| Objective: Atherosclerosis is a severe thereat to health and often starts with endothelium injury.Endothelial cells are a layer of active cells,whose integrity and biological characteristics are of great importance.Besides Atherosclerosis,endothelial cells participate in a lot of significant biological and pathological processes,such as angiogenesis,contraction and dilation of blood vessels,coagulation,inflammation and many more.Angiogenesis refers to the process of sprouting from existing vessels as well as forming new blood vessel networks,which is extremely complicated.This process is dependent on the balance between pro-angiogenic and anti-angiogenic factors.Once the balance is broken,the vascular system will be activated,causing overactivation or inhibition of angiogenesis which leads to pathological events.The role of stem cells in angiogenesis has always been a research hotspot,but the mechanism has not been fully understood,particularly related to regulating target cells by stem cells through paracrine function.Human amniotic epithelial cells(hAECs),as one kind of stem cells,can paracrine a large number of biological active factors such as growth factors and chemokines,which is an important means of regulating the function of target cells.The purposes of our study :(1)In vitro isolate hAECs from human amnion membranes,and perform phenotypic identification and purity analysis.(2)Detect the cytokines in the conditioned midium of hAECs(hAEC-CdM),and perform enrichment analysis of the related signaling pathways.(3)Investigate the biological potential and behavior of hAEC-CdM of different concentrations on proliferation,migration and angiogenesis of cultured human aortic endothelial cells(hAoECs).(4)Investigate the related molecular mechanism of the conditioned midium(CdM)of hAECs on human aortic endothelial cells.Methods:(1)Human amniotic epithelial cells(hAECs)were isolated from amnion membranes by 0.25% trypsin and analyzed for their phenotypic characteristics by immunofluorescence staining.(2)LC MS/MS was performed to test the proteins in the collected hAEC-CdM,and GO Analysis and KEGG Pathway Analysis were performed to analyse the LC MS/MS results.(3)We designed ECM group as control,and ECM with different concentrations of hAEC-CdM as test groups,marked as Control group,0.5×CdM group,1×CdM group and 2×CdM group.For inhibitor assays we set 4 groups:ECM group(Control),1×CdM group,inhibitor group(SB431542 or K02288)and CdM+inhibitor group.(4)We compared the effects of different concentrations of hAEC-CdM on hAoECs proliferation by means of CCK-8 assays after stimulated by hAEC-CdM for 24,48 and 72 hours.(5)We compared the effects of different concentrations of hAEC-CdM on hAoECs migration by means of scratch assays after stimulated by hAEC-CdM for 12 and 24 hours.(6)We compared the effects of different concentrations of hAEC-CdM on hAoECs angiogenesis by means of matrigel assays after stimulated by hAEC-CdM for 24 h.(7)After stimulated by different concentrations of hAEC-CdM,we performed western blot to test the protein expression levels of activated TGF-? signaling pathway of hAoECs.Results:(1)The isolated hAECs are of polygon shape,and after proliferation they turned into paving stone shape.We used passage 2 for identification and the assays.Immunofluorescence results showed that hAECs highly expressed CK19 and did not express vimentin,and the purity was above 90%.(2)Our LC MS/MS results identified273 proteins in hAEC-CdM and 88 signaling pathways were involved.GO analysis showed that hAEC-CdM mainly regulated the metabolic process,endocytosis process and phosphorylation process of target cells.KEGG pathway analysis showed that hAEC-CdM mainly affected the PI3K-Akt pathway,mTOR pathway and TGF-? pathway of target cells.(3)CCK-8 assays: After stimulated by hAEC-CdM for 24 hours,there was no difference of hAoECs proliferative ability between test groups and control group(p>0.05).After stimulated by hAEC-CdM for 48 hours,there was no difference of hAoECs proliferative ability between 0.5×CdM group and control group(p>0.05),while1×CdM group and 2×CdM group both showed better proliferative ability than control group(p<0.05).After stimulated by hAEC-CdM for 72 hours,hAoECs of each test group showed better proliferative ability than control group(p<0.05).The higher concentration of hAEC-CdM,the better proliferative ability of hAoECs.(4)Scratch assays: After stimulated by hAEC-CdM for 12 hours,there was no difference of hAoECs migration ability between 0.5×CdM group and control group(p>0.05),while 1×CdM group and2×CdM group both showed better migration ability than control group(p<0.05).After stimulated by hAEC-CdM for 24 hours,hAoECs of each test group showed better migration ability than control group(p<0.05).So the higher concentration of hAEC-CdM,the better migration ability of hAoECs.(5)Matrigel assays: After stimulated by lyophilized powder solution for 24 hours,we performed matrigel assays to evaluate the angiogenesis ability of hAoECs.At 6 hour point,control group gradually had tube formation.Both 0.5×CdM group and 1×CdM group showed more tube formation than control group,and there was more tube formation of 1×CdM group than 0.5×CdM group,which means low concentraion of hAEC-CdM promoted hAoECs tube formation.(6)Western blots after stimulated by different concentrations of hAEC-CdM:(1)ALK5-Smad3-Snail-Postn signaling pathway: There was no significant difference of ALK5 and Smad3 expression between 0.5×CdM group,1×CdM group,2×CdM group and control group(p>0.05).Expression levels of p-Smad3,Snail and Postn of 0.5×CdM group and 1×CdM group were significantly higher than control group(p<0.05),while that of 2×CdM group were significantly lower than control group(p<0.05).(2)ALK1-Smad5-Snail-Postn signaling pathway: There was no significant difference of ALK1 and Smad5 expression of 0.5×CdM group,1×CdM group,2×CdM group and control group(p>0.05).Expression levels of p-Smad5,Snail and Postn of 0.5×CdM group and 1×CdM group were significantly higher than control group(p<0.05),while that of 2×CdM group were significantly lower than control group(p<0.05).(7)Western blots after interfered by inhibitors:(1)Western blots of p-Smad3 and its downstream genes after interfered by SB431542: After interfered by SB431542,there was no significant difference of Smad3 expression between SB431542 group and control group(p>0.05),while the expression levels of p-Smad3,Snail and Postn were significant lower than control group(p<0.05).p-Smad3,Snail and Postn expression levels of 1×CdM group were significantly higher than control group(p<0.05),which means 1×CdM promoted the above proteins expression.After disturbed by SB431542,the expression levels of p-Smad3,Snail and Postn of(CdM+SB)were significantly lower than 1×CdM group(p<0.05),which means SB431542 inhibited the promotion of 1×CdM on the above protein expression.(2)Western blots of p-Smad5 and its downstream genes after interfered by K02288: After interfered by K02288,there was no significant difference of Smad5 expression between K02288 group and control group(p>0.05),while the expression levels of p-Smad5,Snail and Postn were significant lower than control group(p<0.05).p-Smad5,Snail and Postn expression levels of 1×CdM group were significantly higher than control group(p<0.05),which means 1×CdM promoted the above proteins expression.After disturbed by K02288,the expression levels of p-Smad5,Snail and Postn of(CdM+SB)were significantly lower than 1×CdM group(p<0.05),which means K02288 inhibited the promotion of 1×CdM on the above protein expression.(8)After applying inhibitors:(1)After cultivating hAoECs by 1×CdM for 24 h,we applied matrigel assays.Control group started to have tube formation at 6h point,and 1×CdM group had more tube formation than control group.(2)After applying SB431542 inhibiting Smad3 phsophorylation,at 6h point,SB431542 group had no tube formation,while CdM+SB431542 group had much less tube formation than 1×CdM group,which means inhibition of Smad3 phsophorylation could inhibite the promotion of hAEC-CdM on hAoECs angiogenesis.(3)After applying K02288 inhibiting Smad5 phsophorylation,at6 h point,K02288 group had no tube formation,while CdM+K02288 group had much less tube formation than 1×CdM group,which means inhibition of Smad5 phsophorylation could inhibite the promotion of hAEC-CdM on hAoECs angiogenesis.Conclusion:(1)hAECs were successfully isolated and cultured in vitro,with high expression of CK19 and no expression of vimentin,purity of more than 90%.hAECs could rapidly proliferate in vitro culture conditions.(2)Our LC MS/MS results identified273 proteins in hAEC-CdM and 88 signaling pathways were involved.GO analysis showed that hAEC-CdM mainly regulated the metabolic process,endocytosis process and phosphorylation process of target cells.KEGG pathway analysis showed that hAEC-CdM mainly affected the PI3K-Akt pathway,mTOR pathway and TGF-? pathway of target cells.(3)hAEC-CdM promoted hAoECs proliferation and migration.(4)Low concentrations of hAEC-CdM promoted hAoECs angiogenesis by activating its TGF-?signaling pathway.High concentration of hAEC-CdM might inhibit hAoECs angiogenesis by inhibiting the downstream genes Postn and Snail expression of TGF-?signaling pathway of hAoECs. |
PDF Full Text Request