| Background:Pulmonary arterial hypertension(PH)is a chronic disease that ultimately progresses to right-sided heart failure and death,defined clinically as mean pulmonary arterial pressure>25mmHg at rest with normal left atrial pressure,which is characterized by small pulmonary dysfunction and structural remodellingandright ventricular(RV)failure.Abnormal pulmonary vascular cell proliferation plays a central role in the occurrence and development of PH.Currently,there were a lot of drugs used to improve the clinical symptoms of PH patients;however,these treatments cannot reverse the pulmonary vascular remodelling process and prevent PH development.Therefore,novelapproaches are urgeat the proliferation and angiogenesis of PAEC are underlying chanismntly needed.The pathogenesis of PH is complicated,and the damage of pulmonary artery endothelial cell(PAEC)is a crucial early onset.Reports showed ths for the pathomelogical features of PH.Thus,revealing the potential molecular and cellular mechanism of endothelial cell angiogenesis underlies PH is very important and can help to explore new treatment strategies.Wnt5a is one of a member of the Wingless(Wnt)and activates non-canonical or canonical Wnt signalling pathways through specific coupling of different receptor.Down-regulation of Wnt5a could promote hypoxia-induced pulmonary vascular smooth muscle cell(PASMC)proliferation,and loss of Wnt5a could reduce the formation of new vessels in PH.1 Thus,the production of Wnt5a is likely to become a new way to prevent the vessel loss in PH.Cells isolated from Wharton's jelly,referred to as umbilical cord matrix stromal(UCMS)cells,express mesenchymal stromal cell(MSC)surface markers,self-renew and are multipotent(differentiate into bone,fat,cartilage,etc.)in vitro.Human UCMSC lacks of an apparent host immune response,8 human UCMSderived exosomes(MSC-EXO)isoneofthemaintherapeuticvesicles,workfromourgroup confirmed that intravenous delivery of MSC-EXO could inhibit experimental PH vascular remodelling and right ventricular impairments.MSC-EXO is a promising potential target for new therapies of PH,the mechanism was not very clear.Aim:The aim of the study was to explore the mechanism of MSC-EXO to protect against experimentally induced PH in vivo and vitro.Methods:1.Isolation of MSCs from human umbilical cord Wharton's Jelly with as previously described with some modifications.15 The immunotyping characterization of hUCMSCs occurred at passage 3-4 using humanspecific antibodies CD34,CD45,CD73,CD90,CD105 and HLA-DR(BD Biosciences Pharmingen,San Diego,CA)by a fluorescence-activated cell sorter(FACS,BD FACSAria ?).Cell differentiation ability was performed bydifferentiationmedia and supplements(Cyagen US Inc).osteogenesis,adipogenesis and chondrogenesis-inducing differentiation medium.2.Collection and identification of EXOsomes derived from mesenchymal stem cells:When reached 90%confluences,the adherent cells were incubated in medium with 5%exosome-depleted FBS(ExoDP-FBS,SBIEXO-FBS-50A-1)for 24hours,and 5-8 passage hUCMSC were used for experiments.The conditioned medium was centrifuged at 4? at 300g for 10minutes at 2000g for 10minutes and finally at 10000gfor 30minutes to remove the cells and debris,followed by centrifugation of the supernatant at 100000g at 4 ° C for lhour.MSC-EXO were resuspended in PBS and filtered with a 0.22?m microfiltration membrane,centrifuged again in PBS at 100000g for 1hour to collect the exosomes.The protein concentration of hUCMSC-EXO was determined using a bicinchoninic acid(BCA)assay kit.Transmission electron microscope(TEM)was used to detect the morphology of MSC-EXO according to the manufacturer's instructions.Briefly,the prepared exosomes were stained with phosphotungstic acid solution and then performed under a Hitachi-9000 TEM system.The exosome markers CD63,CD81,TSG101 and ALIX were analysed by Western blot.?-actin expression was used as an internal control.3.We established rat PH model through a single intraperitoneal injection of MCT(50mg/kg;Sigma,St.Louis,MO,USA).25?g of MSCEXO in 200?L PBS via tail vein injection form 21 to 23days.15 Four weeks later,there are 3 rats in MCT group and 2 rats in MSC-EXO group deaths.The surviving experimental animals were divided into 3 groups(n=10,7,and 8):Control(saline-treated)group,MCT group,and MSC-EXO group.Four weeks later,the rats were anesthetized with pentobarbital(30mg/kg,ip,Sigma-Aldrich)and inserted with a 3F-Miller micro-tip catheter via the right jugular vein into the right ventricle(RV)to obtain theheartrate(HR),cardiacoutput(OC)and right ventricular systolic pressure(RVSP).Post-operation,the heart was obtained quickly,and the weight ratio of the right ventricular(RV)to left ventricle(LV)plus the septum(LV+S)and the right ventricular weight was calculated to quantify the right ventricular hypertrophy.The lung tissues were fixed and embedded in paraffin,and the serially sections at a thickness of 4-5 ?m were stained with haematoxylin-eosin stain.The vascular wall thickness(WT),vascular external diameter(ED),vascular wall area(WA)and total vascular area(TA)to calculate WT%(WT/ED)and WA%(WA/TA)were measured as previously study.On the other hand,the serially sections were stained with Masson's trichrome to measure the evaluation of the degree of fibrosis.The average of the 10 highpower fields(hpf)was randomly selected,and positively stained areas were padded with a single colour and converted into pixels through optical density(OD)calibration.Immunohistochemistry was used to analyse the expression of CD31;on the other hand,we detected the expression a-smooth muscle actin(SMA)in lung section.Briefly,after blocking with 5%bovine serum albumin for 30minutes at room temperature,the lung sections were incubated overnight at 4? with anti-CD31(AF3628),?-SMA(ab21027)and V-E cadherin(CST#14472).Then,sections were further incubated with a second antibody for 2hours at room temperature.Subsequently,the 3,3'-diaminobenzidine(DAB)dye was added to visualize the antibodies.For immunofluorescence,the sections were followed by 1-h incubation in the dark with florescence isothiocyanate-conjugated secondary antibody.Images were taken with ZEISS LSM800 confocal microscope(Tokyo,Japan).All experiments were performed by two examiners blinded to treatment assignment.In vitro test:(1)culture and identification of cultured pulmonary artery smooth muscle cells(PASMCS):endothelial progenitor cells smooth muscle growth supplement,100mg/mL penicillin and 100 international units/mL streptomycin were cultured in M200 complete medium supplemented with low serum growth supplement or smooth muscle growth medium supplemented with the following substances.The culture medium was changed every 2-3 days,and the fused cells(>80%)were digested with 0.05%trypsin.Detection of smooth muscle cell marker ?-SMA to identify cells.(2)Establish hypoxic pulmonary artery smooth muscle cells and pulmonary artery endothelial cells model:Pulmonary artery smooth muscle cells and pulmonary artery endothelial cells were incubated in serum-free medium for 24 hours and divided into normal group(cultured under 21%O2,5%CO2 and 74%N2),hypoxic model(cultured under 3%O2,5%CO2 and 92%N2)and EXOsomes group(3%O2,N2)Under the condition of 92%N2 and adding 100 ?g/mL MSC-EXO,the cells were cultured at 37? for 24 hours,48 hours and 72 hours respectively,and the cells were collected for experiment.The expression of Wnt5a in the model was detected by immunofluorescence after 72 hours.(3)The Wnt5a gene was silenced by transfection of Wnt5a siRNA gene,and the inhibition efficiency of mRNA level and protein level was detected.(4)After 48 hours and 72 hours of hypoxia exposure,the apoptosis of endothelial progenitor cells was detected by flow cytometry AV-PI staining.Western blot and PT-PCR were used to detect the protein and mRNA expression of anti-apoptosis gene Bcl2,pro-apoptosis gene caspase-3 and Bax.(5)To test the effect of MSC-EXO on hypoxia-induced cell proliferation:the proliferation of PASMC and PAEC was determined by BrdU method,and the expression of PCNA protein of PAEC was detected by Western blot.(6)PAEC tubule formation test and Transwell test were used to detect the effect of MSC-EXO on angiogenesis and migration ability of PAEC cells treated with hypoxia(3%).(7)The effect of MSC-EXO on the mesenchymal transition of hypoxic PAEC cells:The levels of CD31,VE-cadherin and ?-smooth muscle actin were detected by immunofluorescence and Western blot.(8)Western blot was used to detect the expression of protein related to BMPR signaling pathway.Results:1.MSCs isolated by histocyte adherence method were inoculated into culture flask,and the cells adhered to grow in strip shape after 24 hours.After 7 days,about 90%of the cells were fused,and the shape was long spindle.The results of flow cytometry analysis showed that the cultured primary cells expressed CD-90(94.9%),CD-73(91.5%)and CD-105(87.5%),but did not express CD-34(0.7%),CD-45(0.4%)and HLA-DR.After 3 weeks of osteogenic induction culture in vitro,the formation of round calcium nodules was observed by alizarin red staining After 2 weeks of adipogenesis induction culture in vitro,red lipid droplets were formed by oil red O staining After three weeks of chondrogenic induction culture in vitro,blue-purple chondrocytes were observed by toluidine blue staining.2.Observed under microscope,EXOsomes are particles of different sizes,which are disc-shaped and round,with a diameter of about 50?150 nm;Western Blot showed that the EXO of HUMSCs expressed CD63?CD81?TSG101 andALIX;After MSC-EXO was co-cultured with pulmonary artery endothelial cells for 24 hours and 48 hours,the uptake of MSC-EXO by pulmonary artery endothelial cells was observed under confocal fluorescence microscope.3.General condition and survival rate of animals:After subcutaneous injection of monocrotaline for one week,the rats in the experimental group decreased their eating and activities,and their reactions were slow,their hair was obscure and dull,and their breath was shortness.The above characteristics of rats in control group were not obvious.The above characteristics of rats in EXO group improved after MSC-EXO injection for 3 weeks.However,the symptoms of MCT rats were further aggravated.There were 3 rats in MCT group and 2 rats died in EXO group.Right ventricular systolic pressure(RVSP)and right ventricular/left ventricular plus ventricular septal ratio in MSC-EXO group were significantly lower than those in MCT group(P<0.05).There was no significant difference in HR and CO between MCT group and EXO group.However,RVSP and right ventricle/(left ventricle+right ventricle)in EXO group were significantly lower than those in MCT group(P<0.05).Masson staining showed that the degree of right ventricular fibrosis in EXO group was significantly lower than that in MCT group(P<0.05).Compared with the control group,the pulmonary arterioles in MCT group were obviously thickened and narrowed by HE staining.The percentage of calculated vessel wall thickness(WT%)and vessel wall area(WA%)in MCT group were significantly higher than those in control group,while EXO group was significantly lower than that in MCT group(P<0.05).Masson trichromatic staining showed that there was obvious collagen deposition in pulmonary interstitium of MCT group,while collagen deposition in MSC-EXO treatment group was obviously reduced(P<0.05).Immunohistochemistry showed that the expression of CD31 protein in lung tissue of MCT group was significantly lower than that of control group,and that of EXO group was significantly higher than that of MCT group.Immunofluorescence and western blotting were used to detect endothelial cell interstitial markers in lung tissue.The results showed that compared with the control group,the expression of ?-SMA in MCT group increased significantly,while the protein expressions of CD31 and VE-cadherin decreased significantly.Compared with MCT group,the protein expression of CD31 and VE-cadherin in EXO group increased significantly,while the level of?-SMA decreased significantly(P<0.05).Compared with the control group,the expression of TGF-?1 and Smad2/3 in MCT group was significantly higher,while the expression of BMPR2 and Smad1/5/8 was significantly lower(P<0.05).The expressions of TGF-?1 and Smad2/3 in EXO group were lower than those in MCT group,while the expressions of BMPR2 and Smad 1/5/8 were higher than those in MCT group(P<0.05).The protein expression levels of Wnt5a,?-catenin and cyclin D1 were detected by western blot.We found that wnt5a protein level decreased significantly in MCT group,but the levels of ?-catenin and cyclin D1 were higher than those in control group.Compared with MCT group,Wnt5a protein level was higher and ?-catenin and cyclin D1 level was lower in EXO group(P<0.05).4.(1)Microscopically,the cells grew in a long spindle shape,and immunofluorescence showed strong red fluorescence in the cytoplasm,which was arranged in a filiform pattern parallel to the longitudinal axis of the cells,while the oval nucleus in the center of the cells was not stained.(2)we analyzed the effect of MSC-EXO on the expression of Wnt5a in PASMC and PAEC model damaged by hypoxia by immunofluorescence.the results showed that the expression of Wnt5a decreased significantly in PAEC and PASMC induced by hypoxia after 72 h of hypoxia exposure,and the decrease was recovered in MSC-EXO group(P<0.05).(3)To study the role of Wnt5a pathway in bone marrow mesenchymal stem cells EXO,Wnt5a gene was silenced by transfection of Wnt5a siRNA gene.Transfection of Wnt5a siRNA leads to the decrease of Wnt5a expression.in PAEC and pamcs,the inhibition efficiency at mRNA level is about 96.4%and 92.8%,and the inhibition efficiency at protein level is 50.8%and 70.4%,respectively.(4)After 48 hours and 72 hours of hypoxia exposure,the apoptosis of endothelial progenitor cells was detected by flow cytometry AV-PI staining.Compared with the control group,the percentage of apoptotic cells in hypoxia group gradually increased,while the percentage of apoptotic cells in MSC-EXO group was lower than that in hypoxia group(P<0.05).Compared with MSC-EXO,the percentage of apoptotic cells in Wnt5a siRNA transfection group increased significantly(P<0.05).The results of western blot and PT-PCR showed that after hypoxia for 72 h,compared with the control group,the mRNA and protein expression level of anti-apoptosis gene Bcl2 decreased,and the pro-apoptosis genes caspase-3 and Bax increased in hypoxia group Compared with the control group,the mRNA and protein expression level of anti-apoptosis gene Bcl2 increased and pro-apoptosis genes caspase-3 and Bax decreased in MSC-EXO group.However,the mRNA and protein expression level of anti-apoptosis gene Bcl2 decreased,and pro-apoptosis genes caspase-3 and Bax increased in Wnt5a siRNA transfection group,that is,Wnt5a siRNA transfection increased apoptosis.(5)The proliferation of PASMC and PAEC was determined by BrdU method.Compared with the control group,hypoxia can significantly increase the proliferation of PASMC and PAEC(P<0.05).Compared with hypoxia exposure,MSC-EXO can significantly reduce the proliferation of PASMC and PAEC after hypoxia exposure for 48 hours and 72 hours(P<0.05).Compared with MSC-EXO group,transfection of Wnt5a siRNA increased the proliferation of PASMC and PAEC(P<0.05).The expression of proliferating cell nuclear antigen PCNA protein in PAEC was detected by Western blot.The results showed that the level of PCNA protein in hypoxia group was significantly higher than that in control group after 72 hours of hypoxia treatment.Compared with hypoxia group,PCNA protein level in MSC-EXO group decreased significantly(P<0.05).Compared with MSC-EXO group,the level of PCNA protein in Wnt5a siRNA transfection group was higher(P<0.05).(6)The results of PAEC tubule formation test showed that after 72 hours of hypoxia,compared with the control group,the number of hypoxic tubes and capillary network branches decreased significantly.Compared with hypoxia group,the tube formation and capillary network branches in MSC-EXO group were significantly enhanced However,when the cells were transfected with Wnt5a siRNA,the number of tubes and capillary network branches decreased significantly(P<0.05).Transwell test showed that compared with the control group,the number of migrating cells in hypoxia group decreased significantly;Compared with hypoxia group,the number of migrating cells in MSC-EXO group increased significantly(P<0.05).Compared with MSC-EXO group,the number of migrating cells in Wnt5a siRNA transfection group decreased significantly.(7)Effect of MSC-EXO on Interstitialization of hypoxic PAEC Cells Immunofluorescence and Western blot results showed that compared with the control group,the levels of CD31 and VE-cadherin in hypoxic group were lower,while the level of ?-smooth muscle actin was higher.Compared with hypoxia group,the levels of CD31 and VE-cadherin in MSC-EXO group were higher,while the level of?-smooth muscle actin was lower(P<0.05).Compared with MSC-EXO group,the levels of CD31 and VE-cadherin were lower,but the level of ?-smooth muscle actin was higher after transfection of Wnt5a siRNA.(8)The expression of BMPR signaling pathway related protein was detected by Western blot:compared with the control group,the expression of BMPR2,P/T-Smad 1/5/8,BMPR4 and BMPR9 protein in hypoxia group decreased(p<0.05).Compared with hypoxia group,the protein expressions of BMPR2,P/T-Smad 1/5/8,BMPR4 and BMPR9 in MSC-EXO group were significantly increased(P<0.05),while the protein expressions of P/TSmad2/3 and TGF-?1 were significantly decreased(p<0.05).The expression of BMPR2,P/T-Smad1/5/8,BMPR4 and BMPR9 decreased,while the expression of P/TSmad2/3 and TGF-?1 increased after transfection of Wnt5a siRNA(P<0.05).Conclusion:1.MSC-EXO can significantly reduce right ventricular systolic pressure(RVSP)and right ventricular hypertrophy index induced by MCT in rats,and alleviate pulmonary vascular remodeling and pulmonary fibrosis.2.MSC-EXO can reduce apoptosis of endothelial progenitor cells induced by hypoxia,inhibit proliferation of PASMC cells induced by hypoxia,and promote angiogenesis and migration ability of PAEC cells treated by hypoxia.3.MSC-EXO can inhibit vascular remodeling of pulmonary hypertension,which may be achieved by regulating Wnt5a and/or BMPR2 signaling pathway,and then inhibiting EndMT process. |
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