| BackgroundDiabetes is one of the most severe public health problems in the 21st century.China has become the country with the most significant number of diabetic patients.Compared with adults without diabetes,75%of individuals with diabetes die of cardiovascular complications.Studies have shown that patients with coronary heart disease and type 2 diabetes have a higher degree of coronary artery involvement and stenosis,more diffuse lesions,which significantly increases clinical treatment difficulty.Percutaneous coronary interventions(PCI)are an effective treatment for coronary heart disease with diabetes currently.However,with the improvement of PCI treatment,diabetes has been an independent risk factor for PCI complications,poor short-term and long-term prognosis.In-stent restenosis(ISR)incidence in patients with diabetes increased significantly after PCI,which limited the prognosis of patients.In-stent restenosis(ISR)is a complex disease process involving multiple mechanisms.It is a core problem that worsens the prognosis of patients with cardiovascular complications of type 2 diabetes.Many studies have confirmed that intimal hyperplasia is the primary pathological process of in-stent restenosis.Smooth muscle cells(SMCs)proliferation is an essential pathophysiological signature of intimal hyperplasia,and it was regarded as a vital development mechanism of in-stent restenosis.Rapamycin and paclitaxel drug-eluting stents developed to inhibit smooth muscle cell proliferation can reduce in-stent restenosis incidence to a certain extent.However,there was a "late catch-up phenomenon," and the rate of ISR was 10%with new-generation DES.It suggests that smooth muscle cell proliferation may be the leading intermediate process of intimal hyperplasia rather than the underlying mechanism.Besides,the mechanism of diabetes initiates and accelerates intimal hyperplasia after PCI has not yet been elucidated.Thus,to prevent the development of ISR,it is necessary to explore the initiation process of intimal hyperplasia under diabetes and find intervention targeting it.In terms of vascular structure,endothelial cells,which play a role as the natural barrier between the vessel wall and various pathological stimuli in the blood,are the key to protecting smooth muscle cells from pathological stimuli factors.Endothelial cells may play a core role before smooth muscle cells in the process of intimal hyperplasia.Endothelial cells are damaged during stent implantation.Endothelial dysfunction plays a critical role in the development of atherosclerosis.Perioperative drug therapy has protected the integrity of endothelium to the greatest extent.Nevertheless,SMCs continue to be stimulated and proliferate even the structure of endothelium is complete.Researchers have recently found that endothelial cells can transform into mesenchymal cells under stimulation,named Endothelial-Mesenchymal Transition(EndMT).The research has given us some enlightenment.EndMT is defined as endothelial cells lose their specific antigens under certain physiological or pathological conditions and acquire mesenchymal cell antigens.Simultaneously,their biological characteristics change significantly,and they develop intense proliferation,migration,and contraction abilities.EndMT has been involved in the occurrence of many diseases related to intimal hyperplasia and atherosclerosis promotion.Nevertheless,it has not been involved in preventing and treating restenosis after PCI.Therefore,EndMT may play a vital role in the occurrence of ISR.Recent research has confirmed that various external stimuli factors play an essential role in promoting EndMT,including high glucose,inflammation,hypoxia,lipid metabolism disorder,oxidative stress,mechanical tension,and smoking.However,the pathogenesis of diabetes mellitus,atherosclerosis,and stent restenosis is complex and involves the synergistic effects of all factors mentioned above.Exosomes can carry many proteins,nucleic acids,and lipids,integrate the source cells' practical stimulus information and realize the cascade amplification and long-distance transmission of signals effectively.Information carried and transmitted by Exosomes is closely related to their cells/tissues origin.Adipocyte-derived exosomes(AdExos)contain protein and nucleic acid related to adipose tissue inflammation and insulin resistance,which could induce various biological functions by acting on target cells.They may be an essential bridge linking insulin resistance adipose tissue with diabetic arteries and an essential carrier to integrate various stimulation factors in the body and induce EndMT.Sonic hedgehog(Shh),one of the Hedgehog protein homologous forms,is the most widely expressed and active protein.Shh is a crucial protein involved in embryonic development.Studies have shown that Shh activates the Snail signaling pathway to promote epithelial-mesenchymal transition(EMT)in the tumor.While EndMT is a particular type of EMT.Further researches confirmed that the Snail signal pathway is also a critical signal pathway that regulates the formation of EndMT.In summary,we propose the following hypothesis:In diabetes,AdExos with Shh increase and are taken up by endothelial cells.AdExos activate the EndMT process,resulting in endothelial cells undergoing a phenotypic transition,promoting intimal hyperplasia,which eventually leads to in-stent restenosis.Objectives1.To confirm that AdExos promoted endothelial-mesenchymal transition through Shh,exacerbate intimal hyperplasia,and in-stent restenosis in diabetes at the animal level.2.To explore the characteristics of AdExos promoting endothelial-mesenchymal transition at the cellular level,verify that AdExos activate the Snail signaling pathway of endothelial cells and promote EndMT through Shh,which provides new targets for the prevention and treatment of in-stent restenosis after PCI in patients with type 2 diabetes.Methods1.Culture,induction,and differentiation of 3T3-L1 preadipocytes and construction of insulin resistance modelFirstly,we induced 3T3-L1 preadipocytes to mature adipocytes with the combination of insulin,3-isobutyl-1-methylxanthine,and dexamethasone.Then,we established an insulin resistance model of adipocytes by incubated with high glucose and insulin levels-we cultured cells with exosomes-free serum and collected cells supernatant(control group and insulin resistance group).2.3T3-L1 adipocytes were transfected with Shh adenovirusBased on insulin resistance,we transfected 3T3-L1 adipocytes with Shh shRNA and overexpression adenovirus.After culturing with exosomes-free serum,we collected cell supernatant.We got the insulin resistance group,Shh shRNA of insulin resistance,Shh overexpression of insulin resistance,and vector of insulin resistance groups of adipocytes supernatant.3.Isolation,extraction,and characterization of AdExosExosomes in the supernatant of adipocytes were isolated and extracted by overspeed differential centrifugation.We got AdExos from the control group(Ctrl-AdExos),insulin resistance group(IR-AdExos),Shh interference insulin resistance group(IR-AdExosshh-),Shh overexpression insulin resistance group(IR-AdExosshh+),and vector insulin resistance group(IR-AdExosvector).Then,transmission electron microscopy,dynamic Dynamic Light Scattering(DLS),flow cytometry,and Western blot were used to identify the characteristics of AdExos.4.Construction of type 2 diabetic restenosis of ApoE-/-mice modelFour weeks of male ApoE-/-mice,adaptive feed for one week,fast for 12-14 hours before intraperitoneal glucose tolerance test(IPGTT).Then mice were randomly divided into high-fat diet and normal diet groups.Six weeks later,the IPGTT assay was performed again.Mice in the high-fat diet group with insulin resistance were given a one-time intraperitoneal injection of STZ(standard dose was 75mg/kg),and mice in the normal diet group were given the same dose of sodium citrate buffer for intraperitoneal injection.After feeding with the original diet for two weeks,an IPGTT test and random blood glucose test were conducted again.Mice with random blood glucose level?11.1mmol/L and polydipsia,polyuria,and polyphagia were included in the type 2 diabetes group.The restenosis model was established in type 2 diabetic mice and control mice using cuff implantation.Mice were injected with normal saline,IR-AdExos,and IR-AdExosshh-through the tail vein,respectively.The mice were divided into normal diet restenosis group(Chow),normal diet+IR-AdExos restenosis group(Chow+IR-AdExos),normal diet+IR-AdExosshh-restenosis group(Chow+AdExossh-),diabetic restenosis group(DM),diabetes+IR-AdExos restenosis group(DM+IR-AdExos),and diabetes+IR-AdExossh-restenosis group(DM+IR-AdExoshh-).5.AdExos uptake by blood vessels in miceTo verify that AdExos could be uptake by mouse blood vessels,we used PKH26 dye to label AdExos.We observe the uptake of AdExos in vivo via immunofluorescence.6.Histological staining was performed to analyze restenosisHE,Verhoeff and immunohistochemical staining were performed on the femoral artery to evaluate the restenosis.7.EndMT in restenosis was detected by immunofluorescenceCD31 and SM22?,CD31 and Vimentin,VE-cadherin and SM22?,VE-cadherin and Vimentin Immunofluorescence co-localization were used to detect the occurrence of EndMT in the restenosis site.8.Isolation and extraction of mouse aortic endothelial cells.Verification of the uptake of AdExos by endothelial cells.To study the effect of AdExos on EndMT of endothelial cells,we carried out cell experiments.Firstly,we isolated and extracted mouse aortic endothelial cells via the enzymatic digestion method.Then endothelial cells were incubated with PKH26-labeled AdExos.The uptake of AdExos by endothelial cells was observed under a microscope.9.AdExos incubated mouse aortic endothelial cells to detect EndMTThe obtained AdExos from different sources(Ctrl-AdExos,IR-AdExos,IR-AdExosshh-,IR-AdExosshh+,and IR-AdExosvector)and recombinant proteins of TGF-?and SHH were incubated with mouse aortic endothelial cells.Immunofluorescence staining and western blot were used to evaluate the expression level of endothelial markers and interstitial markers and the occurrence of EndMT.10.Western blotWe collected mouse aortic endothelial cells incubated by AdExos and recombinant proteins and extracted proteins in the molecular mechanism.Western blot was used to detect Shh expression,endothelial cells,stromal cell markers,and signaling pathway molecules' expression.Results1.The construction of insulin resistance model of 3T3-L1 adipocytesMore and larger lipid droplets appeared in the cytoplasm after the induction and differentiation of 3T3-L1 preadipocytes into mature adipocytes.After 24 hours of stimulation of mature adipocytes with high glucose and high insulin,the expression of IRS1 in the total protein of adipocytes was significantly decreased.In contrast,the expression of phosphorylation IRS1 at Ser307 was significantly increased.The phosphorylation level of Akt was considerably reduced.The expression of GLUT4 in adipocyte cell membrane protein decreased while that of GLUT4 in cytoplasm protein increased,indicating that GLUT4 cell membrane translocations decreased in insulin resistance condition.In the state of insulin resistance,lipid deposition in adipocytes increased.These results suggest that we have successfully established the insulin resistance model of 3T3-L1 adipocytes.2.Isolation,extraction,and characterization of AdExosIt was found that under transmission electron microscopy,AdExos was a membrane-coated vesicle with a diameter of 30?100nm.The results of the dynamic Dynamic Light Scattering show that the diameter range of AdExos is 30-100nm.Western blot and flow cytometry showed that AdExos were positive for CD63,TSG101,and CD81 but negative for GRP94 and Calnexin.3.Construction of type 2 diabetic restenosis ApoE-/-mouse modelApoE-/-mice were fed on a high-fat diet combined with a one-time injection of STZ to construct the type 2 diabetes model.Diabetic mice had random blood glucose levels?11.1 mmol/L,polydipsia,polyuria,and polyphagia.Based on this model,the restenosis model was constructed by femoral artery cuff implantation.4.Diabetic adipose tissue-derived exosomes enriched Shh moleculesThe Shh molecule expression in adipose tissue-derived exosomes in the control group and the diabetic group was detected by Western blot.The results showed that the level of Shh in adipose tissue-derived exosomes in the diabetic group was significantly higher than that in the control group,which proved that diabetic adipose tissue-derived exosomes were enriched with Shh molecule.5.AdExos uptake by blood vessels in miceUnder immunofluorescence microscope,we observed that PKH26 labeled AdExos showed red and distributed in the range of mouse femoral artery endothelial cells,which proved that femoral vessels could successfully uptake AdExos.6.IR-AdExos promoted restenosis by carrying ShhHigh-resolution microscopic ultrasound was used to detect femoral artery'peak systolic velocity(PSV),and HE staining was used to analyze restenosis.The results of the two methods were combined to evaluate restenosis.The results showed that the femoral artery's restenosis level in the diabetic group was significantly worse than that in the normal diet group.Compared with the normal diet group or diabetes group,restenosis was further aggravated in the normal diet+IR-AdExos group or diabetes+IR-AdExos group.However,the femoral artery restenosis of mice in the normal diet+IR-AdExosshh-group was lighter than that in the normal diet+IR-AdExos group,and the degree of the femoral artery restenosis of mice in the diabetic+IR-AdExosshh-group was lighter than that in the diabetic+IR-AdExos group.7.IR-AdExos promoted EndMT at the site of femoral artery restenosis by carrying ShhImmunofluorescence co-localization was used to detect the occurrence of EndMT at the site of restenosis.In immunofluorescence co-localization,endothelial cells expressing both endothelial cell markers(CD31,VE-cadherin)and mesenchymal cell markers(SM22?,Vimentin)were identified as cells undergoing EndMT.The results showed that endothelial markers decreased and mesenchymal markers increased,suggesting the occurrence of EndMT.The IR-AdExos injection can further promote the occurrence of EndMT,while the effect of IR-AdExos knocking down Shh on promoting EndMT is weakened,which proves that the Shh molecule on IR-AdExos is the main molecular target of IR-AdExos promoting EndMT.8.The primary mouse aortic endothelial cells were extracted successfully,and AdExos uptake in vitroWe successfully extracted mouse aortic endothelial cells by enzymatic digestion.The immunofluorescence staining proved that the extracted endothelial cells were positive of endothelial cell marker CD31,which proved that the extraction of mouse aortic endothelial cells was successful.PKH26-labeled AdExos were co-incubated with mouse aortic endothelial cells,and red fluorescence was observed in the cytoplasm under the microscope,indicating that AdExos was absorbed by mouse aortic endothelial cells.9.AdExos promoted EndMT in endothelial cellsThe obtained AdExos from different sources(Ctrl-AdExos,IR-AdExos,IR-AdExosshh-,IR-AdExosshh+,and IR-AdExosvector)and recombinant proteins of TGF-?and SHH were incubated with mouse aortic endothelial cells.We detected the occurrence of EndMT in endothelial cells by immunofluorescence co-localization and western blot.In immunofluorescence co-localization,endothelial cells expressing both endothelial cell markers(CD31,VE-cadherin)and mesenchymal cell markers(?-SMA,FAP,FSP-1)were identified as cells undergoing EndMT.In western blot,the decreased expression of endothelial cell markers and the increased expression of mesenchymal cell markers proved the occurrence of EndMT.Compared with the Ctrl-AdExos group,EndMT increased in the IR-AdExos group.Compared with the IR-AdExos group,the occurrence of EndMT in the IR-AdExosshh-group did not increase significantly,while the level of EndMT in the IR-AdExosshh+ group increased.These results suggested that IR-AdExos promoted the development of EndMT in endothelial cells by carrying Shh molecules.10.Molecular mechanism of IR-AdExos promoting EndMT in restenosisCompared with the control group,Snail and Slug's level significantly increased in the IR-AdExos group.The above results proved that the EndMT promoting effect of IR-AdExos was exerted through the interaction of Shh on Snail and Slug molecules downstream of IR-AdExos.Conclusions1.In insulin resistance,adipocytes were rich with lipid,and adipocyte-derived exosomes were enriched with Shh molecules.2.IR-AdExos armed with Shh facilitates the development of EndMT in mouse aortic endothelial cells through the Shh/Snail/Slug signaling pathway.3.IR-AdExos enriched with Shh can promote the EndMT of vascular endothelial cells,which may be one of the vital mechanisms of restenosis in diabetic mice.IR-AdExos are expected to be a new target for further prevention and treatment of diabetic ISR.BackgroundDiabetes mellitus is one of the major chronic diseases threatening human health in the 21st century.Recently,the prevalence of diabetes in China has increased rapidly,and China has become the country with the most significant number of diabetes patients worldwide.Acute coronary syndrome caused by ruptured atherosclerotic plaque is the leading cause of cardiovascular events in diabetic patients.However,the mechanism of severe coronary artery disease and the high incidence of vulnerable plaque in diabetic patients has not been fully elucidated.Evidence-based medicine showed that active control of blood glucose could reduce the microvascular disease incidence in patients with diabetes.However,it failed to prove that intensive hypoglycemic therapy can reduce cardiovascular events in type 2 diabetic patients.This suggested that blood glucose and insulin levels are only metabolism indicators rather than the cause of severe coronary artery lesions and a high incidence of vulnerable plaques in diabetic patients.Therefore,it is urgent to explore the underlying mechanism of increased macrovascular complications in diabetic patients.Previous studies have found that pathological angiogenesis often occurred in atherosclerotic plaques,especially in vulnerable plaques and plaques vulnerable parts,which is closely related to plaque stability.Pathological angiogenesis is a critical link in the formation of vulnerable atherosclerotic plaques.Evidence showed that pathological angiogenesis was related to acute coronary syndrome caused by atherosclerotic plaques.These plaques are fragile and prone to rupture,which can lead to vascular obstruction.Hemorrhage in the plaque and pathological angiogenesis promote atherosclerotic instability plaques by recruiting white blood cells.Type 2 diabetes and atherosclerosis are both chronic inflammatory diseases.In type 2 diabetes,lipids are continuously stored in adipocytes,and as adipocytes gradually got bigger,dysfunctional adipocytes and adipokines appear.The secretion of adipokines is disfunction.Many pro-inflammatory factors are synthesized and released,triggering a systemic metabolic,inflammatory response,and insulin resistance,thus accelerating plaque progression and promoting vulnerable plaques.Besides,studies have found that adipocytes can secrete endothelial-specific mitogens and angiogenic growth factors involved in angiogenesis.However,the exact mechanism of how dysfunctional adipose tissue promotes the angiogenesis in plaques and thus promotes the occurrence and development of plaques has not yet been discovered.Recently,researchers believe that exosomes are essential carriers of information transmission between adipose tissue and blood vessels,providing us with clues.Exosomes are secreted by various cells(such as platelets,endothelial cells,monocytes)and carry many proteins,nucleic acids,and lipids closely related to their cell/tissue source functions.They could realize signal cascade amplification and long-distance transmission.Among all exosomes in the body,the relationship between adipocyte-derived exosomes(AdExos)and the occurrence and development of diabetic atherosclerosis and angiogenesis has attracted increasing attention.Hulsmans believed that AdExos was a vital messenger that transmits the inflammation and oxidative stress produced by adipose tissue to blood vessels,thereby causing endothelial damage and inducing atherosclerosis.However,AdExos has not been reported to affect the changes in plaque stability and pathological angiogenesis in the plaque.Studies have found that AdExos was rich in fibronectin and laminin,conducive to the adhesion,migration,and infiltration of circulating AdExos to the endothelial cells.Under diabetic chronic low-grade inflammation,the permeability of damaged endothelial cells increases,and inflammatory factors(IL-6,IL-8),chemokines(MCP-1),and adhesion factors(VCAM-1,E-selectin)are up-regulated,becoming suitable for AdExos adhesion and infiltration in circulation.Besides,AdExos carries a wealth of miRNAs that promote angiogenesis,such as miR 221,miR 27b,miR 21,and miR 17-92.As an endocrine organ,adipose tissue is the link between multiple organs.Hedgehog protein has three homologous forms in mammals:Sonic hedgehog(Shh),Indian Hedgehog(Ihh),and Desert hedgehog(Dhh),among which Shh is the most widely expressed and has the highest activity.Hedgehog signaling promotes aortic formation through VEGF during zebrafish embryonic development.In Shh-deficient mice,the developing lung cannot normalize vascularization.Hedgehog signaling regulated angiogenesis by controlling the expression of many proangiogenic factors,including VEGF-a,VEGF-b,VEGF-c,and AngII.Hedgehog promotes angiogenesis by binding to a 12-fold transmembrane receptor protein-Patched.Under normal circumstances,Patched receptors bind to another 7-fold transmembrane receptor protein-Smoothened(Smo)and inhibit the effect of Smo.When the Hedgehog protein binds to the Patched receptor,it releases its inhibitory impact on Smo and initiates signal transduction.Smo is the information converter of the Hedgehog signaling pathway.It transmits Hedgehog signals from outside into the cell and activates the Gli transcription factor.Above all,we propose a hypothesis:in diabetic conditions,the amount of AdExos from the dysfunctional adipose tissue released into circulation increases.AdExos binds to the Patched receptor on endothelial cells through the Hedgehog protein on it,promoting the formation of pathological angiogenesis,accelerating plaque progression,and the construction of vulnerable plaques.Objectives1.To analyze the proangiogenic properties of AdExos at the cellular level.2.To verify whether AdExos promotes angiogenesis by activating the Hedgehog signaling pathway.Methods1.Culture,induction,and differentiation of 3T3-L1 preadipocytes and construction of insulin resistance modelFirstly,we induced 3T3-L1 preadipocytes to mature adipocytes with the combination of insulin,3-isobutyl-1-methylxanthine,and dexamethasone.Then,we established an insulin resistance model of adipocytes by incubated with high glucose and insulin levels-we cultured cells with exosomes-free serum and collected cells supernatant(control group and insulin resistance group).2.3T3-L1 adipocytes were transfected with Shh adenovirusBased on insulin resistance,we transfected 3T3-L1 adipocytes with Shh shRNA and overexpression adenovirus.After culturing with exosomes-free serum,we collected cell supernatant.We got the insulin resistance,Shh shRNA insulin resistance,Shh overexpression insulin resistance,and vector insulin resistance groups of adipocytes supernatant.3.Isolation and extraction of AdExosExosomes in the supernatant of adipocytes were isolated and extracted by overspeed differential centrifugation.We got AdExos from the control group(Ctrl-AdExos),insulin resistance group(IR-AdExos),Shh interference insulin resistance group(IR-AdExosshh-),Shh overexpression insulin resistance group(IR-AdExosshh+),and vector insulin resistance group(IR-AdExosvector).4.Isolation and extraction of mouse aortic endothelial cells.Verification of the uptake of AdExos by endothelial cells.To study the effect of AdExos on EndMT of endothelial cells,we carried out cell experiments.Firstly,we isolated and extracted mouse aortic endothelial cells via the enzymatic digestion method.Then endothelial cells were incubated with PKH26-labeled AdExos.The uptake of AdExos by endothelial cells was observed under a microscope.5.AdExos stimulated mouse aortic endothelial cells to detect changes in endothelial cell functionThe obtained AdExos from different sources(Ctrl-AdExos,IR-AdExos,IR-AdExosshh-,IR-AdExosshh+,and IR-AdExosvector)and recombinant proteins of VEGF and SHH were incubated with mouse aortic endothelial cells.Immunofluorescence staining of PCNA and KI67 was used to evaluate endothelial cells' proliferation function.The cell scratch assay was used to detect the endothelial cells' migration function.6.Tube formation experiment to detect the proangiogenic properties of AdExosThe obtained AdExos from different sources(Ctrl-AdExos,IR-AdExos,IR-AdExosshh-,IR-AdExosshh+,and IR-AdExosvector)and recombinant proteins of VEGF and SHH were incubated with mouse aortic endothelial cells.Tube formation assay was used to evaluate the angiogenesis ability of endothelial cells.The number of the junction and the branch length were observed.7.The role of the Hedgehog signaling pathway in angiogenesisAs for the molecular mechanism,cyclopamine,a Hedgehog protein blocker,was given to block the Hedgehog signaling pathway.The tube formation assay detected the Tube formation function of endothelial cells.Endothelial cells were incubated by Hedgehog protein blocker-cyclopamine and Gli inhibitor-Gant61.The expression of signaling pathway molecules was detected by western blot.Results1.The primary mouse aortic endothelial cells were extracted successfully,and AdExos uptake in vitroWe successfully extracted mouse aortic endothelial cells by enzymatic digestion.The immunofluorescence staining proved that the extracted endothelial cells were positive of endothelial cell marker CD31,which proved that the extraction of mouse aortic endothelial cells was successful.PKH26-labeled AdExos were co-incubated with mouse aortic endothelial cells,and red fluorescence was observed in the cytoplasm under the microscope,indicating that AdExos was absorbed by mouse aortic endothelial cells.2.IR-AdExos promoted the proliferation and migration function of mouse aortic endothelial cellsThe obtained AdExos from different sources(Ctrl-AdExos and IR-AdExos)and recombinant proteins of TGF-? and SHH were incubated with mouse aortic endothelial cells.PCNA and KI67 immunofluorescence staining results showed that:Compared with the control group,the expression of PCNA and KI67 in the Ctrl-AdExos group increased the expression of PCNA and KI67 in IR-AdExos increased significantly.VEGF was used as a positive control for promoting angiogenesis.The results showed that the expression of PCNA and KI67 of endothelial cells in the VEGF group increased significantly,indicating that IR-AdExos and VEGF can substantially enhance the cell proliferation function of endothelial cells.The cell scratch assay results showed that compared with the control group,the cell migration rate of the Ctrl-AdExos group increased.The cell migration rate of IR-AdExos increased significantly.The VEGF group's cell migration rate increased significantly,indicating that IR-AdExos and VEGF can dramatically enhance endothelial cells' migration function.4.AdExos promote Tube formation in mouse aortic endothelial cellsCtrl-AdExos,IR-AdExos,and recombinant proteins of TGF-? and SHH were incubated with mouse aortic endothelial cells.Tube formation assay results showed that compared with the control group,the junction and the branching length in the Ctrl-AdExos group increased.The number of the junction and the branching length in the IR-AdExos group increased.The junction and the branching length of endothelial cells in the VEGF positive control group increased,indicating that IR-AdExos and VEGF can significantly enhance endothelial cells' tube formation function.5.Shh intervention of IR-AdExos affects the proliferation and migration function of mouse aortic endothelial cellsThe obtained AdExos from different sources(IR-AdExos,IR-AdExosshh',IR-AdExosshh+,and IR-AdExosvector),recombinant proteins of TGF-? and SHH were incubated with mouse aortic endothelial cells.The results of PCNA and KI67 immunofluorescence staining showed that compared with the IR-AdExos group,the expression of PCNA and KI67 in endothelial cells in the IR-AdExosshh-group decreased.The expression of PCNA and KI67 in the endothelial cells of the IR-AdExosshh+group increased significantly,indicating that Shh is an essential molecule of IR-AdExos promoting endothelial proliferation.The cell scratch assay results showed that the cell migration rate in the IR-AdExosshh-group was reduced compared with the IR-AdExos group.The cell migration rate of endothelial cells in the IR-AdExossh+group increased significantly,indicating that Shh is an essential molecule for IR-AdExos to promote endothelial cell migration.6.Shh intervention of IR-AdExos affects the Tube formation function of mouse aortic endothelial cellsThe obtained AdExos from different sources(IR-AdExos,IR-AdExosshh-,IR-AdExosshh+,and IR-AdExosvector),recombinant proteins of TGF-? and SHH were incubated with mouse aortic endothelial cells.Tube formation assay results showed that compared with the IR-AdExos group,the junction and branching length of endothelial cells in the IR-AdExosshh-group were reduced.The junction and the branching length in the IR-AdExosshh+group increased significantly,indicating that Shh is an essential molecule for IR-AdExos to promote endothelial cell angiogenesis.7.The role of Hedgehog signaling pathway in AdExos promoting angiogenesisThe Hedgehog inhibitor-cyclopamine,IR-AdExos,IR-AdExosshh-,IR-AdExosshh+,IR-AdExosvector,and VEGF were added to mouse aortic endothelial cells and human umbilical vein endothelial cells.Tube formation assay results showed that the junction and the branching length in the IR-AdExos+cyclopamine group were significantly reduced,indicating that Hedgehog is the crucial molecule of AdExos promoting angiogenesis.8.The expression of AdExos' proangiogenic signaling pathwayHedgehog protein blocker-cyclopamine,Gli inhibitor-Gant61,and IR-AdExosshh+were added to mouse aortic endothelial cells and human umbilical vein endothelial cells.Western blot results showed that in the IR-AdExosshh+ group,the expression of Gli was significantly increased.In the cyclopamine and Gant61 group,the expression of Gli was reduced considerably,proving that AdExos promoted angiogenesis through the Patched/Gli signaling pathway.Conclusions1.IR-AdExos could enhance the proliferation,migration,and Tube formation of mouse aortic endothelial cells and promote angiogenesis.2.IR-AdExos promoted cell function of mouse aortic endothelial cells via Shh.3.IR-AdExos promoted angiogenesis through the Patched/Gli signaling pathway. |
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