| Introduction:Hypertension is the leading risk factor for cardiovascular disease,which represents the top cause of morbidity and mortality worldwide.Despite extensive recent studies discussing the multifactorial nature of hypertension,it is important to further explore its pathogenesis and search for new therapeutic targets.Among the numerous etiologies of hypertension,endothelial dysfunction is a common cause and is considered a precursor to the development of micro-and macro-vascular complications.The endothelium is a selective permeable barrier between the bloodstream and the outer vascular wall,but also functions as a critical homeostatic organ,fundamental for regulating vascular tone and blood pressure.AMP-activated protein kinase(AMPK)plays an important role in various physiological and pathological processes.AMPK downregulates the expression of caveolin-1 and increases endothelial nitric oxide synthase(eNOS)activity.However,the mechanisms involved in regulating AMPK expression remain to be explored.Epigenomics is one of the latest approaches used in hypertension research.Sp1 and Sp3 are members of the specificity protein/Kruppel-like factor(Sp/KLF)transcription factor family,which comprises proteins with three zinc fingers and recognizes G-rich promoter elements(GC-box and the related GT-box).Sp1 and Sp3 are ubiquitously expressed in mammalian cell types.Sp1-knockout mice did not survive beyond embryonic day 10.5 and showed a broad range of abnormalities.Sp3-knockout mice were growth retarded,which resulted in death prenatally or at birth due to complications including cardiac malformations.In vivo,deletion of the Sp genes revealed distinct functions of Sp1 and Sp3;however,they have also shown overlapping functions in the early developmental stages in the hematopoietic system.Considering the significance of Sp1/Sp3 in cardiovascular development,their role in endothelial cells and related vascular diseases require further investigation.The endothelium is increasingly becoming a surrogate endpoint of the therapeutic approach against cardiovascular risk factors,as demonstrated by its inclusion among markers of organ damage.Several cardiovascular drugs,including angiotensinconverting enzyme inhibitor(ACEI),ameliorate endothelial dysfunction by their supposed pleiotropic and ancillary properties.ACEIs are important therapeutic agents widely used for treating cardiovascular and renal diseases,with proven effects in patients worldwide.ACEIs inhibit AngⅡ release and bradykinin inactivation;however,the clinical benefits of these actions have not been completely elucidated.Apart from their effects on ACE,ACEIs have a direct effect on the bradykinin B1 receptor to elevate NO release from endothelial cells,although the detailed mechanism is unclear.Here,we aimed to elucidate whether captopril,a clinical ACEI,exerts its effects by targeting endothelial Sp1 and Sp3.Objective:1.To explore the role and mechanisms of endothelial Sp1/Sp3 in hypertension and endothelial dysfunciton.2.To explore the role of Sp1 and Sp3 transcription factors in the underlying mechanism of captopril.3.To investigate of the cellular pathway through which captopril modulates the activity of Sp1 and Sp3 transcription factors.Methods:1.AnimalsTo ablate Sp1 and Sp3 specifically in the endothelium(Sp1/Sp3ECKO),Sp1fl/fl and Sp3fl/fl mice were crossbred with VE-CAD-CreERT2 mice to obtain VE-CADCreERT2+/Sp1fl/fl/Sp3fl/fl mice(dKO),which were intraperitoneally injected with tamoxifen(50 mg/kg)for 5 consecutive days.Littermate VE-CAD-CreERT2/Sp1fl/fl/Sp3fl/fl mice were treated with the same dose of tamoxifen as controls(CTR).2.Animal experiments group(1)Male C57BL/6J mice aged 10-12 weeks were randomly divided into 2 groups with 5 mice in each group:AngⅡ group and Saline group.AngⅡ group was pumped with AngⅡ(1000ng/kg/min)for 28 days.Saline group was pumped with saline.(2)8-week-old male dKO mice and littered-control CTR mice were selected and divided into 2 groups with 12 mice each:CTR group and dKO group.(3)8-week-old male C57BL/6J mice were randomly divided into 2 groups with 10 mice in each group:MITA group and vehicle group.MITA group was injected with mithramycin(0.4 mg/kg)twice a week for 3 weeks,and vehicle group was injected with vehicle twice a week for 3 weeks.(4)8-week-old male dKO mice and littered-control CTR mice were injected with 100μL(4×1010 viral particles)control adenovirus(Ad-LacZ)or continuously activated AMPK virus(CA-AMPK)through the tail vein forming 4 groups with 5 mice each:CTR+Ad-LacZ group,CTR+CA-AMPK group,dKO+Ad-LacZ group,dKO+CA-AMPK group.(5)8-week-old male dKO mice and littered-control CTR mice were intraperitoneally injected with captopril(10 mg/kg)or vehicle every day for 4 weeks forming 4 groups with 5 mice each:CTR+vehicle group,CTR+captopril group,dKO+vehicle group,dKO+captopril group.(6)8-week-old male dKO mice and littered-control CTR mice were injected with AngⅡ(1000ng/kg/min)or saline continuously by embedding pump.Some mice were given captopril(10 mg/kg)at the same time forming 4 groups with 5 mice each:Saline+CTR group,Saline+dKO group,AngⅡ+CTR group,AngII+dKO group,AngⅡ+CTR+captopril group.3.Ang Ⅱ-induced hypertension in miceMale mice were used in AngⅡ-induced hypertension model by subcutaneous infusion of Ang Ⅱ at a dose of 1000 ng/kg/min or saline using osmotic minipumps for 28 days.4.Blood pressure measurementsSystolic,diastolic,and mean blood pressure of conscious mice was recorded indirectly and non-invasively by using a tail-cuff system.Mice were acclimated to the system for 7 consecutive days before blood pressure measurement.Data were calculated as the mean blood pressure of 3 sequential days,and blood pressure was measured at least 3 times in each animal.5.Measurement of vascular reactivity of mesenteric arteriesThe second-order mesenteric arteries were isolated from adult male mice,mounted on an Automated Multi Wire Myograph System(#630MA,DMT,Aarhus,DK).Endothelium dependent relaxation was induced by Ach in NE pre-contracted segments with or without L-NAME.Cumulative concentration response curves in response to SNP were used to assess the endothelial-independent relaxation of vessels.6.EchocardiographyEchocardiography was performed with a Micro-Echocardiography system.2D images and M-mode tracings were recorded from the short-axis view at the high posterior wall thickness,ejection fraction and fractional shortening.7.Mouse lung endothelial cells(MLECs)isolationMLECs were isolated from adult mice.Briefly,lungs were harvested,shredded,and digested with 0.1%collagenase in PBS.MLECs were isolated by immunoselection with CD31-conjugated magnetic beads and ICAM-2-conjugated magnetic beads.8.Mouse aortic endothelial cells(MAECs)isolationThe aortas were opened longitudinally and cut into small pieces about 1-2mm2 and plated with the intima side down in a fibronectin-coated culture dish.9.NO detection(Nitrite/Nitrate detection)NO production was determined by detecting the nitrite level using Griess reagent.The first step converted nitrate to nitrite by using nitrate reductase.The second step used Griess Reagent to convert nitrite to a deep purple azo compound.The production of NO in both cell ly sate and serum was measured at an absorbance of 540 nm.10.eNOS activity detectioneNOS activity of endothelial cells was measured by using the Nitric Oxide Synthase Activity Assay Kit.We measured the fluorescence of samples and standard curve on a microplate reader at Ex/Em=360/450 nm.Protein concentration was determined by the BCA method.eNOS activity was expressed as pmol/mg protein/min.11.Histological analysisArtery samples were fixed in 4%paraformaldehyde solution for paraffin embedding.Frozen tissues were embedded in the O.C.T.compound.Sections were blocked in 5%goat serum and then incubated with primary antibody at 4℃overnight.After washing with phosphate buffered saline,samples were stained with DAB or incubated with Alexa Fluor-labeled secondary antibody to measure the levels of Sp1,Sp3,AMPKα,caveolin-1.12.Western BlotCells were lysed in RIPA lysis buffer.Proteins were resolved in 10%SDS-PAGE gels and transferred to PVDF membrane.Membranes were blocked for 2 hours at room temperature in 5%fat-free milk and incubated with primary antibodies at 4℃ overnight.After TBST washing,membranes were incubated with secondary antibody for 1 hour at room temperature.After TBST washing,bands were scanned and analyzed by using Amershanm Imager 680 detecting the levels of Sp1,Sp3,eNOS,p-eNOS(Ser1177),AMPKα1,AMPKα2,p-AMPKα(Thr172),caveolin-1,P16,P21,ULK1,p-ULK1(Ser555),P62,LC3Ⅰ/Ⅱ,caspase3,cleaved-caspase3.13.Co-immunoprecipitation(Co-IP)To preclear the sample,the samples were incubated with of Protein A&G magnetic beads for 1 hour at 4 ℃ with constant rotation.Simultaneously,antibody was incubated with magnetic beads at 4℃ overnight with constant rotation.The magnetic beads were then washed 4 times with IP buffer,and immunoprecipitated proteins were eluted from magnetic beads by 1x loading buffer.The immunoprecipitated proteins were subject to Western blot analysis.14.Chromatin immunoprecipitation(ChIP)assayChIP assays were performed using the SimpleChIP Enzymatic Chromatin IP kit according to the manufacturer’s protocol.Cells were incubated with 1%fresh paraformaldehyde to crosslink the histone/transcription factor complexes with DNA.The nuclei pellets were digested with Micrococcal Nuclease followed by sonication.The chromatin was immunoprecipitated with antibodies against Sp1,Sp3,HDAC1,HDAC2,HDAC6,p300 and CBP.The protein/DNA complexes were immunoprecipitated by ChIP grade protein G magnetic beads with rotation for 2h at 4℃.The DNA was purified and then amplified by quantitative real-time PCR with the following primers targeted to the AMPKα1 or AMPKα2 promoter.15.Luciferase reporter assayLuciferase reporter transfection and dual luciferase assay were performed using the Promega Dual-Luciferase(?) Reporter Assay System.In brief,HUVECs or 293T cells were seeded in 24-well plates and transfected by using Lipofectamine 2000 with report vector inserted with indicated target sequences;empty vector was the control.PRL-TK vector was co-transfected as an internal control.16.RT-PCRTotal RNA was extracted from cells by using Trizol reagent and reverse transcribed.RT-PCR was performed with the SYBR qPCR Mix and a CFX-96 system.The relative expression of AMPKα1 or AMPKα2 in various groups was calculated with 2-ΔΔCt methodology.17.Flow cytometryCell apoptosis after AngⅡ stimuli was determined by using the FITC Annexin ⅤApoptosis Detection Kit.Flow cytometry was performed by using a FACSCalibur flow cytometer and analyzed by FlowJo.18.Human mesenteric artery samplesHuman mesenteric artery samples were obtained from the Department of Bariatric and Metabolic Surgery,General Surgery,Qilu Hospital,Shandong University.All procedures that involved human samples were approved by the Ethics Committee of Qilu Hospital of Shandong University.For immunofluorescence analysis,Sp1 and Sp3 were detected in endothelium.19.StatisticsData are expressed as means ± SEM,and results were analyzed by using GraphPad Prism 8.0(GraphPad Software,San Diego,CA).Normality assumption of the data distribution was assessed with the Kolmogorov-Smirnov test.For normally distribution,Student t test was used to compare 2 groups.Differences between multiple groups with one variable were determined by one-way ANOVA followed by Bonferroni’s post-hoc test.To compare multiple groups with more than one variable,two-way ANOVA followed by Bonferroni’s post-hoc test was used.Statistical significance was set at P<0.05.Results:1.Sp1 and Sp3 are reduced in the arteries of hypertensive patients and angiotensin Ⅱ(Ang Ⅱ-induced hypertensive miceIn the endothelium of mesenteric arteries from patients with hypertension and arterites from AngⅡ-induced hypertensive mice,Sp1/Sp3 were notably downregulated.AngⅡ,ET-1 and H2O2 markedly decreased Sp1 and Sp3 expression.2.Sp1/Sp3 depletion in EC aggravates hypertension and endothelial dysfunction in miceWe constructed endothelial specific Sp1/Sp3 knockout mice(dKO).Sp1/Sp3 deficiency in endothelium resulted in elevated blood pressure,impaired vasodilation,and cardiacremodeling.3.Sp1 and Sp3 positively regulate AMPKα1 and AMPKα2 transcriptionSp1 and Sp3 upregulated AMPKα1/AMPKα2 and Sp1/Sp3 could directly bind to the AMPKα1/AMPKα2 promoters and promote their transcription.4.Sp1 and Sp3 improve endothelial function via AMPKαSp1/Sp3 could influence endothelial cell senescence,autophagy and apoptosis via AMPKα.The inhibition of Sp1/Sp3 by MITA led to hypertension and impaired endothelial function,which was consistent with the findings in dKO mice.5.Administration of CA-AMPK decreases blood pressure and alleviates endothelial dysfunction in Sp1/Sp3ECKO miceThe SBP and MBP of CTR and dKO mice decreased treated with CA-AMPK.Achinduced endothelial relaxation responses in CTR and dKO mice were ameliorated following CA-AMPK administration.AMPKα plays an important role in the Sp1/Sp3-mediated regulation of endothelial function and blood pressure.6.Endothelial Sp1 and Sp3 are implicated in the effects of captoprilCaptopril lowered blood pressure,improved endothelium-dependent relaxation,increased the serum nitrite/nitrate level by enhancing eNOS activity in CTR mice,but not in dKO mice.7.Captopril activates Sp1 and Sp3 via bradykinin B1 receptorCaptopril increased Sp1/Sp3 protein level and binding with the AMPKα1/AMPKα2 promoters,which was inhibited by B1 receptor antagonist treatment.8.HDACl-mediated Sp1/Sp3 deacetylation is responsible for captopril-induced AMPKα1/AMPKα2 transcription activationAngⅡ/ET-1/H2O2 promoted Sp1/Sp3 degradation via the ubiquitin-proteasome pathway.Inhibition of HDAC1 reversed the increased protein levels of Sp1/Sp3 induced by captopril and greatly increased the ubiquitination of Sp1/Sp3.Conclusions:1.Endothelial specific Sp1/Sp3 knockout contributes to endothelial dysfunction and hypertension.2.Sp1/Sp3 positively regulate AMPKα1/AMPKα2 transcription.3.Endothelial Sp1/Sp3 are implicated in the effects of captopril4.Captopril regulates the acetylation and ubiquitination of Sp1/Sp3.Introduction:The vascular system is a dynamic network of blood vessels that constantly provides the growing organs in an embryo and the healing wounds with gases,liquids,nutrients,signaling molecules,and cells throughout the vertebrate body.Endothelial cells(ECs)that line the inner surface of the blood vessels undergo a series of coordinated events,including cell activation,proliferation,migration,differentiation,branching,anastomosis,lumen formation,and remodeling into arteries and veins from preexisting ones,which is termed angiogenesis.Insufficient angiogenesis contributes to ischemic disorders such as ischemic heart disease or critical limb ischemia;however,excessive and imbalanced angiogenesis leads to malignant,inflammatory,and retinal diseases.Elucidating the molecular mechanisms of angiogenesis is critical for developing effective therapeutics aimed at vascular disorders.Notch family,which is robustly expressed in vascular system,consists of four receptors(Notch1-4),three delta-like ligands(DLL1,3,and 4),and two jagged-like ligands.During angiogenesis,DLL4 binding induces the proteolytic cleavage of Notchl to release the Notch intracellular domain(NICD),which translocates into the nucleus to combine with its co-activators,thereby regulating the transcription of several genes related to angiogenesis,such as HEY1,HES1,and DLL4.NOTCH 1-deficient mice died before embryonic day 10.5(E10.5)because of a failure in cardiovascular development.Another crucial pathway for angiogenesis is vascular endothelial growth factor(VEGF)signaling,which is repressed by Notch signaling.Although the role of Notch signaling in angiogenesis is clear,the regulation of Notch family members is not fully understood.Sp1 and Sp3 are highly related zinc-finger proteins that belong to the family of specificity protein(Sp)/Krüppel-like factors.Sp1/Sp3 play crucial roles in eukaryotic transcriptional machinery by binding to the GC-rich promoters of target genes.Sp1 down-regulated the expression of Notch signaling via lncRNA ZFAS1.Notch ligand Ser is a target of Sp1,and mediates in part the growth-promoting function of Sp1.Thus far,the role of endothelial Sp1/Sp3 in angiogenesis in vivo has not been explored.Angiotensin-converting enzyme(ACE)is a dipeptidyl carboxypeptidase that catalyzes the conversion of inactive angiotensin Ⅰ to angiotensin Ⅱ and inactivates bradykinin(BK).ACE inhibitors(ACEIs)are widely used to treat cardiovascular diseases.In addition,ACE inhibition increases the capillary density in stroke-prone spontaneously hypertensive rats and promotes angiogenesis in ischemic rabbit hindlimbs.The proangiogenic effect of ACEI in vivo was abolished by blockage or deletion of the BK B2 receptor.However,the downstream signaling of ACEI in angiogenesis in the ECs is unknown.Angiotensin(Ang)Ⅱ activates Notch signaling via Ang Ⅱ type 1 receptor(AT1).Renin-angiotensin system(RAS)seems to be closely related to Notch signaling.Additionally,previous work has reported that ACEI improved VEGF level in experimental diabetes or in in a mouse model of retinopathy.However,there are findings suggesting that ACEI has a potential to inhibit tumor growth due to suppression of VEGF-induced angiogenesis,demonstrating that the function of ACEI in this setting remains unclear and requires further investigation.Therefore,given the recent findings,we sought to investigate whether ACEI regulates angiogenesis via Notch and VEGF signaling.Objective:1.To explore the role and mechanisms of endothelial Sp1/Sp3 in angiogenesis.2.To explore the role of Sp1/Sp3 transcription factors in the underlying mechanism of ACEI.3.To explore the processes by which ACEI modifiy Sp1/Sp3.Methods:1.AnimalsTo ablate Sp1 and Sp3 specifically in the endothelium(Sp1/Sp3ECKO),Sp1fl/fl and Sp3fl/fl mice were crossbred with VE-CAD-CreERT2 mice to obtain VE-CADCreERT2+/Sp1fl/fl/Sp3fl/fl mice(dKO),which were intraperitoneally injected with tamoxifen(50 mg/kg)for 5 consecutive days or tamoxifen(1 mg/mL,50 μL)by intraperitoneal injection from P2 to P4.Littermate VE-CAD-CreERT2/Sp1fl/fl/Sp3fl/fl mice were treated with the same dose of tamoxifen as controls(CTR).In the same way,we obtained VE-CAD-CreERT2+/Sp1fl/fl(Sp1ECKO)and VE-CAD-CreERT2+/Sp3fl/fl(Sp3ECKO).2.Animal experiments group(1)8-week-old or P5 male dKO mice and littermates of control CTR mice were selected and divided into 2 groups with 5 mice each:CTR group and dKO group.(2)8-week-old male dKO mice and littered-control CTR mice were injected intraperitoneally with perindopril(3 mg/kg/d)every day;dKO and CTR mice were selected,and 10μL perindopril(1 mg/mL)was injected intraperitoneally from P2 to P4 to forming 4 groups with 5 mice each:CTR+vehicle group,CTR+ACEI group,dKO+vehicle group,dKO+ACEI group.(3)8-week-old male dKO mice and littered-control CTR mice were injected intraperitoneally with perindopril(3 mg/kg/d),P22077(15 mg/kg/d)or P5091(15 mg/kg/d)every day;dKO mice and CTR mice were injected intraperitoneally from P2 to P4 with 10μL perindopril(1 mg/mL),10μL 22077(5 mg/mL)or 10μL P5091(5 mg/mL)forming 4 groups with 5 mice each:vehicle group,vehicle+ACEI group,P22077+ACEI group,P5091+ACEI group.(4)8-week-old male dKO mice and litter control CTR mice were selected and injected intraperitoneally with DAPT(60 mg/kg/d)every day;dKO mice and litter control CTR mice were injected 10μL DAPT(20 mg/mL)intraperitoneally from P2 to P4 to forming 4 groups with 5 mice each:CTR+vehicle group,CTR+DAPT group,dKO+vehicle group,dKO+DAPT group.3.Mouse lung endothelial cells(MLECs)isolationMLECs were isolated from adult mice.Briefly,lungs were harvested,shredded,and digested with 0.1%collagenase in PBS.MLECs were isolated by immunoselection with CD31-conjugated magnetic beads and ICAM-2-conjugated magnetic beads.4.Mouse retinal endothelial cells(MLECs)isolationEyes were removed from euthanized mice,and retinas were collected in cold phosphate buffered saline(PBS).Retinas were then digested with collagenase typeⅡ and separated into single cells.Fluorescence-Activated Cell Sorting(FACS)was used to isolate CD31+ cells and exclude CD45+cells for further study.5.Histological analysisSamples were fixed in 4%paraformaldehyde solution for paraffin embedding.Frozen tissues were embedded in the O.C.T.compound.Sections were blocked in 5%goat serum and then incubated with primary antibody at 4℃ overnight.Samples were stained with DAB or incubated with Alexa Fluor-labeled secondary antibody to measure the levels of Sp1、Sp3、CD31、HIF-1α、NOTCH1、VEGFR2.Cells were,fixed using 4%PFA,permeabilized with 0.1%Triton X-100 in PBS,blocked with 10%goat serum,and incubated with the corresponding antibodies.After PBS wash,cells were incubated with Alexa fluor-conjugated secondary antibody to measure the level of USP7.6.Western BlotCells were lysed in RIPA lysis buffer.Proteins were resolved in 10%SDS-PAGE gels and transferred to PVDF membrane.Membranes were blocked for 2 hours at room temperature in 5%fat-free milk and incubated with primary antibodies at 4℃ overnight.After TBST washing,membranes were incubated with secondary antibody for 1 hour at room temperature.After TBST washing,bands were scanned and analyzed by using Amershanm Imager 680 detecting the levels of Sp1、Sp3、DLL4、NOTCH1、NICD、VEGFR2、p-VEGFR2(Tyr1175)、p-VEGFR2(Tyr1059)、p-ERK1/2(Thr202/Tyr204)、ERK1/2、p-PLCyl(Tyr783)、PLCγ1、USP7、Ubiquitin.7.Co-immunoprecipitation(Co-IP)To preclear the sample,the samples were incubated with of Protein A&G magnetic beads for 1 hour at 4 ℃ with constant rotation.Simultaneously,antibody was incubated with magnetic beads at 4℃ overnight with constant rotation.The magnetic beads were then washed 4 times with IP buffer,and immunoprecipitated proteins were eluted from magnetic beads by 1x loading buffer.The immunoprecipitated proteins were subject to Western blot analysis.8.Chromatin immunoprecipitation(ChIP)assayChIP assays were performed using the SimpleChIP Enzymatic Chromatin IP kit according to the manufacturer’s protocol.Cells were incubated with 1%fresh paraformaldehyde to crosslink the histone/transcription factor complexes with DNA.The nuclei pellets were digested with Micrococcal Nuclease followed by sonication.The chromatin was immunoprecipitated with antibodies against Sp1 and Sp3.The protein/DNA complexes were immunoprecipitated by ChIP grade protein G magnetic beads with rotation for 2h at 4℃.The DNA was purified and then amplified by quantitative real-time PCR with the following primers targeted to the NOTCH1 promoter.9.Luciferase reporter assayLuciferase reporter transfection and dual luciferase assay were performed using the Promega Dual-Luciferase(?) Reporter Assay System.In brief,HUVECs or 293T cells were seeded in 24-well plates and transfected by using Lipofectamine 2000 with report vector inserted with indicated target sequences;empty vector was the control.PRL-TK vector was co-transfected as an internal control.10.RT-PCRTotal RNA was extracted from cells by using Trizol reagent and reverse transcribed.RT-PCR was performed with the SYBR qPCR Mix and a CFX-96 system.The relative expression of HES1、HEY1、DLL4、CD31、VE-cadherin in various groups was calculated with 2-ΔΔCt methodology.11.Mouse retinal angiogenesis assayEyes from postnatal day 5 pups were fixed in 4%paraformaldehyde.Retinas were dissected and permeabilized in PBS containing 1%Triton X-100.Then the permeabilized retinas were incubated in 5%BSA.Retinas were stained with isolectin B4-Alexa Fluor 594.Whole mounts were photographed using a fluorescence microscope.12.Hindlimb ischemia modelIn brief,a portion of the femoral artery was exposed and ligated both proximally and distally using 6-0 silk sutures,and the vessels between the ligation sites were excised.We measured ischemic(left)and non-ischemic(right)limb blood flow ratios using a noninvasive laser Doppler imaging system at baseline(day-1)and immediately after HLI(day 0),with subsequent imaging on days 7,14,21,and 28 post-HLI.13.In vivo matrigel plug assayA mixture containing 500 μL Matrigel,50 ng/mL VEGF,and 30 U/mL heparin was subcutaneously injected into the dorsal surface of the mice,leading to the formation of a solid plug.Mice were sacrificed after 14 days and the matrigel plugs were retrieved for analysis.14.Mouse skin wound healing modelMice were anesthetized with 1.5%to 2%isoflurane in oxygen,and their back skin was wounded using a 5 mm biopsy punch without injuring the underlying muscle.Wounds were digitally photographed at days 0,3,5,and 7,and the wound diameters were measured using ImageJ to determine the wound closure rate.15.Mouse aortic sprouting assayThe thoracic aortas were isolated,trimmed of all extraneous tissues,and flushed with PBS via the lumen to remove all the blood.Aortic rings(1-mm long)were embedded in matrigel and cultured in Opti-MEM supplemented with 10%FBS and VEGF(20 ng/mL)in a humidified incubator at 37℃,5%CO2 for approximately 6 days.16.Tumor transplantation model Lewis lung carcinoma(LLC)cells(4 × 105 cells),B16 tumor cells(1 × 106 cells),and MC-38 cells(8 × 105 cells)were suspended in 200 μL of Hank’ s balanced salt solution and injected subcutaneously into the murine flank.Tumor size was measured with digital calipers on days 8,10,12,14,and 16.17.In vitro tube formation assayCells were serum-starved overnight in a medium containing 0.5%FBS.Matrigel(250 μL/well)was added to a 24-well plate,and 1 × 105 cells were plated on the Matrigel.After 4 h,tubes were visualized and images were captured using a phasecontrast inverted microscope.18.Human gastrocnemius samplesHuman tissue biopsies from gastrocnemius were obtained from 10 patients(7 male and 3 female)in Qilu Hospital of Shandong University.Five patients with CLI and 5 patients without CLI were included in this study.All patients gave written informed consent to participate in the study.All procedures involving human samples were approved by the Ethical Committee of Qilu Hospital of Shandong University.All relevant ethical regulations were followed in this study.19.StatisticsData are expressed as means± SEM,and results were analyzed by using GraphPad Prism 8.0(GraphPad Software,San Diego,CA).Normality assumption of the data distribution was assessed with the Kolmogorov-Smirnov test.For normally distribution,Student t test was used to compare 2 groups.Differences between multiple groups with one variable were determined by one-way ANOVA followed by Bonferroni’s post-hoc test.To compare multiple groups with more than one variable,two-way ANOVA followed by Bonferroni’s post-hoc test was used.Statistical significance was set at P<0.05.Results:1.Endothelial Sp1/Sp3 is required for angiogenesis in vivoSp1/Sp3 were reduced in the the blood vessels in gastrocnemius samples obtained from patients with CLI.dKO mice displayed a significant reduced angiogenesis in retina,HLI,wound closure and tumor models.2.ACEI increases angiogenesis via Sp1/Sp3 in ECsACEI treatment promoted angiogenesis in retina,HLI,matrigel plug,aortic ring and tube formation assays in CTR mice,but not in dKO mice.3.ACEI stabilizes Sp1/Sp3 protein and promotes angiogenesis via USP7USP7 knockdown significantly impaired Sp1/Sp3 stabilization.Two selective USP7 inhibitors,P22077 and P5091 decreased angiogenesis in retina,HLI,matrigel plug,aortic ring and tube formation assays in ACE treated mice.4.USP7 interacts with Sp1/Sp3 protein and decreases Sp1/Sp3 ubiquitinationUSP7 stabilizes Sp1/Sp3 by removing the K48-linked poly-ubiquitin.USP7 plays a critical role in ACEI-mediated upregulation in the transcriptional function of Sp1/Sp3 by increasing the accumulation of them.5.HDAC1 inhibits Sp1/Sp3 ubiquitination by deacetylating Sp1-lys703/Sp3lys551HDAC1-mediated deacetylation of Sp1/Sp3 in the presence of ACEI leads to their increased association with USP7.6.ACEI increases nuclear localization of USP7 independent of HDAC1 activityACEI induced the USP7 translocation into the nucleus independent of HD AC 1 activity.CK2 inhibition attenuated the increased protein level of Sp1/Sp3 induced by ACEI and blocked USP7 translocating into the nucleus.7.Endothelial Sp1/Sp3 promotes angiogenesis via Notchl-VEGFR2 signalingSp1/Sp3 deletion in ECs led to a significant increase in the levels of Notch 1,NICD,and its target gene.DAPT treatment significantly increased angiogenesis in retina,HLI assays in both CTR and dKO mice.ACEI affects the activation of VEGFR2 via inhibiting Notchl signaling rather than directly activating VEGFR2.8.Sp3 enhances Sp1-mediated transcriptional repression activity of NOTCH1 in ECsWe observed that both Sp1 and Sp3 could bind to the NOTCH1 promoter and inhibited its transcription.Sp3-mediated inactivation of Notchl signaling depends on the presence of Sp 1.Conclusions:1.Endothelial specific Sp1/Sp3 knockout leads to impaired angiogenesis.2.Sp1/Sp3 facilitate angiogenesis through suppression of NOTCH1 transcription.3.Endothelial Sp1 and Sp3 are involved in the beneficial effects of ACEI promoting angiogenesis.4.ACEI stumulate Sp1/Sp3 through activating USP7. |
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