The Molecular Mechanism Of Protective Role Of Glucagon-like Peptide-1 And Its Receptor In Cardiovascular

Globally,one-third of the total annual deaths in the world are attributable to cardiovascular diseases(CVD).The morbidity and mortality of CVD are still on the rise.The total hospitalization expenses of CVD are also increasing rapidly.The raising burden of CVD in China has become a major public health problem,and the prevention and treatment of CVD can not be delayed.As an incretin prptide,glucagon like peptide 1(GLP-1)was directly secreted by intestinal L cells,and has been widely used in the clinical treatment of type II diabetes.In recent years,it has found that GLP-1 has a good protective effect on the cardiovascular system.However,the detailed mechanism of GLP-1 in CVD is still unclear.In this study,spontaneously hypertensive rats(SHR)were used as modelss of myocardial hypertrophy ans vascualr remodeling in vivo.Angiotensin II(Ang ?)induced H9C2 cardiomyocytes and rat aortic vascular smooth muscle cells(RASMC)respectively to construct myocardial hypertrophy and vascular remodeling models in vitro.The positive effect and mechanism of GLP-1 in CVD were discussed from two aspects of cardiac hypertrophy and vascular remodeling.The main research contents and results are listed as follows:(1)The mechanism of GLP-1 in reserving myocardial hypertrophy.In an animal model of myocardial hypertrophy in vivo,the effect of GLP-1analogue Liraglutide and the dipeptidly peptidase IV inhibitor Alogliptin on myocardial hypertrophy were detected by BL-420 Data Acquisition and Analysis System,HE staining,Masson staining and transmission electron microscope.The results showed that Liraglutide and Alogliptin decreased heart rate,heart weight,dampened the hypertrophic responses,reduced the expression of collagen fibers,and prevented the hyperplastic and enlargement of mitochondria,promoted the regular arrangement of the heart,kept the morphology of the myocardial cells intact.In the model of myocardial hypertrophy in vitro,different concentration of GLP-1(5n M,10 n M,20 n M)treated H9C2 cell to detecting the role of GLP-1 in myocardial hypertrophy.The Immunofluorescence and Western Blotting experiments results showed that GLP-1 reduced the size of H9C2 cardiomyocytes and down-regulated the expression of myocardial hypertrophy marker genes(ANP,BNP,?-MHC)significantly induced by Ang ? in a concentration-dependent manner.Protein kinase A(PKA)and Rho-associated coiled-coil protein kinase 2(ROCK2)inhibitors H89 and Y27632 could depress the reversing effect of GLP-1in reversing cardial hypertrophy.The above results revealed the molecular mechanism of GLP-1 reversing myocardial hypertrophy: GLP-1 reversed myocardial hypertrophy through reducing the myocardial hypertrophy related protein expression via c AMP/PKA/Rho A/ROCK2 signaling pathway.It is revealed that ROCK2 could be used as a therapeutic target against myocardial hypertrophy.(2)The mechansim of GLP-1 reversing vascular remodeling.In an model of vascular remodeling in vivo.The HE staining,Masson staining and Western Blotting were used to detect the effects of Liraglutide and Alogliptin on the thickness of blood vessel wall,lumen of vascular,vascular collagen fibers and extracellular matrix(ECM).The results showed that Liraglutide and Alogliptin could alleviate the vascular wall thickening,blood lumen stenosis and ECM disorder of SHR.In an model of vascular remodeling in vitro.The influence of GLP-1 on the proliferation and cell cycle of RASMC were detected by MTT,Cell Cytometry and Western Blotting assay.The results showed that GLP-1 inhibited the abnormal proliferation of RASMC induced by Ang ? in a concentration-dependent manner.The results of Cell Wound Scratch Assay showed that GLP-1 suppressed the abnormal migration of RASMC induced by Ang ? in the same way.The treatment of GLP-1R antagonist Exendin 9-39 eliminated the inhibitory effect of GLP-1 on the abnormal proliferation and migration of RASMC induced by Ang ?.The results of Western Blotting showed that GLP-1R could attenuate the expression of col1a1,col3a1,MMP9,MMP2 and MMP1 in ECM.However,the knockout or inhibitor BB-94 of matrix metalloproteinase 1(MMP1)treatment strengthened the inhibitory effect of GLP-1 on the abnormal proliferation and migration of RASMC induced by Ang ?.The inhibition of extracellular regulated protein kinase 1/2(ERK1/2)signaling pathway further enhanced the down-regulation of GLP-1 on MMP1.In addition,GLP-1prevented the nuclear import of nuclear factor kappa-B(NF-?B)induced by Ang ?.These results showed that molecular mechanism of the beneficial effects of GLP-1on vascular remodeling: GLP-1 inhibited the ERK1/2-NF-?B signaling pathway by binding with GLP-1R,and then down-regulated the expression of MMP1,which prevented the abnormal proliferation and migration.It was benfited to reversing the thickening of vascular wall caused by vascular remodeling.(3)The study on the molecular mechanism of Ang ?-induced the nuclear export of GLP-1R.Immunofluorescence and immunohistochemistry were used to detect the subcellular localization of GLP-1R in vascular remodeling models in vivo and in vitro,respectively.The results showed that GLP-1R subcellular localization is mainly concentrated in the nucleus in normal physiological conditions and resting state of RASMC.While the subcellular localization of GLP-1R was transferred from the nucleus to the cytoplasm under pathological conditions of vascular remodeling and Ang ? stimulates RASMC.It was determined that the 8-17 amino acid sequence of GLP-1R is the NES sequence of GLP-1R by using Bioinformatics and gene mutation experiments.The treatment of the classic nuclear export inhibitor LMB could inhibit the nuclear export induced by Ang ?.The nuclear export of GLP-1R induced by Ang ? was inhibited when NES is absent.Co-immunoprecipitation results also show that Ang ? promoted the binding of GLP-1R with the nuclear transport receptor CRM1.In the absence of NES,the binding of GLP-1R and CRM1 induced by Ang ? disappears.In the model of Ang ?-induced vascular remodeling in vitro,several common signal pathway inhibitors of the Ang ?/AT1 R signal pathway were used to detect the subcellular localization of GLP-1R for exploring the molecular mechanism of GLP-1R nuclear export caused by Ang ?.The results showed that Ang ? promotes the nuclear export of GLP-1R by activating the PI3K/PDK1/Akt/PKC? signaling pathway.The regulation of PKC? on GLP-1R nuclear export was further analyzed.The results showed that Akt promotes PKC? nuclear import,and PKC? phosphorylates the Ser416 site of GLP-1R in the nucleus,which causing GLP-1R binds with CRM1,and triggering GLP-1R nuclear export phenomenon occurred.Once PKC?-specific inhibitors treated the RASMC induced by Ang ?,the nuclear import of PKC? is inhibited,and nuclear export of GLP-1R also disappeared.However,the nuclear export of GLP-1R was inhibited and the binding between the GLP-1R and CRM1 was disappeared when GLP-1R Ser416 is mutated to Ala,even though GLP-1R and PKC? co-localized in the nucleus.When Ser416 mimics phosphorylation(S416D),GLP-1R S416 D could still bind to CRM1 to promoteing nuclear export.In conclusion,this part is the first elaborates the specific molecular mechanism of nuclear export of GLP-1R under the pathophysiological state of Ang ?-induced vascular remodeling: Ang ? activates the PI3K/PDK1/Ak/PKC? signaling pathway by binding to the AT1 R receptor,and phosphorylated PKC? enters the nucleus,which phosphorylated GLP-1R Ser416 site,and the phosphorylated GLP-1R undergoes nuclear export by binding to CRM1.(4)The molecular mechanism of GLP-1R subcellular localization regulated proliferation and migration of RASMC.We silenced the endogenous GLP-1R of RASMC cells,and then artificially constructed GLP-1R deletions lacking NES and nuclear localization sequences(NLS),and localized in the nucleus or cytoplasm,respectively.The effects of GLP-1R subcellular localization on the proliferation and migration of RASMC were checked by MTT,Western Blotting and Cell Scratch.The results show ED that the cytoplasmic localization of GLP-1R significantly promoted the proliferation and migration of RASMC.In addition,we also found that the nuclear export of GLP-1R induced by Ang ? inhibited by LMB,and the the proliferation and migration of RASMC induced by Ang ? was also weakened.We knocked out and overexpressed C14orf166 to test its effect on Ang ? promoting the proliferation and migration of RASMC.The results showed that the decrease of C14orf166 expression inhibited the the proliferation and migration of RASMC inducec by Ang ?,and the up-regulation of C14orf166 expression strengthened further the proliferation and migration of RASMC induced by Ang ?.However,Ang ? had no effect on the protein expression and m RNA level of C14orf166.It indicated that C14orf166 does not play a role by regulating the level of expression.We further tested the interaction between C14orf166 and GLP-1R.Co-immunoprecipitation results showed that C14orf166 and GLP-1R can form a complex before Ang ? intervention.Once Ang ? stimulated,the complex dissociates.We also found that C14orf166 does not bind with GLP-1R(S416/D)that mimics the phosphorylation state,but it could bind with the dephosphorylated mutant GLP-1R(S416/A).When PKC? activity is inhibited,it will reverse the dissociation effect of Ang ? on the C14orf166 and GLP-1R complex.The molecular mechanism of GLP-1R nuclear export regulating the proliferation and migration of RASMC: Under normal physiological condition,the combination of GLP-1R and C14orf166 in the nucleus inhibits the proliferation and migration of RASMC.Once Ang ? treatment,the complex formed by GLP-1R and C14orf166 was dissociation,GLP-1R undergoes nuclear export,which resulting in the loss of inhibitory effect on C14orf166.C14orf166 acts as a factor promoting cell proliferation,leading to the proliferation and migration of RASMC.In summary,this study discusses the positive role and specific molecular mechanisms of GLP-1 in myocardial hypertrophy and vascular remodeling.In the process of GLP-1 reversing vascular remodeling,we observed for the first time that the transfer of GLP-1R subcellular localization from the nucleus to the cytoplasm,and detailed the molecular mechanism of Ang ? inducing GLP-1R nuclear export.The reason why the nuclear export of GLP-1R triggers the abnormal proliferation and migration of RASMC was further discussed.This work provides a new research direction for the in-depth analysis of the function of GLP-1R,and provides a theoretical basis and basis for the prevention and treatment of GLP-1 and GLP-1R in cardiovascular diseases.