| 1 BackgroundIt has been widely accepted that diabetes was the risk equivalents of coronary atherosclerotic heart disease(CHD).Multiple guidelines in different countries have accepted type 2 diabetes as the main risk factor for cardiovascular disease(CVD)events.According to the results of numerous clinical trials and meta-analyses,diabetic patients with cardiovascular disease present earlier,wider and heavier pathological changes but the prognosis is poorer.Current evidence-based medical evidence shows that strict control of fasting glucose and glycosylated hemoglobin can significantly reduce the incidence of diabetic microvascular complications,but the effect for diabetic macrovascular complications remains controversial.Intensive glucose-lowering group with higher mortality showed the change of blood glucose and insulin levels not been the core element of diabetic patients with the acute coronary syndrome.Therefore,exploring other factors apart from blood glucose and finding underlying mechanism increasing risk of cardiovascular complications in diabetic patients has great significance to the prevention and treatment of diabetes vascular lesions.Many animal and clinical trials illustrate the clear and close ties between adipose tissue and cardiovascular disease.Bariatric surgery can reduce adipose tissue dysfunction and the risk of cardiovascular disease.Adipose tissue dysfunction in diabetic patients is characterized by chronic,subclinical state inflammation.Dysfunctional adipose tissue releases adipokine to induce chronic,subclinical inflammation state,ultimately lead to a vicious cycle between adipose and circulation.However,the route of adipokine getting into the circulation,mechanism of target cells absorbing adipokine are unclear.Microvesicles are the extracellular membranous capsules with different size,released by cells,which contain rich proteins,nucleic acids and lipids.They derive from endothelial cells,macrophages,and platelets,even tumor cells,and mediate the signal communication between organizations and organizations,cells and cells.According to the size of the microvesicles and generation mechanism,microvesicles are classified into eoxsome(size,30-100 nm),Microvesicle(100-1000 nm),and Apoptotic body(1-5 microns),etc.Exosome is been studied thoroughly.Exosome may involve antitumor immune escape,tumor metastasis,tissue repair,communication between nerve cells,pathogen immune,embryonic development and process of epigenetic regulation and other life and disease course.Recent studies have found that Exosome plays an important role in cardiovascular homeostasis.Some reported that mesenchymal cells,macrophages,adipocyte can actively secrete Exosome(adipose derived Exosome,ADE).ADE can not only participate in activated macrophages mediated inflammation,help tissue repair,but also be involved in the formation of tissue fibrosis.Nevertheless,the composition of ADE derived from inflamed adipose tissue in diabetes remains unclear.What are the main functions of the ingredients,which life and disease process dose ADE participate in?For exploring the above issues,we extract ADE from the normal mice,obesity diabetic mice(ob/ob mice)and the lean diabetic mice(STZ＋high-fat feeding induction),using a variety of means to detect the physical and chemical characteristics of Exosome.Protein mass spectrometry and bioinformatics analysis are used to analyze the composition and function of protein in diabetic ADE.These works would lay the foundation for further exploring the relationship between ADE and cardiovascular disease in diabetes conditions2 Objectives1.To explore strategies for separation and purification of ADE;2.To analysis of composition of diabetic ADE protein;3.Analysis of ADE protein function using methods of bioinformatics.3 Method1.Collecting ADE from visceral adipose tissue in diabetic and non-diabetic mice and human;2.Isolation and purification of ADE with differential centrifugation,density gradient ultracentrifugation(heavy water sucrose cushion)and sedimentation agent(ExoQuick);3.Detecting the characteristic of the marker proteins,marker enzymes,total protein,and micromorphometric by western blotting,dynamic light scattering and electron microscopy morphometric;4.Analysis of ADE protein components by mass spectrometry;5.Analysis of ADE adipokines by database comparison;6.Bioinformatics analysis for ADE protein function;7.Compare non-diabetic and diabetic subjects ADE adipokines and related clinical factors by western blotting and ELISA methods8.Identification of adipokines spatial orientation in ADE with immune gold labeling and western blotting.4 Results1.ADE isolation and purification methods1.1 Comparison for output:In differential methods,differential centrifugation yieldedmost Exosome marker protein-CD63,maker enzyme-Acetyl-CoA;1.2 Comparison for purity:By comparison of marker enzyme-Acetyl-CoA and total protein,differential centrifugation produced ADE with highest purity.In addition,TEM results represent ADE from differential centrifugation with lowest background,while ADE from Exoquick accompanied with particulate matter pollution in highelectron density;1.3 Comparison for particle size:Differential centrifugation produced Exosome in 30-100nm and had smallest diameter difference between each batch;heavy water sucrose cushion obtained Exosome in 30-120nm,but containing some 100-400nm particle in some batches;Exoquick obtained Exosome in 15-130nm,but had relative largest diameter difference between each batch;1.4 The effect of incubation time of adipose tissue to ADE output:Compare theincubating time of adipose tissue among 15min,30min,60min.2h and 3h,the results showed that 6h incubation produced most total protein,Exosome marker proteins CD63 and marker enzyme Acetyl-CoA;1.5 The effect of incubation time of adipose tissue to ADE purity:The results showed no significant difference among those incubation time periods whether 6h,15min,30min,60min,2h and 3h;2.Analysis protein components of diabetic ADE2.1 Diabetic ADE protein profiles:After 34901 peptide compared to the database,the threshold value is set at p<0.00575,ADE was identified into 1331 kinds of protein;Compared with Exosome characterized protein database ExoCarta-Top 100,ADE contained 87 kinds of marker proteins,without the VCP,HSPA5,SLC3A2,CCT2,TUBA1B,ANXA6,TUBA1C,TFRC,HIST2H4A,ITGA6,HIST1H4B and YWHAH;2.2 Adipokines in diabetic ADE:There were Dipeptidyl peptidase 4,Retinol-binding protein,Resistin,Pigment epithelium derived factor,Adiponectin-five adipokines in diabetic ADE,and DPP4 peptides match up to the total number of matches bits 49,the focus match 23.2.3 Clustering of diabetic ADE Protein:David was used to identify functional protein clustering.The results showed that main function of the protein ADE include tricarboxylic acid cycle,proteasome core complex,GTP binding,oxidoreductase activity,annexin,contractile fiber,protein import into nucleus,regulation of T cell mediated cytotoxicity,neutral lipid metabolic process,nucleoside binding,positive regulation of adaptive immune response,glycogen metabolic process,mitochondrial respiratory chain complex i,nicotinamide nucleotide metabolic process,lipoid acid binding,and ATP synthesis coupled proton transport(P<0.007).2.4 Pathway cluster of diabetic ADE protein:The pathway ADE involved was mainly related with binding proteins,nucleic acids in combination,and calcium channel function.2.5 Comparison of DPP4 content in ADE derived non-diabetic and diabetic mice(WT vs.ob/ob),human visceral fat:Compared with WT mice,DPP4 in ob/ob ADE significantly increased.In order to clarifying the expression of DPP4 in other diabetic mice model,excluding the impact of leptin gene,we established a model of STZ-induced diabetic mice.Compared with WT mice,DPP4 in ADE derived from STZ-induced diabetic mice also significantly increased.In order to explore its clinical relations,we collected 19 cases of human omental visceral adipose tissue(9 cases of diabetes and 10 normal controls),isolated and purified humanized ADE.ELISA results showed that DPP4 in ADE derived from patients with diabetes was significant higher than normal controls.2.6 The relationship among DPP4 in ADE,adipose cell size and fasting blood glucose levels:To explore related clinical factors,we collected clinical and laboratory data from of 19 patients described above,the adipose cell size(r = 0.60,p<0.01),fasting plasma glucose(r = 0.50,p<0.01)were positively correlated with DPP4 content in ADE.The sex,age,weight,smoking history,drinking history,and history of coronary heart disease,history of hypertension,diabetes mellitus,diabetes duration,TC,TG,HDL-C and LDL-C have no significant relationship with DPP4content in ADE.2.7 Membrane and contents fraction analysis in ADEDPP4 can be cleaved intracellular-trans membrane domain and the extracellular domain.Extracellular domain as soluble form,can be released into body fluids.After cracking crushed ADE,ADE was separated into the membrane and contents component.The results showed that the contents of the component did not contain DPP4.2.8 DPP4 immune gold labeled in situ electron microscopy for diabetic ADEWe used the immune gold to label DPP4 in ADE.Electron microscopy showed that PBS control group had no binding gold particles,but diabetic ADE membrane surface blinded to gold particles significantly.5 Conclusion(1)Differential centrifugation is the most suitable method for the isolation and purification of ADE stably;(2)Diabetic ADE was riched in DPP4 and other adipokines,its content was related with fasting blood glucose levels and adipose cell size;(3)Protein in diabetic ADE may be involved in energy metabolism,biological processes,material synthesis and immune regulation.1 BackgroundAmong all of the identified risk factors,diabetes mellitus increased the risk of cardiovascular disease up to 4-fold.However,whether hypoglycemic therapy alone reduces cardiac events in subjects with established diabetes remains controversial.There are several strategies that reduce cardiovascular morbidity and mortality in diabetic subjects.Current invasive or noninvasive treatments for this disease process,including surgical bypass and percutaneous interventions,are limited by restenosis and the formation of neointima.By applying a drug-eluting stent in coronary ar;teries,diabetes is still a risk factor for restenosis..Emerging data obtained from studies within the last decade suggest that diabetes induced chronic local inflammation in adipose tissue and the production of proinflammatory cytokines.A recent study showed that adipose tissue Exosome-like vesicles mediate the activation of macrophages and induce proinflammatory cytokines and insulin resistance.Exosomes are extracellular,membrane-bound vesicles that are heterogeneous in size(ranging from 30 to100 nm).The components expressed in Exosomes affect cellular signaling and transport processes,as well as regulate epigenetic modulations,metabolic memory,angiogenesis,lipid metabolism and immunology in cells and organs.However,the role of adipose tissue-derived Exosomes(ADE)in neointima formation after vessel injury and its effect on VSMCs migration and proliferation,which are key processes in neointima formation,is not fully understood.Dipeptidyl peptidase-4(DPP4)is a novel adipokine that participates in insulin resistance in an autocrine and paracrine fashion.Substantial DPP4 activity is also found in plasma and other body fluids due to the soluble form of DPP4.DPP4 inhibitors are widely used as a second-line antidiabetic drug,usually in combination with metformin.In contrast,much less is known about the cardiovascular biology of DPP4 in neointima formation.In the present study,we aimed to investigate whether ADE also plays a role in neointima formation in a mouse model,and the effect of DPP4 in diabetic neointima.2 Objectives1.Explore the role of diabetic ADE in neointima;2.Illustrate the role of the ADE loading DPP4 on vascular smooth muscle cell proliferation,migration and neointima;3.Explore molecular mechanism of neointima induced by ADE.3 Methods1.Animal Model:mouse carotid intima injury model;2.Cell Model:primary mouse aortic vascular smooth muscle cells(3-5 passage cells);3.Effect of ADE on neointima:Mouse with carotid intima injury was injected diabetic and non-diabetic ADE by tail vein injection;4.Effect of ADE on vascular smooth muscle cell proliferation and migration:Scratch test,Transwell test and immunofluorescence of Ki67 were used to evaluate proliferation and migration of vascular smooth muscle cells;5.PKH26 fluorescent dye labeled ADE tracer experiment;6.Effect of DPP4 loaded in ADE on neointima:hydrochloric acid sitagliptin and exenatide treated ADE induced neointimamouse model to observe the effect of DPP4 inhibitors and GLP1 analogs;7.Vascular smooth muscle cells were added ERK1/2 and NF-κB inhibitor to explore the intracellular pathway:8.IPGTT,IPITT,blood lipid and glucose function and basic physiological parameter were measured to evaluate the effect on glucose and lipid metabolism in mice.4 Results4.1 Impact of ADE on Neointima After Vessel InjuryIn vivo vascular injury model,no significant difference was observed in terms of neointima area,lumen area and neointima/media ratio between control mice and mice with WT ADE injection.In contract,ob/ob ADE significantly increased neointimal lesions than WT ADE at day 14.Furthermore,proliferating cells in injured carotid arteries in the injured carotid arteries were significantly increased in WT mice injected by ob/ob ADE compared with WT ADE at day 14.To determine whether ADE is taken up by VSMCs in the vessels,we labeled ob/ob ADE with PKH26 dye before injecting them intravenously into mice at vascular injury 7 days.The right carotid artery was harvested 48h after the injection of labeled ADE.The red labeled ADE cumulated in the tunica media as presented.Exosome uptake assay in vitro also was indicated in vitro assay.4.2 Effect of ADE on VSMCs proliferation and migrationTo verify that ADE has a positive effect on VSMCs proliferation and migration,we isolated the VSMCs from aortas of non-diabetic C57BL/6 mice.The Ki67 immunofluorescence stain was performed for cell proliferation assay.As shown in Figure 2A,the proliferation of SMCs was significantly augmented by diabetic ADE.Moreover,VSMCs had increased migratory ability when supplemented with diabetic ADE.4.3 DPP4 enriched in ADEThe mass spectrometry identified five adipocytokines in ADE from db/db mice.Among these adipocytokines,DPP4 is significantly potent of promoting cell proliferation.Immunogold labeling ADE of ob/ob mice,electron microscopic observation showed that DPP4 was present in purified diabetic ADE.To investigate the location of DPP4 in ADE,we extract the membrane fractionation from cytoplasm protein and possible soluble protein pollution outside exosome.Immunoblot analysis confirmed that DPP4 was located in membrane but not in luminal space,and there were no soluble DPP4 contaminants in isolated and purified exosome.As expected,ob/ob ADE carried more DPP4 protein.To study if the DPP4 enriching in ADE generally occurred in other mouse models and was not only restricted to ob/ob mice,we used the STZ induced diabetic mouse to validate our findings.The DPP4 was also significantly increased in ADE in STZ induced diabetic mice.4.4 Treatment of mice with sitagliptin or exenatide after ADE injectionIn diabetic subjects,DPP4 plays a important role in the catalytic degradation of incretins such as glucagon-like peptide-1(GLP-1).To determine whether ADM promotes neointima through GLP-1 pathway,the mice injected with ob/ob ADM were administrated with GLP-1 receptor agonist(exenatide)and DDP4 inhibitor(sitagliptin).Even though exenatide improved insulin sensitivity,there were minor differences in the neointima between the mice with exenatide compared with controls.In the course of in vivo experiments,similar results were observed.However,sitagliptin represented significant suppression for neointima in mice stimulated by diabetic ADM.Sitagliptin significantly decreased neointima/media ratio in diabetic ADM stimulated mice.The Ki-67 positive cell ratio was lower among in vivo and in vitro experiments.The lower-outer surface of transwell was reduced significantly,4.5 Effect of ADE on TLR4-ERK/NF-κB Signaling PathwayTLR4 was the key ligand of exosome activating downstream signals.To study the relationship of ADM and neointima formation,we examined the effect of ADM in TLR4 KO mice carotid injury model.The experiment showed that TLR4 knockout could abolish the diabetic exosome mediated neointima.Previous studies suggested NF-κB and AMPK pathway might be involved in anti-proliferation effect of DPP4i.The immunoblot analysis of injured right carotid artery also presented unregulated phosphorylation of p65and ERK1/2 in mice adopting diabetic ADE.To establish a link between ADE and intracelluar p65 and ERK,we used caffeic acid phenethyl ester and FR180604 to block the intracellular signal pathway.Pre-treatment with caffeic acid phenethyl ester and FR180604 partially decreased p65 and ERK1/2 phosphorylation in response to diabetic ADE,and abolish the elevating proliferation and migration of VSMCs.4.6 Clinical RelevanceTo explore the clinical relevance,we conducted a pilot experiment by recruiting subjects with chronic cholecystitis or benign diseases in cholecyst(exclude acute systemic inflammation or malignant tumor)to our studies.DPP4 in ADE is significantly higher in diabetic subjects compared with control subjects.Furthermore,DPP4 contents in ADE correlate with adipocyte diameter and free blood glucose positively.5 Conclusion(1)Diabetic ADE contains more DPP4,can significantly enhance the proliferation and migration of vascular smooth muscle via TLR4-NF-κB/ERK1/2 pathway,increased neointima;(2)DPP4 inhibitors significantly attenuated diabetes ADE exacerbated neointima,GLP1 analoguehad no significant effect;(3)ADE communicating inflammatory adipose tissue and injury vessels may become a new target for the treatment of diabetic cardiovascular complication.1 BackgroundDiabetic macrovascular disease is the common pathology basement of the complication of diabetes-coronary heart disease,stroke and peripheral vascular disease.Adipose tissue in diabetic patients plays a pivotal role as an important endocrine and paracrine organ in diabetes and its macrovascular complications.Recent studies found that perivascular adipose tissue(PVAT)closed to macrovessels is closely related to diabetic macrovascular complications such as atherosclerosis.Framingham and Multi-Ethnic Study of Atherosclerosis studies have found that epicardial PVAT volume was an independent risk factor for cardiovascular and atherosclerosis.Therefore,some scholars have suggested that the increase of PVAT volume on behalf of the blood vessels "local obesity" may be associated with diabetes or metabolic syndrome related atherosclerotic disease more closely.Subsequent studies have confirmed that "local obesity" of PVAT appears to enhance the expression of adipose local inflammation and adipokines.And these local adipokines are more likely to reflect the severity of coronary artery disease compared with various plasma biomarkers in the circulation.However,the mechanism of production and regulation of adipokines and the mode across the vessel wall is still unclear.Macrophage polarization is a key link between the inflammation of the adipose and metabolic disorders in diabetic adipose tissue.Macrophages exacerbate adipose cell dysfunction,and dysfunctional adipose cells released inflammatory mediators activate macrophages in return.Two macrophage activation states have been defined in rodents and humans:"classically activated"(M1)cells have pro inflammatory effect or functions,and "alternatively activated"(M2)cells have anti-inflammatory properties.The existence of these different activation states implies prominent roles in different phases of an immunological response,i.e.,inflammation and tissue remodeling.M1 cells are identified by high expression of the enzyme inducible nitric oxide(NO)synthase,a potent respiratory burst,and secretion of proinflammatory cytokines such as tumor necrosis factor-a(TNF-a)and interleukin-12(IL-12).The activation is induced by two signals,one toll-like receptor agonist,such as lipopolysaccharide(LPS),and one cytokine receptor—mediated signal,e.g.,interferon-g(IFNy).Peroxisome proliferator-activated receptor y(PPARy)is an important transcription factor for regulating adipose remodeling and macrophage polarization.PPARy can activate transcription of multiple genes involved regulating adipose tissue function and macrophage polarization.The clinical studies also found that the number of M2 cells in the adipose tissue was increased in diabetic patients taking PPARy activator(thiazolidinediones)while the Ml/M2 ratio decreased.However,the regulatory mechanism of PPARy in perivascular adipose tissue and related macrophages remains unclear.TRB3 is an important regulator of diabetes mellitus metabolism and macrophage function.Previous studies have found that TRB3 is involved in the apoptosis ofmacrophages in diabetes mellitus.After TRB3 gene silencing,apoptosis of macrophage in plaque was reduced.At the same time,it has been found that TRB3 is involved in mediating the disorder of diabetic adipocytes,and overexpression of TRB3 can significantly inhibit the transcriptional activity of PPARy.However,TRB3/PPARy pathway in the role of macrophages and inflammation in the perivascular adipose tissue has not been reported.Combined with the above studies,macrophage polarization related inflammation in"local obesity" PVAT,is a key factor between the PVAT and atherosclerosis.TRB3 gene expression in diabetic patients was significantly increased,and PVAT inflammation in two important cells-adipocytes and macrophages function is closely related.Its downstream PPARy may be involved in regulating macrophage inflammation in perivascular adipose tissue.However,the mechanism of PVAT macrophage polarization in diabetes is unclear,and the role of TRB3/PPARy pathway in perivascular adipose inflammation remains investigation.To explore the above-mentioned problem,we established a diabetic mouse carotid artery adipose transplantation model.Adoptive immune polarized macrophages,local silencing TRB3 gene,PPARy agonist intervention were used to explore the role of TRB3/PPARy pathway in PVAT macrophage polarization and atherosclerotic plaque in diabetes.2 Objectives1)To establish the model of diabetic perivascular adipose transplantation and explore the relationship between PVAT and atherosclerosis plaques in diabetic mice;2)To investigate the effects of macrophage polarization on inflammation of PVAT and plaque vulnerability via adoptive immune therapy of polarized macrophages in vivo;3)To clarify the role of TRB3-PPARy pathway in macrophage polarization-mediated PVAT inflammation and plaque formation via intervention of pioglitazone and local silencing TRB3.3 Method1)The model of PVAT transplantation was established on the basis of diabetic atherosclerotic model mice;2)The effects of carotid PVAT transplantation on the systemic glucose and lipid metabolism,insulin resistance,adipose content and energy metabolism were examined.3)Ultrasonography and pathologic methods were used to detect carotid artery atherosclerosis and vascular remodeling after carotid perivascular adipose transplantation;4)After adoptive immune by polarized macrophages in diabetic mice receiving carotid perivascular adipose transplantation,inflammation in PVAT and atherosclerotic plaque burden and vulnerability were detected.5)Given the PPARy activator(pioglitazone)treatment,inflammation in PVAT and atherosclerotic plaque burden and vulnerability in diabetic mice were detected;6)By ultrasound-guided perivascular adipose was injected with TRB3-shRNA virus,inflammation in PVAT and atherosclerotic plaque burden and vulnerability in diabetic mice were detected.4 Results1)Diabetic PVAT Transplantation Model:the transplanted PVAT present neovascularization and nerve fiber markers,without nectosis;2)PVAT Transplantation had no significant effect on the systemic glucose and lipid metabolism,adipose content and eneygy metabolic rate in control diet and diabetic mice;3)PVAT Transplantation did not increase the carotid plaque load in control mice,but in diabetic mice,the PVAT Transplantation increased with the progression of the carotid plaque burden and the vascular remodeling index(artery intima media thickness,IMT and pulse wave velocity,PWV);4)Diabetic carotid PVAT transplantation increased carotid plaque burden and vulnerability,and promoted perivascular adipose inflammation and M1 cell polarization:5)M1 macrophages adoptive immune increased carotid plaque burden and vulnerability,and increased perivascular adipose and M1 cell polarization,M2 macrophages adoptive immune had opposing effects.6)PPARy activator pioglitazone treatment in diabetic mice reduce the systemic plaque burden,and carotid plaque burden and vulnerability,moreover alleviated perivascular adipose inflammation and increased M2 macrophage ratio.7)Local silence of TRB3 expression in perivascular adipose can reduce the perivascular adipose transplantation increased carotid plaque burden and vulnerability,relieved the PVAT inflammation,and promote macrophages to M2 polarization;8)Immunoprecipitation assay confirmed that TRB3 interacts with PPARy in protein-protein interaction.5 Conclusions1)The expression of TRB3 in the perivascular adipose increase in diabetic mice,and negatively regulated PPARy activity;2)Diabetes promotes the M1 polarization macrophages in PVAT,aggravate PVAT and adjacent intravascular inflammation;3)TRB3 silencing and PPARy activation could significantly inhibit macrophage polarization-mediated perivascular adipose inflammation,reduce plaque burden and stabilize plaque. |
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