Establishment Of Intimal Hyperplasia Model Of LDLR Knockout Hamster And Study On Glucose Metabolism Characteristics Of Vascular Smooth Muscle

Objective: Vascular remodeling is a key pathological process in many cardiovascular diseases,which is mainly caused by the proliferation of vascular smooth muscle cells(VSMCs)leading to vascular wall thickening and lumen narrowing.Phenotype switching of VSMCs is the main cause of vascular remodeling diseases,and its mechanism is still worthy of further research.Recent studies have found that the phenotype switching of VSMCs is driven by a metabolic switch which including aerobic glycolysis,fatty acid oxidation,and amino acid metabolism.VSMCs switch from a quiescent “contractile” phenotype to a highly proliferative “synthetic” phenotype accompanied by increased aerobic glycolysis during atherosclerosis,and VSMCs can alter their metabolism to fulfill the bioenergetic and biosynthetic requirements.Recent studies have found that glycolysis and pentose phosphate pathways are significantly enhanced in atherosclerotic plaques.The expression and activity of key enzymes in glucose metabolism play an important role in the phenotype switching of VSMCs,such as hexokinase 2(HK2),pyruvate kinase M2(PKM2),lactate dehydrogenase A(LDHA)and phosphofructokinase 1(PFK1).In this study,proteomics analysis was performed on the intimal hyperplasia model in LDLR+/-hamster and proliferative VSMCs stimulated by PDGF-BB,focusing on the changes of glucose metabolization-related enzymes in vascular remodeling,and exploring the mechanism of PFK1 in VSMC proliferation.Methods: WT,LDLR+/-and LDLR-/-hamsters were used to screen and establish an animal model of intimal hyperplasia after artery injury in LDLR+/-hamsters.B-ultrasound,HE staining and Immunofluorescence staining were used for detection and verification in small animals to determine the most appropriate animal model.Proteomics analysis was performed on the intimal hyperplasia model in LDLR+/-hamster and proliferative VSMC stimulated by PDGF-BB.The detected significant differential proteins were analyzed by Gene Ontology(GO)classification and KEGG Pathway enrichment analysis,and the significant differential proteins of interest were identified and verified by Western Blot and immunofluorescence in frozen section tissues.si PFKL was used to knock down the target protein PFKL,crosslinking assay was used to detect the tetramer structure of PFK1,and CCK8 and cell count were used to detect the role and function of PFK1 in VSMC.The proliferative marker PCNA was also detected by Western Blot.Finally,we used deacetylase inhibitors and si HDAC6/8 to regulate the acetylation of PFK1,and the acetylation of PFK1 was verified by immunoprecipitation.Results: 1.Establishment and identification of intimal hyperplasia model in LDLR+/-hamster 1.1 The peak blood flow decreased significantly after ligation of the left common carotid artery in hamsters Pulse Doppler imaging showed that the peak blood flow velocity decreased rapidly after ligation of the left common carotid artery in WT、LDLR+/-and LDLR-/-hamsters.Compared with WT hamsters,LDLR+/-and LDLR-/-hamsters showed a more significant decrease in peak blood flow velocity after ligation.The above results indicate that after ligation of the left common carotid artery,the blood flow of the hamster left carotid artery is blocked,and the peak blood flow decreases significantly.Moreover,the loss of LDLR has an impact on the decrease of the peak blood flow.1.2 After ligation of the left common carotid artery,the wall thickness of the left carotid artery of hamsters increased significantly B-ultrasound results of small animals showed that with the extension of ligation time,WT,LDLR+/-and LDLR-/-hamsters showed significant difference in vascular wall thickness at each time point after ligation compared with that before ligation.In addition,14 d after ligation,there was significant difference in the thickening of the left common carotid wall between LDLR+/-and LDLR-/-hamsters compared with WT,while there was no statistical significance between LDLR+/-and LDLR-/-hamsters.HE staining results also showed that the intima of WT,LDLR+/-and LDLR-/-hamsters was significantly thickened with the extension of ligation time.Moreover,the intima/media ratio(I/M)of LDLR+/-and LDLR-/-hamsters was significantly increased compared with the WT group.Immunofluorescence staining also showed that PCNA expression increased significantly after ligation of the left common carotid artery.Since LDLR+/-hamsters can quickly show symptoms of intimal hyperplasia after vascular injury,the intimal hyperplasia model in LDLR+/-hamster was selected for subsequent proteomics.1.3 Left common carotid artery ligation had no effect on heart function and right common carotid artery in hamsters B-ultrasound results showed that cardiac ejection fraction(EF)of WT LDLR+/-and LDLR-/-hamsters changed slightly with the extension of ligation time,but the data are not statistically different.In addition,ECG monitoring results of hamsters showed that after 14 days of ligation,the QT interval of WT LDLR+/-LDLR-/-hamsters was slightly prolonged,but the data are not statistically different,and then returned to normal levels.In conclusion,ligation of left common carotid artery has no significant effect on hamster heart function.Pulse Doppler imaging and B-ultrasound analysis showed that the peak blood flow velocity of the right common carotid artery in WT,LDLR+/-,and LDLR-/-hamsters was not affected by ligation of the left common carotid artery,and the thickness of the blood vessel wall was also not affected.In conclusion,ligation of the left common carotid artery in hamsters has no significant effect on cardiac function and the shape and function of the right common carotid artery.2.Proteomic analysis of proliferative VSMCs and intimal hyperplasia model in LDLR+/-hamster.2.1 Proteomic analysis of the proliferative VSMCs In GO classification,differentially expressed proteins in proliferative VSMCs mainly related to biological process(BP)such as nucleotide phosphorylation,cofactor and coenzyme metabolic process,carbohydrate metabolic process,ribose phosphate and monocarboxylic acid metabolic process,carboxylic acid,organic acid and oxoacid metabolic process,etc.Protein classification by cellular component(CC)revealed that most proteins were implicated in MCM complex,stress fiber,DNA packaging complex,contractile actin filament bundle,actomyosin and extracellular matrix.Molecular Function(MF)classification showed that most differentially abundant proteins were related to lyase activity(carboxy lyase,carbon-carbon lyase),ligase activity,oxidoreductase activity and isomerase activity.KEGG pathway enrichment analyses were used for differentially expressed proteins,and the results are shown that the most significantly altered pathways were involved in glucose metabolism pathway(glycolysis,gluconeogenesis and pentose phosphate pathway)and biosynthesis of amino acids.At the same time,the metabolism of carbon,galactose,fructose,mannose,pyruvate and various amino acids(tryptophan,valine,leucine,isoleucine,cysteine,methionine,glycine,serine,threonine,alanine,aspartate,glutamate)is also significantly affected.In conclusion,various metabolic pathways are abnormal in PDGF-BBinduced proliferative VSMC,which played a very important role in the survival,proliferation,migration and phenotypic transformation of VSMC.2.2 Proteomic analysis of intimal hyperplasia model in LDLR+/-hamster Proteomic analysis of intimal hyperplasia model in LDLR+/-hamster showed that there were many differentially expressed proteins in intimal hyperplasia vessels mainly related to BP,such as glycosphingolipid metabolic process,ATP hydrolysis coupled transmembrane transport,regulation of complement activation and regulation of acute inflammatory response,etc.Protein classification by CC revealed that most proteins were implicated in glycoprotein complexes(especially in dystrophin-associated glycoprotein complex)and ribosome(cytosolic ribosome and ribosomal subunit).MF classification showed that most differentially abundant proteins were related to ATPase activity(calcium-and cation-transporting ATPase activity,hydrogen- exporting ATPase activity).Subsequent KEGG analysis revealed that the most significantly altered pathways were involved in glycosphingolipid biosynthesis and glycosaminoglycan degradation,as well as significant effects on vascular smooth muscle contraction,dilated cardiomyopathy(DCM)and hypertrophic cardiomyopathy(HCM).2.3 Proteomic analysis and validation of proliferative VSMCs and intimal hyperplasia model in LDLR+/-hamster We compared the differential proteins in intimal hyperplasia model in LDLR+/-hamster and the proliferative VSMCs.Venn diagram showed that 130 differentially expressed proteins appeared in the two models simultaneously,including 67 up-regulated proteins and 63 down-regulated proteins.We performed GO and KEGG analysis of these common differential proteins.The classification of differential proteins by BP showed that many proteins were abnormal in carbohydrate metabolism.CC classification showed differences in actin cytoskeleton composition.MF classification showed that differential proteins were enriched in cell adhesion function.KEGG enrichment dot bubble showed that the differential proteins were mainly concentrated in metabolic pathways(such as pentose phosphate pathway,glycolysis and gluconeogenesis),accounting for 25% of the analyzed proteins.The glycolytic pathway showed up-regulation of phosphofructokinase 1 of liver type(PFKL),phosphoglycerate mutase(PGM),enolase,and Lactate dehydrogenase A(LDHA),while downregulation of pyruvate dehydrogenase(PDH).PFKL and LDHA are the key enzymes in the glycolysis,so Western Blot and immunofluorescence analysis were used respectively to examine their abundant in VSMC and hamster carotid artery slices.Consistent with the LC-MS/MS data,Western Blot assay showed that the levels of PFKL and LDHA were significantly up-regulated in proliferative VSMCs.Similarly,in LDLR+/-hamsters,immunofluorescence analysis results showed that PFKL and LDHA expression were significantly increased in left common carotid artery after 21 days ligation,compared with the control group.3.PFK1 deacetylation can enhance its enzyme activity and promote the proliferation of VSMC 3.1 The expression and activity of PFKL were increased in proliferative VSMCs PFK1 consists of three types of subunits: liver(PFKL),muscle(PFKM),and platelet(PFKP).Western Blot analysis showed that the expression of PCNA in VSMC treated with PDGF-BB increased significantly,while the expression of SM22α and α-actin decreased significantly.VSMCs switch from a quiescent “contractile” phenotype to a highly proliferative “synthetic” phenotype.The expression of PFKL was significantly increased in VSMC treated with PDGFBB for 24 hours,while the expression of PFKP did not change significantly.The activity of PFKL in proliferative VSMCs was detected by glutaraldehyde crosslinking assay,and verified by PFK1 activity detection kit.The results showed that in proliferative VSMCs,the PFKL tetramer form was significantly increased,and the activity of PFKL was also increased.These results indicated that PFKL expression and activity were enhanced in the proliferative VSMCs.3.2 Knockdown of PFKL expression inhibited the growth of VSMC When knockdown the PFKL by si PFKL,the expression of PCNA in VSMC was significantly decreased,and the number of VSMC was also decreased,even though treated with PDGF-BB.When PFKL was knockdown,VSMC viability was significantly decreased and the production of lactic acid was significantly inhibited.These results suggest that knockdown of PFKL expression can inhibit glycolysis and VSMC proliferation significantly.3.3 Inhibition of PFK1 acetylation can promote its activation The results of post-translational modification showed that the acetylation of PFK1 decreased significantly when treated with PDGF-BB.Deacetylase inhibitor TSA can reverse the PDGF-BB induced decline in PFK1 acetylation,and the tetramer form of PFK1 was significantly reduced,the activity of PFK1 was also significantly decreased.When treated with TSA,the expression of PCNA,the number of VSMC,and the viability of VSMC were significantly decreased.In addition,the production of lactic acid in VSMC was significantly decreased when treated with TSA,suggesting that acetylation of PFK1 can significantly inhibit glycolysis and proliferation of VSMC,possibly mediated by inhibition of PFK1 enzyme activity.3.4 HDAC6 can catalyze the deacetylation of PFK1 The deacetylase inhibitor TSA inhibits the deacetylation of PFK1,and there is a similar effect when treated with si HDAC6.The activity of PFK1 in VSMC was inhibited significantly when treated with si HDAC6,and the expression of PCNA,cell number,and cell vitality was also significantly decreased,and lactic acid production was significantly decreased.These results suggest that HDAC6 can enhance the enzyme activity of PFK1 and the glycolysis of VSMC by inhibiting the acetylation of PFK1,thus promoting the proliferation of VSMC.Conclusions: 1.Intimal hyperplasia model in LDLR+/-hamster is a good animal model to study vascular remodeling diseases.2.In proliferative VSMCs and intimal hyperplasia model in LDLR+/-hamster,the differential proteins were mainly concentrated in the glucose metabolic pathway,and the expressions of PFKL and LDHA were significantly increased.3.In proliferative VSMCs,HDAC6 regulates deacetylation of PFK1 enhances its enzyme activity,thus promoting aerobic glycolysis and proliferation in VSMC.