A Study Of The Role And Mechanism Of Angiogenesis On Diabetic Atherosclerosis And Adipose Tissue Dysfunction

BackgroundAtherosclerotic Cardiovascular Disease(ASCVD)can be considered the most important cause of death in diabetic population.In type 2 diabetes mellitus(T2DM),the number of vessels in the vasa vasorum(VV)is increased and associated with plaque rupture.Inflammation is a potent driving factor that induces W angiogenesis.Chronic low-grade inflammation in adipose tissue has been considered to be a crucial event in accelerating diabetic atherosclerosis,but how adipose tissue inflammation aggravates vulnerable plaques by increasing VV angiogenesis is not clear.Although the Canakinumab Anti-inflammatory Thrombosis Outcomes Study(CANTOS)provides compelling evidence that the targeting of inflammation is effective in preventing recurrent cardiovascular events,the researchers note an increase In fatal infection for approximately one in every 1,000 patients treated,and previous studies on anti-inflammatory approaches have yielded negative results.Due to the large number of factors influencing the development of atherosclerosis and due to non-specific cell targeting by these factors,the effect of adipose tissue inflammation on the promotion of atherosclerosis is exceedingly unlikely to be understood using the levels of any one factor.Recently,the possibility that EVs(extracellular vesicles)interact with specific target cells has been suggested,and adipocyte-derived EVs contain a variety of adipocytokines.Thus,adipocyte-derived exosomes(IRADEs)may be a more important vector of transmission,which may explain accelerated atherosclerosis in T2DM.Exosomes(50-100 nm)are microvesicles that are derived from multivesicular bodies(MVBs)and are released into the extracellular environment after MVB fusion with the cytomembrane.Recent studies have demonstrated that exosomes can mediate intercellular communication under normal and pathological conditions.By transporting proteins,mRNAs and miRNAs,exosomes,as vehicles of information,may alter the behaviours of recipient cells.Numerous studies have shown that exosomes have pathophysiological effects on atherosclerotic plaque destabilization.However,whether exosomes are responsible for VV angiogenesis remains unknown.Angiogenesis rising from the VV may interconnect diabetic macroangiopathy and microangiopathy.Accumulative evidence suggests that angiogenesis from the VV is associated with accelerated atherosclerosis and plaque vulnerability.Recently,several studies have preliminarily shown that exosome-induced angiogenesis involves various mechanisms and that exosomes from ADSCs(adipose stromal cells)are involved in angiogenesis.These studies suggest that IRADEs may integrate the information of adipose tissue inflammation and transmit this information to the vascular adventitia VV,thereby increasing VV angiogenesis and plaque vulnerability.Among the molecular signals involved in angiogenesis,the Hedgehog(HH)pathway is featured prominently.Sonic hedgehog(SHH)is the most widely expressed hedgehog signal molecular.The lack of SHH is lethal to embryo.Several recent observations highlight a fact that by activating perivascular cells,SHH is involved in the maintenance of adult coronary vasculature.Hedgehogs(HH)are a class of 19-kDa morphogens.HH activates the intracellu.lar pathway by binding recipient cells Patched(Ptch)receptor.Once the Ptch receptor is activated,its inhibition on Smoothened is abrogated and activates signaling via transcription factor Gli,Gli2 and Gli3.The mechanism of the intercellular movement of the lipid-modified hydrophobic SHH molecules has been explained by several models.One of these models is the exosomes as carriers.However,whether SHH protein is responsible for the VV angiogenesis induced by IRADEs remains a question.To determine whether IRADEs are involved in VV angiogenesis and plaque stability,we performed a series of in vitro,ex vivo and in vivo experiments.Objective1.Establishment of insulin resistance adipocyte model to observe IRADEs and SHH expression2.To establish a type 2 diabetes model of atherosclerosis in mice and observe the effect of IRADEs on plaque VV angiogenesis,plaque burden and stability3.Role of SHH in RADEs in W angiogenesis,plaque burden and stabilityMethods1.To design and synthesize siRNA against mouse SHH according to RNAi Principle,then to construct the pAdxsi-GFP-SHH-shRNA.To design and primers of mouse SHH according to Genebank,pShuttle-CMV-RED-SHH was constructed.2.Construction of Insulin Resistance adipocyte model:The 3T3-L1 preadipocytes(ATCC,USA)were cultured in Dulbecco's Modified Eagle's medium(DMEM,Low Glucose,Gibco)containing 10%fetal calf serum(FCS,Gibco),100U/ml penicillin-streptomycin and in a 5%C02 humidified atmosphere.After reaching 100%confluence two days,3T3-L1 preadipocytes were differentiated to adipocytes in a cocktail containing 0.5mM 3-isobuty1-1-methylxanthine(Sigma-Aldrich),10?g/ml insulin(Sigma-Aldrich),and InM dexamethasone(Sigma-Aldrich).48 h later,cells were maintained with DMEM(High Glucose,Gibco)containing 10%FCS and 10?g/ml insulin every second day until day 8.The mature adipocytes were confirmed by light microscopy and oil red O staining.Insulin resistance adipocytes were induced by incubating differentiated 3T3-L1 adipocytes in DMEM containing 25 mM glucose,10%FCS and lOnM insulin for 24h.3.The characterization of IRADEs was measured with electron microscopy,NTA(Nanoparticle Tracking Analysis)and western blot respectively.4.Exosomes uptake Assay in vitro and in vivo according to the manufacturer's protocol(PKH26GL,Sigma-Aldrich).5.The roles of IRADEs in angiogenesis were demonstrated by aortic ring and matrigel plug assay.6.Plaque burden,plaque stability and angiogenic factor expression were evaluated by ultrasonography,immunohistochemistry and western blot respectively.Results1.Generation of the diabetic atherosclerosis mouse modelInsulin resistance in mice was confirmed with the(IPGTT).At 9 weeks of age,the blood glucose levels were significantly higher in the DM group than in the Chow group at baseline at all time points tested except at 0 minutes(p<0.05).However,at 11 weeks of age,the blood glucose levels were significantly higher in the DM group than in the Chow group at all time points tested(p<0.05).Similarly,at 9 and 11 weeks of age,the AUC for the glucose level of the DM group mice was higher than that of the Chow group(p<0.05).As expected,the body weight was significantly higher in the DM group than in the Chow group at 9 weeks of age but not at 11 weeks of age(p<0.05).At 24 weeks of age,the IPGTT results showed that glucose tolerance was significantly lower in the DM+IRADEs and Chow+IRADEs groups than in the DM and Chow groups,respectively(p<0.05).After the silencing of SHH expression in IRADEs,the IPGTT results showed no significant changes(p<0.05).Several metabolic indices were substantially altered in the diabetic mice.Compared to the Chow group,the levels of total triglycerides,total cholesterol,low-density-lipoprotein cholesterol,and high-density-lipoprotein cholesterol were significantly increased in the DM group(p<0.05).No significant differences were found between the IRADEs treatment groups(including Chow+IRADEs and DM+IRADEs group)and no treatment(including Chow and DM group).After SHH expression in IRADEs was silenced,these metabolic indices showed no significant changes.2.Characterization of IRADEsThe ultrastructure of the exosomes were observed by using transmission electron microscopy and Nanosight.The results revealed that the IRADEs had a cup-shaped morphology with a diameter range of 30?100 nm,reaching a peak size of 80 nm.The expression levels of exosomes markers CD63 and TSG101 were confirmed by western blot.The purity of the exosomes was tested by examining the expression of two negative markers for exosomes,namely,Calnexin and Grp94.These data indicated that the exosomes were successfully isolated from the culture medium.Next,we analysed the number of isolated IRADEs from vehicle-and SHH-transfected cells to examine whether SHH protein expression changed IRADEs production.Interestingly,SHH protein expression did not affect IRADEs production.The quantitated results showed that the concentration of the isolated IRADEs was approximately 7.5×109/ml(p<0.05).3.Construction of the insulin resistance adipocyte model and confirmation of the presence of SHH proteins on IRADEsTo determine whether insulin resistance could be established in adipocytes,the IRS-1 phosphorylation level was determined by western blot with the phosphor-specific IRS-1(Ser307 and tyr632)antibody.Phosphorylation was induced with high glucose and high insulin,and the induction occurred in a time-dependent manner.IRS-1 phosphorylation(Ser307)was increased significantly at 6 h,and IRS-1 phosphorylation(tyr632)was decreased significantly at 6 h(p<0.05).Light microscopic observation showed that the translucent "beaded" material in the mature adipocytes was generated intracellularly and that mature adipocytes showed lipid accumulation as determined by Oil red O staining.Next,we investigated whether SHH could be observed on IRADEs.Immunogold labelling for SHH on IRADEs revealed SHH on their surfaces.The specific expression of SHH was examined by western blot in exosomes derived from SHH-overexpressing and SHH-silenced insulin resistance adipocytes cultured in exosome-free medium.As expected,SHH expression on IRADEsSHH+ was increased significantly(p<0.05)and SHH expression on IRADEsSHH_was downregulated significantly(p<0.05).4.IRADE uptake both in vitro and in vivoAn analysis was performed to examine the ability of exosomes to transfer into endothelial cells by examining the uptake of PKH26-labelled IRADEs.After HUVECs were treated with the IRADEs,the IRADEs were internalized in a time-dependent manner.The IRADEs rapidly entered into the cytoplasm within 3 hours and further accumulated at the perinuclear area after 6 hours.Next,we intravenously injected PKH26-labelled IRADEs into diabetic ApoE-/-mice to investigate whether the endothelial cells of atherosclerotic plaque could uptake IRADEs,and the results confirmed that these endothelial cells took up the IRADEs.5.IRADEs contribute to angiogenesis via SHH ex vivoTo determine whether IRADEs could contribute to angiogenesis,we performed matrigel plug assays.Two weeks after the injections,the matrigel plugs were excised,fixed and embedded.The angiogenic response was analysed by determining the total cell number and the endothelial(CD31 positive)area in the matrigel.The total cell number was highest in the matrigel of the VEGF group;lowest in the matrigel of the control group;significantly higher in the IRADEs and IRADEsSHH+ groups than in the IRADEsSHH-group(p<0.05);and significantly higher in the IRADEssSHH+ group than in the IRADEs group(p<0.05).This identical pattern was found among the five groups for the protein expression of CD31,an indicator of angiogenesis.Next,the effect of IRADEs on angiogenesis was observed in aortic ring assays.The number of sprouting microvessel branches from the aortic rings was highest in the VEGF group,lowest in the control group,significantly higher in the IRADEs group and IRADEsSHH+ group than in the IRADEsSHH-group(p<0.05),and significantly higher in the IRADEsSHH+ group than in the IRADEs group(p<0.05).6.SHH carried by IRADEs increases the vasa vasorum angiogenesis of aortic plaques in diabetic Apo-/-miceCompared to the Chow group,the number of VV in the adventitia surrounding the atherosclerosis plaques in the aortic roots was significantly increased in the DM group(p<0.05).The numbers of VV were significantly higher in the DM+IRADEs and Chow+IRADEs groups than in the DM and Chow groups,respectively(p<0.05),and significantly lower in the DM+IRADEsSHH*and Chow+IRADEsSHH-groups than in the DM+IRADEs and Chow+IRADEs groups,respectively(p<0.05).7.SHH contributes to IRADEs-mediated angiogenesis in vitroTo determine the effect of IRADEs on angiogenesis,we performed tube formation assays with both MAECs and HUVECs.We measured the total branching lengths and branches numbers to determine these pro-angiogenic effects.The results showed that the total branching lengths and branches numbers in the IRADEs group(p<0.05)and VEGF group(p<0.05)were significantly higher than in the control group,indicating that the IRADEs exhibited pro-angiogenic effects.To determine the effect of SHH on the pro-angiogenic effects of IRADEs,we overexpressed and silenced SHH on IRADEs.We observed that the total branching lengths and branches numbers in IRADEsSHH+ group(p<0.05)was much higher than IRADEs group while IRADEssSHH-group(p<0.05)was significantly lower.Meanwhile,the treatment of cyclopamine could block the pro-angiogenic effects of IRADEs.Clearly,SHH contributed to IRADEs-mediated angiogenesis.8.IRADEs increase the plaque burden by SHH in vivoHigh-resolution ultrasonography examination revealed atherosclerotic plaques in the arteries of the mice.In our study,the aortic,brachiocephalic and carotid IMTs were significantly increased in the DM group compared to in the Chow group.We found that each of the three parameters was significantly increased in the DM+IRADEs and Chow+IRADEs groups compared with in the DM and Chow groups,respectively(p<0.05),whereas the silencing of SHH on IRADEs significantly reduced the aortic,brachiocephalic and carotid IMTs in the DM+IRADEssSHH-and Chow+ IRADEssSHH-groups compared with in the DM+IRADEs and Chow+IRADEs groups,respectively(p<0.05).Next,the en face lesion and cross-sectional plaque area of the aortic sinus were significantly increased in the DM group compared with in the Chow group(p<0.05).As expected,the en face lesions and cross-sectional plaque areas of the aortic sinuses were increased in the DM group compared to in the Chow group.Each of the two parameters was increased in the DM+IRADE and Chow+IRADE groups compared with the DM and Chow groups(p<0.05),respectively,and these effects were lessened in the DM+IRADEsSHH-and Chow+ IRADEssSHH-groups compared with in the DM+IRADEs and Chow+IRADEs groups,respectively(p<0.05)9.SHH carried by IRADEs increases the vulnerability of aortic plaques in diabetic ApoE-/-miceIn vivo,the relative contents of the aortic components were assessed by immunohistochemistry.The relative contents of lipids and macrophages were significantly increased in the DM group compared with in the Chow group(p<0.05).Further analyses showed that the relative contents of lipids and macrophages were higher in the DM+IRADEs and Chow+IRADEs groups than in the DM and Chow groups,respectively(p<0.05).These effects were lessened in the DM+IRADEsSHH-and Chow+ IRADEsSHH-groups compared with in the DM+IRADEs and Chow+IRADEs groups(p<0.05).In contrast,the relative contents of vascular smooth muscle cells(VSMCs)and collagen displayed opposite patterns among the six groups.Consequently,the plaque vulnerability index was dramatically higher after IRADEs treatment but lower after IRADEsSHH-treatment(p<0.05).10.Effects of SHH carried by IRADEs on angiogenesis-related protein expression in diabetic ApoE-/-mice atherosclerosis plaqueWe examined the expression of proinflammatory cytokines that are directly or indirectly angiogenic,such as TNF-?,IL-6,IL-1 ? and ICAM-1;extracellular matrix-related proteins;and angiogenesis factor VEGF-A.The expression of angiogenesis-related factors were significantly increased in the DM group compared with in the Chow group(p<0.05).Compared with the DM and Chow groups,the DM+IRADEs and Chow+IRADEs groups,respectively,showed significantly increased protein expression levels of proinflammatory and angiogenic factors(p<0.05),whereas these effects were attenuated in the DM+IRADEssSHH-and Chow+IRADEsSHH-groups,respectively(p<0.05).Conclusions1.ADEs carried SHH protein,and the expression of SHH increased in IRADEs;2.HUVECs and the endothelial cells of atherosclerotic plaque could uptake IRADEs;3.IRADEs significantly increased matrigel plug assays in mice and angiogenesis in mouse aortic rings;4.The significant increase of W angiogenesis by IRADEs may be an important mechanism of IRADEs promoting the plaque burden and vulnerability of diabetic ApoE-/-mice.5.The mechanism of IRADEs increasing W angiogenesis may be related to IRADEs promoting the expression of angiogenesis related factors in plaque;6.IRADEs increase VV Angiogenesis is mediated by SHH proteinBackgroundAdipose tissue is considered to be one of the main driving forces for the development of insulin resistance(IR).The vasculature as a key anatomical structure to furnish nutrition to adipose tissues plays a vital role in the pathogenesis of IR.It has been demonstrated that angiogenesis could promote or improve IR via regulating the adipogenesis and energy balance.Adipose tissues consist of white and brown adipose tissues(WAT and BAT).Previous studies only focused on the role of WAT or BAT angiogenesis in IR respectively.The role of different adipose tissue angiogenesis in IR remains unclear.Adipose tissue angiogenesis is closely related to IR.As reported in obese patients,hypertrophic adipocytes in WAT can increase proangiogenic factors secretion and promote angiogenesis which in turn promote adipocytes hypertrophy,thereby creating a vicious circle and aggravating IR.In the metabolically active BAT,angiogenesis can increase energy consumption and improve IR.PPARy is present in endothelial cells and multiple studies demon-strated that PPARy activation suppressed the proliferation of endothelial cells in vitro and their differentiation into tube-like structures.It has been demonstrated that PPAR? plays a major role in regulating endothelial cells apoptosis in the tumor tissue via CD36.Six transmembrane protein of prostate 2(STAMP2)is well known for its anti-inflammatory and metabolic role in maintaining normal glucose tolerance,lipid metabolism and improving IR.Recent studies have shown that STAMP2 is a metalloreductase which may regulate cellular iron in cancer growth,angiogenesis and metastasis.However,the effects of STAMP2,particularly in adipose tissues,on angiogenesis-related development of insulin resistance and the underlying mechanisms are still unknown.STAMP2 knockdown leads to inhibition of adipogenesis by diminishing the expression of PPARy.However,whether STAMP2 regulate the angiogenesis of adipose tissue through the PPAR?/CD36 signalling pathway remains to be further investigated.With the aim of evaluating the effects of STAMP2 on adipose tissue angiogenesis in different adipose tissue of diabetic mouse,we established the type 2 diabetes ApoE-/-/LDLR-/-mice models with STAMP2 gene overexpression in vivo.We studied the effects of STAMP2 on angiogenesis in different regional adipose tissues and determined the molecular mechanism whereby STAMP2 modulates PPARy/CD36 signaling pathways in human umbilical vein endothelial cells.Objectives1.To observe the effect of STAMP2 on angiogenesis in different regional adipose tissues of the type 2 diabetes ApoE-/-LDLR-/-mice models;2.The effect of STAMP2 on HUVECs migration,tube formation and angiogenesis related factor expression in vitro;3.To determine the molecular mechanism whereby STAMP2 modulates PPARy/CD36 signaling pathway.Methods1.Diabetic model and in vivo experiments Three-week-old male ApoE-/-/LDLR-/-mice were fed a high-fat diet(34.5%fat,17.5%protein,48%carbohydrate;Beijing HFK Bio-Technology,China).After 6 weeks,the ApoE-/-LDLR-/-mice underwent an intra-peritoneal glucose tolerance test(IPGTT).Those mice showing IR were injected once with low dose of streptozotocin(STZ,Sigma-Aldrich,st.louis,MO,USA;75-80 mg/kg i.p.in 0.1 mol/L citrate buffer,pH 4.5)intraperitoneally.After 2 weeks,most high-fat diet/STZ-treated mice displayed hyperglycemia,IR,and glucose intolerance,as previously reported.At age 11 weeks,mice with similar degrees of hyperglycemia and body weight were randomly divided into two groups,one will be treated with the STAMP2-expressing adenoviruses,referred to as the DM+STAMP2 group(n=10),and the other treated with the expressing vector control adenovirus,referred to as the DM +Vehicle(n=10;see later for the details of the adenovirus used).The mice fed a normal diet were used as non-diabetic controls,divided into Control +Vehicle(n=10)and Control+STAMP2(n=10)groups.All animal procedures were performed in accordance with animal protocols approved by Shandong University Institutional Animal Care and Use Committee.2.Intraperitoneal glucose tolerance test(IPGTT)Glucose tolerance was assessed by IPGTT after mice fasted for 12-16 h.A bolus of glucose(2 g/kg)was injected intraperitoneally,and blood samples were collected from the tail vein at 0,15,30,60 and 120 min and glucose was measured using a One-Touch Glucometer(Life-Scan,Milpitas,CA).3.Production and administration of adenoviral vector The cDNA of mouse STAMP2(GenBank accession no.BC006651)from SinoGenoMax Company Limited was cloned into the p-Shuttle vector.STAMP2 cDNA was sub-cloned between KpnI and EcoRI of the p-Shuttle expression cassette.Then recombinant pAdxsi adenovirus was constructed using the p-Adxsi Adenoviral System(SinoGenoMax,Beijing,China).After amplification,viruses were purified,tittered,and stored at-80? until used.All mice were injected via the jugular vein with 5×109 plaque-forming units of virus at 20 weeks.Adenovirus transfer was repeated at 22 weeks.The control group was injected with control virus(vehicle).Four weeks after first adenovirus injection,all mice were killed for further study.The recombinant pEnter adenovirus constitutively expressing human STAMP2 was constructed using the pEnter Adenoviral System(no.CH835357 Vigene,Jinan,P.R.China).Then STAMP2 cDNAs from human were inserted into pEnter-CMV vector.The pEnter-EGFP vector adenovirus was used as the control vehicle virus.The recombinant pEnter adenovirus was use for cell transfection.4.Blood analyses At the end of all experiment,we collected the murine samples to measure the levels of fasting blood glucose,total cholesterol,total triglyceride and free fatty acids.5.Histological and morphometric analyses Samples were taken from epididymal,subcutaneous and brown adipose tissue(EWAT,SWAT and BAT).Each adipose tissue sample was cut into 2 pieces.One-half of the samples were fixed in Para-formaldehyde(4%)and embedded in paraffin,and cut into 5mm sections.A single adipocyte was measured with images captured from hematoxylin and eosin-stained sections.Every adipocyte area was assessed under ×400 magnification within adipose tissue,and a mean was obtained by quantitative morphometry with automated image analysis(Image-Pro Plus,Version 5.0;Media Cybernatics,Houston,TX).6.Ex vivo aortic ring angiogenesis assay Aortas of mice were dissected for aortic ring assay as described with some modifications.Briefly,dissected thoracic aortas from treated ApoE-/-/LDLR-/-mice were cut into about 0.5mm long rings.Some rings were transfected with 1×106 PFU/ml STAMP2-overexpressing adenovirus overnight.Then all aortic rings were embedded in matrigel(Cat.No356234,BD,USA).After the matrix polymerize,aortic rings were incubated with 2.5%fetal bovine serum(FBS)Opti-MEM and medium was changed on days 3,6 and 8.The VEGF treatment was supplemented to a final concentration of 30ng/ml.Pictures were captured by use of a Canon Camera linked to a light microscope on day 8 and the number of micro-vessels sprouting was counted.7.Immunohistochemical staining Paraffin sections underwent immunohistochemistry by a microwave-based antigen retrieval method.The sections were incubated with anti-Mouse endomucin(1:200 eBioscience Inc.California USA)and CD31(1:100 ab28364)overnight and then with a matching biotinylated secondary antibody for 30 min at 37?.Negative controls were omission of the primary antibody.The stained sections were developed with diaminobenzidine and counterstained with hematoxylin.The results were viewed under a confocal FV 1000 SPD laser scanning microscope(Olympus,Japan).8.Cell culture Primary human umbilical vein endothelial cells(HUVECs)were purchased from American Type Culture Collection.HUVECs were grown in ECM medium(Sciencell,6076 Corte Del Cedro Carlsbad,USA)supplemented with 10%FBS,100U/ml penicillin,100 ?g/ml streptomycin.Human umbilical vein endothelial cells with in five passages were used in the following experiments.9.SiRNA transfection Transfection was performed with Lipofectamine 2000 reagent(5?L,per well/6-well plate,Invitrogen).Cells were transfected with 100pmol siRNA(siRNA-STAMP2,5?L,per well/6-well plate)or 100pmol negative control siRNA(siRNA-NC,5?L,per well/6-well plate).After incubation for 6 hours,culture medium should be changed.Then the cells were observed under a Laser scanning confocal microscopy(LeicaTCSSP2;Leica).10.Transfection of STAMP2 over-expressing adenovirus The cells were administered virus in 200 MOI.The culture medium was changed after 12 hours.Then the cells were observed under a laser scanning confocal microscopy(LeicaTCSSP2;Leica).11.Cell migration assay Migration of cells was examined by using the wound assay as previously described.HUVECs were treated with siRNA-NC,siRNA-STAMP2,siRNA-STAMP2+RSG(0.1mM,pretreated 1 hour,ab120762),Ad-NC,Ad-STAMP2 or Ad-STAMP2+GW9662(2.5 ?M,pretreated 1 hour,Sigma-M6191)respectively.After 48 hours,confluent cell monolayers were wounded by use of a yellow tip.Detached cells were washed away and then fresh low serum medium was added.After 12 hours of incubation,cells that had migrated across the edge of the wound and into the gap were counted as migrating cells.Images were photographed at 0 and 12 hours after scratching.The closure of the wounded area was analyzed by use of Image-pro plus 6.0.12.Transwell migration assay HUVECs from different groups(siRNA-NC,siRNA-STAMP2,siRNA-STAMP2+RSG,Ad-NC,Ad-STAMP2 or Ad-STAMP2+GW9662)were seeded in the upper chamber and the lower chambers were filled with low serum ECM medium.Cells were allowed to migrate for 4h.Non-migrated cells were removed and migrated cells on the lower side of the membrane were stained with crystal violet.Images were captured in five random fields(x 100).13.Tube formation assay Tube formation assay involved use of growth factor reduced Matrigel(Cat.No356234,BD,USA).Briefly,after matrigel matrix gelled,HUVECs from different groups(siRNA-NC,siRNA-STAMP2,siRNA-STAMP2+RSG,Ad-NC,Ad-STAMP2 or Ad-STAMP2+GW9662)(1×105 cells per well/48-well plate)were suspended in low serum medium.The formation of capillary-like tubes was captured 12h later.The mean tube length was calculated in five random fields(× 100).14.Quantitative real-time RT-PCR Total RNA samples were prepared from HUVECs transfected with siRNA-NC,siRNA-STAMP2,siRNA-STAMP2+RSG,Ad-NC,Ad-STAMP2 or Ad-STAMP2+GW9662 respectively.First strand cDNA was generated by using the first-strand cDNA synthesis kit for reverse transcription-polymerase chain reaction(RR037A Takara).Quantitative RT-PCR was performed with a Light-Cycler system(Roche Diagnostics)according to the manufacturer's instructions.15.Western blot analysis Western blot analysis was as previously described.We used antibodies against STAMP2(1:1000 Cat.No11944-1-AP Protein-tech Group Inc.,Chicago,IL),PPAR?(1:1000 ab191407),CD36(1:1000 ab133625),followed by anti-IgG horseradish peroxidase-conjugated secondary antibody.STAMP2,PPAR?,CD36 protein level was normalized to that of ?-actin(1:1000 Cat.NoZM0003 ZSGB-bio Beijing)or GAPDH(1:1000 Cat.NoTA336621,ZSGB-bio Group Inc.,Beijing)as an internal control.16.Statistical analysis Values are presented as mean±SD.SPSS 17.0(SPSS,Chicago,IL)was used for statistical analysis.Results were compared by one-way ANOVA,followed by Tukey-Kramer post hoc test and independent samples t test.p<0.05 was considered statistically significant.Results1-Overexpression of STAMP2 improves metabolism in ApoE-/-LDLR-/-mice.Consistent with our previous publication,diabetes mellitus(DM)induced by high fat and sugar diet combined with a small dose of STZ in male ApoE-/-/LDLr-/-mice could resemble human diabetes mellitus and STAMP2 overexpression decreased risk factor of insulin resistance in diabetic ApoE-/-LDLR-/-mice.2.WAT and BAT morphologyThis result was consistent with the our formerly report,STAMP2 overexpression reduced adipocyte size in epididymal white adipose tissue(EWAT)and lipid area in brown adipose tissues(BAT).However,overexpression of STAMP2 has no effect on adipocyte size in SWAT.3.STAMP2 gene over-expression inhibits angiogenesis ex vivoWe observed the effect of STAMP2 over-expression on angiogenesis ex vivo by mouse aortic ring assay.The numbers of sprouting micro-vessel branches from aortic rings were reduced with STAMP2 over-expression than control treatment(p<0.05).4.STAMP2 gene over-expression suppresses angiogenesis in EWAT and BATThe density of angiogenesis using endomucin or CD31 as angiogenesis markers were significantly increased in WAT in DM group(p<0.05).DM+STAMP2 group showed significantly decreased angiogenesis density in EWAT.We also found that angiogenesis density was elevated significantly in BAT in the diabetic ApoE-/-/LDLR-/-mice group(p<0.05).DM+STAMP2 group showed significantly decreased angiogenesis density in BAT.Moreover,we found that angiogenesis density in EAT and BAT of Control+STAMP2 group were much less than that of Control+Vehicle(p<0.05).Those changes above were not seen in SWAT in DM+STAMP2 group compared to DM+Vehicle.These data suggested that STAMP2 over-expression can suppress angiogenesis in EWAT and BAT.5.STAMP2 gene over-expression increases expression level of PPARy/CD36 in EWAT and BATConsistent with our previous publication,endogenous STAMP2 expression was significantly decreased in EWAT and BAT in DM+Vehicle group compared with that in Control+Vehicle group(p<0.05).Over-expression of STAMP2 could significantly increase STAMP2 expression in EWAT and BAT in diabetic ApoE-/-/LDLR-/-mice(p<0.05).However,there were no significant differences in STAMP2 expression in subcutaneous white adipose tissues(SWAT)in all groups.With STAMP2 over-expression treatment,the protein expression level of PPARy/CD36 was marked increased in EWAT and BAT(p<0.05).However,the changes above in protein expression level of PPARy/CD36 were not seen in SWAT in Control+STAMP2 and DM+STAMP2.6.The effect of STAMP2 gene on HUVECs migration and tube formation in vitroIn this study,we examined the function of STAMP2 in vitro in the present study.We treated HUVECs with siRNA-NC,siRNA-STAMP2,siRNA-STAMP2+RSG,Ad-NC,Ad-STAMP2 or Ad-STAMP2+GW9662 and examined their effects on angiogenesis-related properties of HUVECs.The migration of endothelial cells to designated sites is a critical step in angiogenesis.Thus,we used wound-healing assay to observe the effects of STAMP2 on cell motility.STAMP2 silencing in the siRNA-STAMP2 group significantly promoted wound closure as compared with the siRNA-NC group,which was completely abolished by RSG(p<0.05).As expected,cell from Ad-STAMP2 group showed significantly lower migration ability compared with from Ad-NC group,which was antagonized by GW9662(p<0.05).This effect of STAMP2 on HUVECs migration was further confirmed by Trans-well assay(p<0.05).HUVECs spontaneously form tubes when plated on Matrigel.Whereas siRNA-NC group formed tube structures on Matrigel surfaces,this tube-forming ability was heightened in HUVECs treated with siRNA-STAMP2(p<0.05).As expected,tube formation was reduced in Ad-STAMP2 compared with Ad-NC group.The effects of Ad-STAMP2 transfection on tube formation were completely reversed by treatment with GW9662,while RSG significantly abolished the roles of siRNA-STAMP2 in tube formation.These results suggested that silence or over-expression of STAMP2 affected cell abilities of migration,tube formation by PPAR?/CD36 signaling pathway.7.The role of STAMP2 in regulating the PPARy/CD36 signaling PathwayWe investigated the role of STAMP2 in regulating the PPARy/CD36 signaling pathway(p<0.05).Western blot analysis showed that the protein level of STAMP2 was lower in the siRNA-STAMP2 group than the siRNA-NC group(p<0.05).After silence of STAMP2,the protein levels of PPARy/CD36 were reduced respectively in siRNA-STAMP2 group compared with siRNA-NC group.Cells were treated with the RSG significantly increased PPARy protein level,while CD36 preotein level was also high-regulated.(p<0.05).These data showed that STAMP2 silencing suppressed the PPARy/CD36 signaling Pathway.As expected,the protein level of STAMP2 was increased in Ad-STAMP2 group compared with Ad-NC group(p<0.05).The protein levels of PPARy/CD36 were increased respectively in Ad-STAMP2 group compared to Ad-NC group.Then we decreased PPARy protein level by using GW9662 and CD36...