Screening And Functional Studies Of Key Gene In Venous Malformation Based On By TMT Proteomics And Zebrafish Model

Background and objectives:Venous malformation(VM)is a common congenital vascular malformations comprising dilated and tortuous veins of variable size with a sparse and disorganized arrangement of vascular smooth muscle cells,which gradually expand and tortuously form clusters with growth and development.It can occur in all parts of the body,with the oral and maxillofacial areas,head and neck,and extremities being the main sites of onset.The main clinical manifestations are swelling,pain,bleeding,and limited range of motion,which eventually form irreversible dysfunction,leading to severe dysfunction or severe bleeding and becoming life-threatening when the lesions involve vital organs.Several pathogenic mutations associated with venous malformation have been recently identified,and attempts have been made to elucidate the pathophysiological pathways involved in their development.Studies have shown that somatic mutations in genes,such as TIE2(TEK),PIK3CA,and MAP3K3,are associated with VM.Based on the mode of inheritance,VM can be divided into disseminated VM and familial venous malformations(VMCM),of which the familial type is more complex and difficult to treat.Several studies have found that many VMCM patients have specific genetic mutations,namely TIE2-R849W.Twenty differentially expressed proteins were selected for further functional screenings by high-content screening(HCS)at the gene level.The differentially expressed gene ACTA2 was identified and validated in the tissues of patients with venous malformations by RNA and protein expression profiles.Cell assays were performed using human microvascular endothelial cells(HMEC-1)to explore the function of the ACTA2 gene.Finally,we established the ACTA2 zebrafish model to observe how mutations affect the development of the caudal venous plexus and to investigate the related mechanism.Materials and methods:1.Samples collection:Ten patients with venous malformations from 2019 to 2020 in the Department of Plastic Surgery of Shandong Provincial Hospital were selected,and surgically excised venous malformation tissues were collected.Ten cases of normal tissues were selected as the control group.The tissue specimens were transferred to liquid nitrogen for preservation.Of these samples,three pairs were used for quantitative proteomics analysis,while all samples were assessed for further validation.Postoperative pathological diagnoses were made by two independent pathologists.The study was approved by the Ethics Committee of Shandong University Provincial Hospital.2.Quantitative proteomics analysis by TMT:Tandem Mass Tag(TMT)was used to screen proteins that were differentially expressed between patients with disseminated venous malformations and healthy people.Each of the control and experimental groups contained three tissue samples that were labeled as Al,A2,and A3 and B1,B2,and B3,respectively.Proteins with expression differences greater than 1.4-fold(upregulated or downregulated)and a P value of less than 0.05 were identified as differentially expressed proteins.Gene Ontology(GO)analysis was performed on the collection of differentially expressed proteins,including biological process(BP),molecular function(MF),and cellular component(CC),to explore the function of the target proteins.The KEGG Orthology(KO)and Links Annotation were used for the pathway enrichment analysis.The target protein sequences were first KO categorized by comparing to the KEGG GENES database,and information on the pathways involved in the target protein sequences was automatically obtained according to the KO categorization.3.High-content screening cell proliferation assay:20 differentially expressed genes were selected based on the results of proteomics and bio informatics analysis.The differently expressed proteins were selected for further functional screenings by high-content screening(HCS)at the gene level.Multi-target RNAi lentiviral vectors(mix)with a green fluorescent protein(GFP)were prepared for transfection of HMEC-1,and the corresponding control vectors were also prepared.20 proteins with significant differential expression were selected for HCS to find key driver genes and characterize the expression of these genes in patients with venous malformations.4.QRT-PCR was used to detect the expression of ACTA2 in pathological tissues of patients with venous malformation and control tissues.5.Human microvascular endothelial cells(HMEC-1)were used to explore the function of the ACTA2 gene in vitro experiments:CCK-8 analysis was used to assess whether ACTA2 could promote proliferation.OrisTM plate wound healing assay was employed to assess the effect of migratory ability of ACTA2.The invasion assay was used to assess the effect of the invasion ability of ACTA2.The cell cycle analysis was used to assess the effect of cell cycle of ACTA2.The effect of angiogenesis of ACTA2 was investigated by tube formation analysis experiment.6.To investigate the vascular formation process affected by ACTA2,a zebrafish model with ACTA2 knockdown was constructed.Antisense MO(GeneTools)were microinjected into fertilized one-cell stage embryos according to standard protocols.The zebrafish ACTA2 gene was targeted by two specific morpholino antisense strategies to prevent either the translation of the zebrafish gene(ATG-MO)or proper splicing of exon2(E2I2-MO).7.To evaluate blood vessels formation in zebrafish,fertilized one-cell flila-EGFP transgenic lines embryos were injected with ACTA2-MO and control-MO.The phenotypes of complete intersegmental vessels(ISVs)(i.e.the number of ISVs that connect the DA to the DLAV),CVP(caudal vein plexus)were quantitatively analyzed.8.Total RNA was extracted from zebrafish and the expression of related molecules was detected by quantitative real-time PCR(qRT-PCR).Statistical analysis was attempted to explore the pathogenesis of the target gene involved in venous malformation.Results:1.A total of 7408 peptides and 801 protein groups were identified in venous malformations and normal tissues.Proteins with expression differences greater than 1.4-fold(upregulated or downregulated)and a P value of less than 0.05 were identified as differentially expressed proteins.As a result,71 differentially expressed proteins were identified,with five of them being upregulated and 66 being downregulated.GO analysis was performed on the differentially expressed proteins,including BP,MF,and CC.In BP analysis,the differentially expressed proteins primarily focused on the biological regulation,cellular process,and regulation of cytoskeleton organization.In MF analysis,differentially expressed proteins primarily focused on actin filament binding,molecular transducer activity,and binding.In CC analysis,differentially expressed proteins primarily focused on the cytosol,organelles,and cell part.KEGG Automatic Annotation Server software was used to annotate the KEGG pathways of the target protein collection,and the results showed that the KEGG pathways of differentially expressed proteins primarily included Salmonella infection,the IL-17 signaling pathway,and regulation of the actin cytoskeleton.2.Five highly and 15 lowly expressed protein genes were selected based on bioinformatic analysis and proteomics results for the RNAi functional screening test.The upregulated genes were CFHR2,MMP2,COMP,F7,and CD248.The downregulated genes were MAPK1,HSPA8,HSP90AA1,MMP9,ACTA2,YWHAB,HRG,TFPI,TFPI,ACTG1,ACTG1,GSTP1,GAPDH,GAPDH,and S100A9.Based on the HCS platform,in vitro knockdown and overexpression of HMEC-1 models were established for the HCS of venous malformations,and 20 selected genes were tested.Lentivirus was used to construct overexpression and knockdown cell models for the corresponding genes.Plasmids were used to construct overexpression cell models for the five upregulated genes.The HCS screening results showed that among the 20 genes to be tested,the shACTA2 group with a fold-change>1.5 was the differentially expressed gene identified in this experiment(proliferation inhibition-positive cell group).3.qRT-PCR was used to detect the expression of ACTA2 in pathological tissues of patients with venous malformation and control tissues.The experimental results are shown in Figure 3AB,in which there was a significantly low expression of ACTA2 in venous malformation tissues relative to that in normal tissues(P<0.05).Western blotting results showed that the protein expression of ACTA2 was lower in venous malformation tissues than in control tissues.4.Considering lower expression of ACTA2 in patients with venous malformation,we performed over-expression of ACTA2 in HMEC1 cells.We constructed LV-ACTA2(27000-1)lentivirus-transfected cells using HMEC1 as the study object,and observed the cells successfully transfected with LV-ACTA2(27000-1)virus overexpressing ACTA2 and negative control virus under a fluorescence microscope after 72 h.The cell cycle assay revealed that the ACTA2 group had increased cells in the G1/G2 phase(P<0.05),and decreased cells in the S phase(P<0.05),while the NC group was just the opposite.The effect of ACTA2 overexpression on vascular endothelial cell migration was detected using OrisTM plate wound-healing,and the results showed that the migration ability of cells was higher than that in the control group(P<0.05).As for the invasion ability of cells,the invasion assay results concluded that the invasion ability was significantly higher(P<0.05).Moreover,we used the Cell Counting assay(CCK-8)to analyze the effect of ACTA2 on the proliferation of HMEC-1.Overexpression of ACTA2 significantly increased the proliferative ability of HMEC-1(P<0.05).These findings indicate that the overexpression of ACTA2 promotes the proliferation,migration,and invasion ability of human vascular endothelial cells.The in vitro angiogenesis assay was used to test whether ACTA2 overexpression leads to the changes relative to the control group.Compared with the normal control group(NC),the angiogenic capacity,including area,length,number of nodes,and number of branches of vessels,were all higher in the overexpression group than in the control group(P<0.05).5.Morpholino knockdown of acta2 causes vascular defects,sprouting angiogenesis,and CVP(caudal vein plexus)formation defects of zebrafish.Compared with control MO,embryos injected with acta2-MO present a lower number of incomplete ISVs and ectopic sprouts.In control embryos,the parachordal vessels(PAV)form normally.While compared with control,MO knockdown acta2 prevents the parachordal vessels(PAV)formation,the precursor to the lymphatic system.Loss of acta2 impairs the formation of the CVP in zebrafish.In contrast,acta2 knock down resulted in specific defects in CVP formation.According to these results,the direct effect of ACTA2 on early angiogenesis was identified in vivo.Morpholino knockdown of acta2 causes vascular defects,sprouting angiogenesis,and CVP(caudal vein plexus)formation defects of zebrafish.6.The knockdown of ACTA2 inhibited the D114/notch 1 signaling pathway,Ephrin-B2 signaling pathway,and vascular integrity-related molecules and activated the Hedgehog(Hh)signaling pathway.The expression of molecules related to each pathway in the control and ACTA2 knockdown groups was detected by qRT-PCR,revealing that dll4,notch1a,notch1b,hey2,efnb2a,ptp-rb,cd146;nuclear receptor subfamily 2 group F member 1a(nr2f1a),and s1pr1,which are associated with the Dll4/notchl signaling pathway,Ephrin-B2 signaling pathway,and vascular endothelial protein tyrosine phosphatase(VE-PTP),were all significantly downregulated.qRT-PCR testing of the knockdown and control groups found that vascular malformation by ACTA2 knockdown may be due to the inhibition of the Dll4/notch1 signaling pathway,Ephrin-B2 signaling pathway,and vascular integrity-related molecules,as well as activation of the Hh signaling pathway.Conclusion:1.In summary,this study screened differentially expressed proteins in venous malformation and normal human tissues by proteomics and identified the most significantly differentially expressed gene,ACTA2,by HCS.Compared with healthy people,the expression of ACTA2 was significantly reduced in patients with venous malformations.2.We established a cell model to overexpress ACTA2 in HMEC-1 and revealed that the abnormalities of cell proliferation,metastasis,invasion,and angiogenesis were reversed by ACTA2 overexpression.3.We also established a zebrafish animal model and performed in vivo experiments,resulting in the finding that knockdown of ACTA2 in zebrafish results in defective vascular development,disrupted vascular integrity,and abnormal development of the caudal vein,QRT-PCR testing of the knockdown and control groups found that vascular malformation by ACTA2 knockdown may be due to the inhibition of the Dll4/notch1 signaling pathway,Ephrin-B2 signaling pathway,and vascular integrity-related molecules,as well as activation of the Hh signaling pathway.ACTA2 may be a potential target for the diagnosis and treatment of disseminated venous malformations.However,the mechanism of crosstalk between cell signaling pathways in vivo is very complex,more work needs to be continued and discussed.