| ObjectiveChronic psychological stress is closely related to the occurrence and development ofthrombotic CCVD,and promotes the occurrence of thromboembolic events.Recently,we have observed that chronic stress increase plasma DPP-4 levels and cause a series of pathophysiological changes,but the role of DPP-4 in chronic stress related thrombosis formation has not been clarified The aim of the study was investigated the role of DPP-4 in stress-related thrombosis formation in mice,focusing on DPP-4,vVW,ADAMTS13,and PAI-1 associated with inflammation and oxidative stress,and to provid new therapeutic target to prevent and manage stress-related thrombotic CCVD.Methods1.To explore the impact of chronic stress on thrombosis formation in mice,8-week-old male mice were randomly divided into one of the stress+thrombus(Stress group)and thrombus alone groups(Non-stress group).Mice in the stress group were placed into an immobilized stress device without eating and drinking for 4 hrs/day,once a day for 14 consecutive days.Mice were allowed contact with each other and left undisturbed as the non-stress control mice.On the 15th day of chronic stress and non-stress,FeCl3-induced thrombus model was established in the left carotid arteries of both groups(15min).The blood,inguinal fat,and carotid artery tissue were collected from both groups of mice for the following experiments:? Evaluation of the weight and length of the carotid artery thrombus;? Carotid artery HE staining and CD31 immunohistochemical staining to evaluate endothelial injury and thrombosis;?Chemiluminescence assay to determine DPP-4 activity in plasma;? Enzyme-linked immunosorbent assay to evaluate the level of PAI-1,vWF and ADAMTS13 in plasma;? Quantitative real-time PCR to evaluate the levels of PAI-1,ADAMTS13,NADPH oxidase components(p22phox,gp91phox,p47phox and p67phox),ICAM-1,VCAM-1,MCP-1,TNF-?,IL-1?,MMP-2,MMP-9,TIMP-1,TIMP-2,CatS,CatK and other target genes;? Western blot method to determine the levels of the target protein(gp91phox,eNOS,Catalase,SOD-1,SOD-2).2.To explore the effect of DPP-4 on FeCl3 induced thrombosis formation in mice under stress conditions,the mice were randomly assigned to the stress-thrombus model group(Stress group)and the stress-thrombus model+ alogliptin group(S-Ana group).Chronic restraint stress model was established in both of group mice,while the S-Ana group was given the DPP-4 inhibitor alogliptin(30 mg/kg,twice daily by gavage)for 14 days.On the 15th day,FeCb-induced thrombosis model was also established in the carotid arteries of2 group mice,and then the related tissues were isolated for biological and morphological analysis as the part 1 study.3.HUVECs were seeded into the 6-well plates and cultured for 24 hrs.After cultured in serum-free EBM-2 for the overnight,the cells were treated by H2O2 at the indicated concentrations(0 ?M,200 ?M,and 400 ?M)for 24hrs.RT-PCR was used to detect eNOS,VCAM-1,ICAM-1,and MCP-1,MMP-2,and MMP-9,gp91phox,P22phox and other target gene expression levels.4.To explore the DPP-4 inhibition-mediated vasculoprotective effect following pretreatment with alogliptin(0?M,10 ?M?and 30 ?M)for 30 minutes,then the HUVECs were cultured in presence of H2O2 at 400?M for 24hrs.RT-PCR was used to detect the levels of ADAMST13,eNOS,VCAM-1,ICAM-1,CatK,CatS,MCP-1,gp91phox and P22phox genes and/or proteins.Result1.Chronic stress promoted FeCl3 induced thrombosis formation on the carotid artery of mice Stress reduced body weight and increased the weight and length of thrombus.HE staining showed that the thrombus-induced carotid arteries had decreased number of CD31+endothelial cells.ELISA results showed that plasma DPP-4 activity and PAI-1,vWF levels were significantly increased,while plasma ADAMTS13 levels were significantly reduced in the stressed mice compared to the non-stressed mice.Quantitative real-time PCR results showed that stress markedly increased the investigated inflammation(ICAM-1,VCAM-1 and MCP-1),oxidative stress(p22phox,p47phox,p67phox,and gp91phox,proteolysis(CatK and CatS,MMP-2,MMP-9,TIMP-1,TIMP-2),and thrombosis(PAI-1)related genes in the injured carotid arteries.In contrast,it reduced eNOS mRNA expression levels.Quantitative western blotting data revealed that stress also enhanced he levels of gp91phox protein and decreased the levels of eNOS,Catalase,SOD-1,and SOD-2 protein in the injured carotid arteries.Similarly,we observed that stressed inguinal adipose tissues had increased levels of the investigated oxidative stress(p22phox and gp91phox)and inflammation(TNF-?,IL-1?),MCP-1,ICAM-1,and VCAM-1).In addition,the numbers of blood white blood cell,neutrophil,and platelets were higher than in that control non-stressed mice.2.The nhibition of DPP-4 significantly improved stress related FeCl3 induced thrombosis formationThe DPP-4 inhibitor anagliptin markedly reduce the weight and length of thrombus.Anagliptin improved the number of CD31+endothelial cells in the injured arteries as well as blood white blood cells,neutrophils,and platelets in the stressed mice.ELISA results showed that anagliptin significantly reduced plasma DPP-4 activity as well as blood PAl-1 and vWF levels and increased ADAMTS13 levels in the stressed mice.Anagliptin treatment resulted a decrease in the levels of the inflammation(ICAM-1,VCAM-1 and MCP-1),oxidative stress(P22phox,P4phox,p67phox,and gp91phox,proteolysis(CatS,CatK,MMP-2,MMP-9,TIMP-1,and TIMP-2)and thrombosis(PAI-1)related genes in the injured arteries.Moreover,the levels of gp91phox=protein were lower and the lvels of eNOS,Catalase,SOD-1,and SOD-2 were higher in the injured carotid arteries of the S-Ana mice than in that of the stress alone mice.Likewise,anagliptin also migrated the levels of the investigated genes(PAI-1,P22phox,gp91phox,TNF-?,IL-1?,MCP-1,ICAM-1 and VCAM-1 in the inguinal fats.3.The DPP-4 inhibitor exhibited a protective effect on H2O2 induced HUVEC injury by inhibiting oxidative stressH2O2 reduced the expression levels of ADAMTS13 mRNA and eNOS protein in HUVECs in a dose-dependent manner.In contrast,it resulted an increase in the mRNA levels of inflammatory chemokines(ICAM-1,VCAM-1,and MCP-1)and proteolytic enzymes(MMP-2,MMP-9,CatS,Catk).Alogliptin dramatically improved these harmful changes in response to H2O2.Conclusion1.Chronic stress increased plasma DPP-4 level and activity,carotid arterial inflammation,oxidative stress production,and proteolysis,leading to imbalance of vWF and ADAMTS13,thereby promoting thrombosis.2.Increased DPP-4 activity caused by chronic stress can enhance subcutaneous adipose inflammation and PAI-1 expression,leading to promoting thrombosis.3.DDP-4 inhibition mitigated inflammation,oxidative stress production,and proteolysis as well as rectified vWF and ADAMTS13 imbalance in carotid arteries and/or adipose,thereby reducing carotid endothelial damage and thrombosis.4.In vitro,DPP-4 inhibitor prevented oxidative stress-induced harmful thrombotic effects in the HUVECs.Thus,DPP-4 inhibition appears to improve FeCl3-induced thrombosis in mice that received stress,possibly via the improvement of ADAMTS13 and oxidative stress,suggesting that DPP4 could become a novel therapeutic target for chronic psychological stress-related thrombotic events in metabolic cardiovascular disorders. |
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