section of our study,we found that vildagliptin inhibits the activation of ER-Stress and NF-κB as well as the proliferation and hypertrophy of VSMCs.Since vildagliptin cannot act on VSMCs directly,we further investigate the effects of ER-Stress and NF-κB signaling on the proliferation and hypertrophy of VSMCs through drug blockade,and the relationship between these two pathways.METHODS: Rat aortic smooth muscle cells were extracted and treated with conventional glucose culture,high glucose culture,taurine blocking ER-Stress and ACHP blocking NF-κB treatment.CCK-8 was applied to detect and analyze the time-dependent cell proliferation.Ed U staining was used to assess the cell proliferation,and patch clamp was used to detect the cell hypertrophy by whole cell membrane capacitance;α-SMA,PCNA,phospho-IKKα/β,phospho-IER1 expression levels were detected by immunoblotting and to analyze the upstream and downstream relationship between ER-Stress and NF-κB interactions.RESULTS: VSMCs blocked in ER-Stress and NF-κB pathway showed different numbers of cells reduction at 24,48,and 72 hours compared with VSMCs treated with high glucose.The proportion of Brdu-positive cells was significantly decreased in this group.The whole cell membrane capacitance was significantly reduced,and the expression levels of α-SMA and PCNA in the cell proteins were significantly decreased.In addition,immunoblotting revealed that the blockade of the ER-Stress pathway caused a significant decrease in the expression of phospho-IER1 and phospho-IKKα/β,whereas blocking the NF-κB pathway only reduced the expression of phospho-IKKα/β.There is no significant effect on the expression of Phospho-IER1.Conclusion: High glucose treatment can induce proliferation and hypertrophy of VSMCs,while drug blockade of ER-Stress and NF-κB pathway can effectively reduce the proliferation activity and hypertrophy of VSMCs.ER-Stress acts as an upstream pathway of NF-κB and its inhibition further reduced the activation of the NF-κB pathway and ultimately inhibited the proliferation and hypertrophy of VSMCs through the regulation of the phosphorylation of phospho-IKKα/β by phospho-IER1.