Mechanistic Studies On The Role Of Sema7A In Endothelial-to-mesenchymal Transition

During embryonic development and disease progression,endothelial cells(ECs)display a considerable plasticity of transition to other cell types,such as endothelial to mesenchymal transition(End MT),in which the ECs lose specific endothelial markers such as CD31,VE-cadherin,and progressively express mesenchymal markers like ?-SMA and fibroblast-specific protein-1(FSP-1).With specific respect to atrioventricular canal and heart-valve development,ECs in the region of the forming atrioventricular canal undergo End MT to give rise to mesenchymal cells that form the endocardial cushion tissue and semilunar valves.In the adult,End MT has emerged as a player in the pathogenesis of chronic fibrotic injuries,such as cardiac fibrosis,kidney fibrosis,and system sclerosis.End MTderived mesenchymal-like cells altered extracellular matrix collagen protein and matrix metalloproteinase(MMP)production,which contribute to fibrosis transition.In the cardiovascular system,End MT exist in atherosclerosis and is associated with plaque instability by altering collagen-MMP balance.In addition,End MT is involved in the pulmonary vascular remodeling,causing pulmonary arterial hypertension.As End MT is common in many pathological lesions,identification of key molecules involved in End MT is highly demanded for the diagnosis and treatment of related diseases.The semaphorin family contains a large number of secreted and membrane-bound proteins,which were originally detected on immunocyte membranes.The semaphorin family initially identified as axon guidance cues signaling through their receptors neuropilins,and plexins.Recently,the semaphorin family were found to participate in a broad of pathophysiological process,including atherosclerosis,vascular inflammatory disease.Semaphorin7A(Sema7A)was discovered based on sequence similarities with the vaccinia virus sema homolog A39 R,and signaling through plexins or integrins to exert functions.Sema7 A has been reported to associate with activity-dependent olfactory synapse formation,pulmonary fibrosis,multiple sclerosis,T-cell-mediated inflammatory responses,and breast tumor progression.Our lab has reported that Sema7 A take part in atherosclerosis via eliciting leukocyte infiltration,monocyte-macrophage retention,platelet hyperreactivity,and neovascularization.Whether Sema7 A is involved in the progress of End MT is unknown,although previous study reported that Sema7 A promotes growth and migration of oral tongue squamous cell carcinoma by regulation epithelial-mesenchymal transition(EMT).In this study,we hypothesized that Sema7 A induced End MT through the TGF/Smad signaling pathway.This study is mainly divided into the following five parts.Part I: The function of Semaphorin 7A in endothelial cells and phenotype tranformationObjective To explore the function of Semaphorin 7A in endothelial cells(ECs),and phenotype transformation.Methods: We firstly generated Sema7A-overexpressed HUVECs(Sema7A-HUVECs)by transducing lentiviral vectors expressing human Sema7 A with a GFP(green fluorescent protein)tag(Lenti-h Sema7A-GFP)into primary HUVECs and performed a RNAsequencing,comparing con335-HUVEC with Sema7A-HUVEC to find the different expression genes.The activated signaling pathways were analyzed by KEGG database.In addition,We performed functional gene ontology(GO)analysis on the up-regulated genes to explore the change of function in ECs after Sema7 A transducting.Then,we observe the cell morphogenesis change in Sema7A-HUVECs.As CD31 is the critical marker of ECs,we detected CD31 through flow cytometry between Con335-HVUECs and Sema7A-HUVECs.In addition,we detect ECs marker-CD31,VE-cadherin,and mesenchymal cell marker ?-SMA and FSP-1 through q-PCR and Western-blot to confirm the function.Results Results showed that Sema7 A overexpression caused the upregulation of a total of 1168 genes and downregulation of 886.Among them,most of the molecules associated with End MT and mesenchymal cells were upregulated in Sema7A-HUVECs,as compared to Con335-HUVECs with a higher endothelial gene expression profile.Con335-HUVECs were oval,round,regulated sizes and forme a typical cobblestone-like appearance,however,Sema7A-HUVECs are long fusiform or irregular under the microscope.Flow cytometry analysis revealed a 48% reduction of CD31+ cells in Sema7A-HUVECs(45.64±2.76%)compared with con335-HUVECs control(87.48±0.36%).Significantly,compared with con335-HUVECs,Sema7A-HUVECs presented lower levels of EC markers CD31 and VEcadherin(VE-cad),and higher levels of mesenchymal cell markers ?-SMA and FSP-1 as shown by q-PCR and Western-blot.Conclusion Sema7 A expression is low in normal ECs,and is associated with inflammation,stress response and phenotype transformation.Sema7 A promotes End MT,which imply a potential therapeutic strategy for End MT-related diseases by targeting Sema7 A.Part ? The role of TGF-?2 and TGF/Smad signaling pathway in Sema7 A induced End MTObjective To explore the role of TGF-?2 and TGF-?/Smad signaling in Sema7 A induced End MT.Methods By q-PCR and ELISA,We detected the expression of TGF-?2 in Sema7 AHUVECs with Con335-HUVECs.Gene set enrichment analysis(GSEA)was used to detected the activated pathway in Sema7A-HUVECs.We assessed Smad3 phosphorylation(p-Smad3),an indicator of TGF-?/Smad signaling activity.In the end,we inhibit TGF-?2 and TGF-?/Smad to verify their function in Sema7 A induced End MT.Results By q-PCR and ELISA,we showed that TGF-?2 was upregulated in Sema7 AHUVECs.Gene set enrichment analysis(GSEA)showed that TGF-?/Smad signaling pathway is enriched in Sema7A-HUVECs.We therefore assessed Smad3 phosphorylation(p-Smad3),an indicator of TGF-?/Smad signaling activity and showed that p-Smad3 was elevated in Sema7A-HUVECs.In contrast,inhibition of TGF/Smad signaling by inhibitor oxymatrine attenuated phosphorylation of Smad3 induced by Sema7 A overexpression.Notably,blocking TGF-?2 by neutralizing antibody T4442 reduced phosphorylation of Smad3 as well,implying an upstream regulation of TGF-?2 on Smad3 in Sema7A-HUVECs.Moreover,inhibition of TGF/Smad signaling by oxymatrine or blocking TGF-?2 by antibody T4442 enhanced CD31 expression and reduced ?-SMA expression in Sema7 AHUVECs.Conclusion Sema7 A overexpression induced End MT potentially by upregulating TGF-?2 and activating TGF-?/Smad signaling.Part ? Mechanism of ATF3 in mediating Sema7 A and TGF-?2Objective To investigate the molecular mechanism of ATF3 in Sema7 A induced End MTMethods Combined with RNA-sequencing data and reports,we predicted that ATF3 are associated with End MT induced by Sema7 A,then we confirmed they expression change in Sema7A-HUVEC through q-PCR and Western-blot.Through Chip and Luciferase reporter gene assay,we detect the regulation between ATF3 and TGF-?2.We respectively used neutralizing antibody T4442 to inhibit TGF-?2,oxymatrine to TGF-?/Smad,and verify they function in Sema7A-HUVECs.Results.In addition to TGF-?2,RNA-seq analysis showed that ATF3 also ranked in the top thirty.Q-PCR and Western-blot showed that ATF3 was upregulated in Sema7 AHUVECs.To ask whether ATF3 regulates TGF-?2 expression in Sema7 A induced End MT,we introduced ATF3-si RNA to Sema7A-HUVECs and showed that both TGF-?2 m RNA level and supernatant protein concentration were downregulated,implying an upstream regulation of ATF3 on TGF-?2 expression.Further study using Chip-q-PCR assays showed a higher TGF-?2 percentage of input in Sema7A-HUVECs compared with Con335-HVUECs.Using luciferase reporter assay,we showed that luciferase activity of reporter gene containing wild-type TGF-?2 promoter(p GLC3-WT)was increased after ATF3 plasmid transfection,while a 6bp mutation(-19 to-12,the upstream of transcriptional start site)of TGF-?2 promoter region in reporter gene(p GLC3-mut)eliminated this change.Furthermore,we observed that ATF3 overexpression in HUVECs upregulated TGF-?2,but not TGF-?1,m RNA expression.We next investigated whether ATF3 participated in Sema7 A induced End MT by regulating TGF-?2 expression.As TGF-?2 activated TGF/Smad signaling to induce End MT in Sema7A-HUVECs,we transfected Sema7A-HUVECs with ATF3-si RNA and found that Smad3 phosphorylation was reduced,indicating that inhibiting ATF3 reduced TGF-?/Smad signaling.Using the same strategy,we showed that inhibition of ATF3 upregulated CD31 and downregulated ?-SMA,indicating an alleviation of End MT.Conclusion Sema7 A overexpression enhances ATF3 expression that in turn promotes TGF-?2 transcription,inducing End MTPart ? The role of integrin ?1 in the End MT induced by Sema7A overexpressionObjective: In preview study,we conformed that Sema7 A impired the function of ECs by binding to integrin ?1.To expore the role of integrin ?1 in the End MT induced by Sema7 A overexpressionMethods We treated Sema7A-HUVECs with a ?1 integrin blocking monoclonal antibody(P5D2),and detected the expression of ATF3,TGF-?2,and End MT.Moreover,we desired rescue experiments to comfirm the link between Sema7A/ integrin ?1 and TGF-?2/Smad3.We transfected ATF3 plasmid into Sema7A-HUVECs that were preincubated with P5D2 to block ?1 integrin activation by Sema7 A,and observed the change of ECs phenotype transformation.Results ?1 integrin blockage reduced Sema7A-induced increase of ATF3 and TGF-?2,Smad3 phosphorylation and End MT as indicated by upregulated CD31 and downregulated ?-SMA in Sema7A-HUVECs.To confirm the role of integrin-?1 and ATF3 axis in mediating Sema7 A induced End MT,we transfected ATF3 plasmid into Sema7A-HUVECs that were preincubated with P5D2 to block ?1 integrin activation by Sema7 A.Results showed that ATF3 overexpression rescued TGF-?2 expression and End MT pre-inhibited by integrin ?1 blockage as shown by the reduced CD31 expression and enhanced ?-SMA expression.Conclusion Sema7 A promotes End MT through integrin ?1-ATF3 axis.Part ? The role of Sema7 A in the End MT induced by disturb flowObjective: In our previous study,we found disturb flow induced by PCL upregulated the expression of Sema7 A in ECs,we used Sema7 A deletion mice,and PCL model to detect the function of Sema7 A on ECs in vivo.Methods Through immunofluorescence staining,we detect carotid intima exposed to disturb flow induced by PCL in Sema7A+/+ and Sema7A-/-mice.Results In wildtype(WT)mice,co-localization of CD31/?-SMA,CD31/FSP-1 or VWF/?-SMA were observed in the endothelium of the carotid artery upon PCL by immunofluorescence staining,while in Sema7 A deficient mice,co-localization of CD31 with ?-SMA or FSP-1 was hardly seen.The total fluorescence intensity analysis showed a reduction of CD31 signal and enhancement of ?-SMA and FSP-1 signal in the endothelium of the carotid artery upon PCL in WT mice,compared with Sema7 A deficient miceConclusion D-flow induced by PCL promotes End MT.Sema7 A expression promotes End MT induced by d-flow in vivo.