The Role Of T-cadherin In Diabetic Vascular Injury And The Mechanisms

BackgroundSince the 21 st century,Diabetes mellitus(DM)has now become a major threat to human health with its incidence in the world rapidly increased.About over 90% of diabetic patients are type 2 diabetes mellitus(T2DM)characterized by insulin resistance.Complications of T2 DM,especially cardiovascular complications,have become an important cause of death of diabetes.Vascular endothelial dysfunction,which is characterized by the decrease of endothelium-dependent vasodilatation,is a major change in NO production as the main features of the pathological changes.As a trigger for atherosclerosis,endothelial dysfunction plays a key role in the development of T2 DM.However,the current effective treatment to improve endothelial injury was very limited,as well as the corresponding clinical application doesn't have made satisfactory progress.Therefore,it is of great significance to elucidate the potential molecular mechanism of vascular endothelial dysfunction in the development of type 2 diabetes mellitus and to find the corresponding therapeutic target for diabetic cardiovascular complications.T-cadherin(T-cad)is a member of the cadherin family.But unlike other family members,it is located on the surface of cell membrane without intracellular domain.T-cad is mainly expressed in the aorta and in the heart.Numerous studies found that T-cad plays an important role in regulating endothelial cell function.Notably,recent research reported that in the early stage of the development of type 2 diabetes,T-cad is more expressed in the region of atherosclerosis than in those of the normal artery.In addition,there are clinical studies said that T-cad expression levels and the incidence of type 2 diabetes are linked.But so far,the knowledge about the role of T-cad in diabetic cardiovascular complications still lacks.The associated research is quite limited,especially for those of in vivo functional research.Therefore,based on our lab's previous work,the present study constructed the model of type 2 diabetes mellitus in mice successfully,and designed to explore the role of T-cad in diabetic cardiovascular injury and its mechanism both in vivo and in vitro.Aims1.To clear the role of T-cad expression for endothelial dysfunction and vascular injury in WT mice and T2 DM mice 2.To explore the underlying mechanism for endothelial dysfunction induced by T-cad deficiency Methods 1.To build the model of T2 DM in mice,we randomized the male C57/BL6 mice into two groups.One is ND group,which we fed with normal diet(10% kcal fat)for 8 weeks.The other one is HFD group,which we fed with high fat diet(60% kcal fat)for 8 weeks.There is no limit for the usage of water.We monitered the body weight and the fasting blood-glucose level every week and tested the fasting blood-insulin level,fasting blood-triglyceride level,total cholesterol level and HOMA-IR index as the makers for the successful model of T2 DM.2.To test whether the abnormality of T-cad expression exists in diabetic mice,we removed the aortas from the WT mice fed with or without the high fat diet.Next,Western Blot(WB)and Realtime PCR(q RT-PCR)were performed to test T-cad expression in vascular tissue.3.To observe the direct impact of the change of T-cad expression to vasorelaxation in mice,we removed the descending aortas from the WT mice and T-cad KO mice,and processed them into vascular rings.Next,we randomized the rings into two groups.One is acetyl-choline(Ach)group,which is an endothelium-dependent vasodilator.The other one is acidified Na NO2 group,which is an endothelium-independent vasodilator.The vasorelaxation was determined with DMT multi-wire myograph system.4.In order to observe the effect of T-cad expression on vasodilatation in mice under high glucose/high fat(HGHF)environment,we removed the descending aortas from the WT mice and T-cad KO mice,and processed them into vascular rings.Next,we utilized prepared HGHF work solution to mimic the HGHF environment(final concentration: HG 25.5mmol/L;HF 300?mol/L)and randomized the rings into two groups: T-cad KO+HGHF and WT+HGHF.The vasorelaxation was determined with DMT multi-wire myograph system.5.In order to further confirm the effect of T-cad expression on vasodilatation in type 2 diabetic mice,we removed the descending aortas from the WT mice and T-cad KO mice which were fed with high fat diet for 8 weeks,and processed them into vascular rings.Next,we randomized the rings into two groups: T-cad KO+HFD 8w and WT+HFD 8w.The vasorelaxation was determined with DMT multi-wire myograph system.6.In order to further explore the underlying mechanism of Tcad expression downregulation on vasodilatation in mice,we removed the vascular tissues from the WT mice and T-cad KO mice.Next,we turned nitrate into NO by using reducing agent.NO accumulation in vascular tissues was measured with nitric oxide analyzer(SIEVERS 280)7.In order to further confirm the effect of Tcad expression downregulation on vasodilatation in mice,we collected the liquid which was left after finishing the vascular ring experiment.NOx Production from aortic segments was tested with nitric oxide analyzer(SIEVERS 280)8.To explore the molecular mechanism of decrease in NO production in vascular tissues,we extracted protein from vascular tissues in WT or T-cad KO mice.Protein levels of p-Akt/Akt,p-e NOS/e NOS was assessed with Western-blot(WB)9.To verify the effectiveness of Akt inhibitors,we cultured HUVECs and randomized them into Akt-inhibitor treatment group and controls.Protein levels of p-Akt/Akt was assessed with Western-blot(WB)10.To further confirm the underlying mechanism of decrease in NO production induced by T-cad expression down-regulation,based on previous experiment,we cultured HUVECs and randomized them into Akt-inhibitor treatment group and controls.Apoptotic cell death of HUVECs was determined by Caspase-3 activity kit.11.To further confirm the underlying mechanism of T-cad expression down-regulation induced vasorelaxation injury through NO bioactivity reduction,we removed the vascular tissues from the WT mice and T-cad KO mice.Quantitation of tissue nitrotyrosine content was determined by ELISA kit.12.To get the evidence supporting T-cad expression down-regulation induced vasorelaxation injury through NO bioactivity reduction,we removed the vascular tissues from the WT mice and T-cad KO mice.Superoxide production was measured with superoxide analyzing system.Results 1.Compared to ND mice,mice fed with HFD for 8 weeks appeared the following change: body weight was increased(P<0.01);Average level of fasting blood-glucose was increased(P<0.01);Average level of fasting blood-insulin was increased(P<0.01);Average level of fasting blood-triglyceride(P<0.05)and total cholesterol were increased(P<0.01);HOMA-IR index was increased(P<0.01).These results showed that the T2 DM model of mice was successfully constructed.2.Protein level of T-cad was decreased in WT mice after fed with High-fat diet(HFD)for 8 weeks(P<0.01).The tendency of m RNA level and protein level were consistent,but there is no significant difference in m RNA level between these two groups (P>0.05).3.There is no significant difference in WT mice in vasorelaxation induced by Ach or acidified Na NO2(P>0.05).But compared to acidified Na NO2 treatment,vasorelaxation treated by Ach in T-cad KO mice was greatly reduced(P<0.05).4.2.5h High glucose-high fat(HGHF)treatment caused reduction in vasorelaxation in aortic rings from both WT and T-cad KO mice.However,compared with WT,concentration-dependent vasorelaxation in T-cad KO mice was greatly decreased(P<0.01).5.Vasorelaxation was reduced in both WT and T-cad KO mice after fed with HFD for 8 weeks.Compared to controls,the decrease in concentration-dependent vasorelaxation was more significant in mice with HFD(P<0.01).6.After nitrate was turned into NO by reducing agent,compared to WT,both NO accumulation and NOx production were reduced significantly in T-cad KO mice(P<0.05),suggesting T-cad expression down-regulation induced decrease in NO production.7.Compared to WT,protein level of p-Akt in T-cad KO mice was greatly reduced,whereas there is no difference in Akt level.Protein level of p-e NOS and e NOS in T-cad KO group were slightly increased,but no significant difference compared with WT(P>0.05).In summary,Akt phosphorylation,not e NOS phosphorylation,was significantly reduced in T-cad KO mice.8.Compared to controls,Akt phosphorylation was significantly reduced(P<0.01),whereas Caspase-3 activity was significantly increased in HUVECs with Akt inhibitor treatment(P<0.05),suggesting apoptosis of endotheial cells was significantly induced after Akt activity inhibition.9.Compared to WT,both nitrotyrosine content and superoxide production were increased significantly in T-cad KO mice(P<0.05),suggesting T-cad expression downregulation induced exacerbation in NO inactivity Conclusion 1.We have successfully constructed T2 DM model of mice and found T-cad expression downregulation in vascular tissues from mice with T2 DM.2.With the help of DMT multi-wire myograph system,we have provided the first in vitro functional evidence that T-cad deficiency induced endothelial dysfunction both in WT and T2 DM mice.3.Mechanism research showed that reduction of NO bioactivity characterized by decrease in NO production and increase in NO inactivity contributes to endothelial dysfunction induced by T-cad expression downregulation.In summary,we have provided the first in vitro evidence that T-cad deficiency caused endothelial dysfunction in T2 DM model vascular segments and partially elucidated the underlying mechanisms.Furthermore,supplanting T-cad deficiency may be a potential therapeutic avenue in the prevention and amelioration of vascular injury in the diabetic population.