Journal: Journal of Advanced Research

Article Title: Transglutaminase 2 modulates inflammatory angiogenesis via vascular endothelial growth factor receptor 2 pathway in inflammatory bowel disease

doi: 10.1016/j.jare.2025.07.002

Figure Lengend Snippet: The expression of TGM2 is increased in enteric endothelial cells of IBD patients and experimental colitis mice. (A) Gene microarray data from our IBD center indicate a significant upregulation in TGM2 expression in IBD patients (HC n = 18, CD n = 12, UC n = 9). (B, C) GEO datasets ( GSE95095 , GSE243625 ) also demonstrate a marked upregulation of TGM2 in IBD patients, particularly at the inflamed area. (D) qPCR revealed TGM2 mRNA levels is upregulated in intestinal tissues from IBD patients (n = 15 in each group). (E, F) qPCR revealed TGM2 mRNA levels is elevated in intestinal tissue from DSS and TNBS colitis model (n = 10 in each group). (G-K) The protein level of TGM2 in IBD and colitis model is assessed by WB. The results show that protein level of TGM2 is ascended in IBD and mouse colitis model (n = 4 in each group). (L) The UMAP of different 7 subgroups of total 165,618 cells. (M) The violin plot shows TGM2 is increased in enteric endothelial cells of IBD patients. (N, O) IHC and IF results show significant increase in blood vessel density along with heightened expression levels of TGM2 (green) specifically within CD31 (red) endothelial cells among IBD patients (white arrow). (P, Q) IHC and IF results show prominent upregulation of TGM2 expression in endothelial cells in DSS-induced colitis (white arrow), but only partial co-localization between TGM2 (red) and CD31 (green) within endothelial cells in TNBS colitis (white arrow). (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Plasma TGM2 levels were quantified by ELISA (ELH-TGM2-1, RayBiotech, USA).

Techniques: Expressing, Microarray

Journal: Journal of Advanced Research

Article Title: Transglutaminase 2 modulates inflammatory angiogenesis via vascular endothelial growth factor receptor 2 pathway in inflammatory bowel disease

doi: 10.1016/j.jare.2025.07.002

Figure Lengend Snippet: TGM2 regulates the angiogenic ability of intestinal vascular endothelial cells. (A, B) Multiple immunofluorescence staining for CD31 (red), TGM2 (green), and Ki-67 (cyan) reveals a significant presence of co-stained Ki-67 and TGM2 double positive vascular endothelial cells in patients with CD and UC (White arrow. A representative image of TGM2 + proliferative vascular endothelial cells is displayed within the white frame.). (C, D) The DSS colitis mice exhibit a significant upregulation in the number of CD31(green), TGM2(red), Ki-67(cyan) positive vascular endothelial cells, whereas this cell is less observed in TNBS colitis mice (White arrow. A representative image of TGM2 + proliferative vascular endothelial cells is displayed within the white frame). (E) The in vitro tube formation assay demonstrated that knockdown of TGM2 significantly attenuated the ability of HIMEC to form tubes, whereas overexpression of TGM2 markedly augmented the tube formation ability of HIMEC. (F, G) EdU proliferation assay showed that TGM2 knockdown significantly inhibited HIMEC proliferation, while TGM2 overexpression significantly enhanced HIMEC proliferation (n = 3 in each group). (H) The Matrigel invasion assay demonstrated that knockdown of TGM2 significantly impeded HIMEC invasion ability, whereas overexpression of TGM2 notable strengthen HIMEC invasion ability. (I, J) The wound healing assay demonstrated a significant reduction in HIMEC migration ability upon inhibition of TGM2, whereas the overexpression of TGM2 significantly enhanced HIMEC migration ability (n = 3 in each group). (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Plasma TGM2 levels were quantified by ELISA (ELH-TGM2-1, RayBiotech, USA).

Techniques: Immunofluorescence, Staining, In Vitro, Tube Formation Assay, Knockdown, Over Expression, Proliferation Assay, Invasion Assay, Wound Healing Assay, Migration, Inhibition

Journal: Journal of Advanced Research

Article Title: Transglutaminase 2 modulates inflammatory angiogenesis via vascular endothelial growth factor receptor 2 pathway in inflammatory bowel disease

doi: 10.1016/j.jare.2025.07.002

Figure Lengend Snippet: STAT1-TGM2-VEGFR2 axis drives inflammatory angiogenesis in IBD. (A) The mRNA transcription of TGM2 was significantly upregulated upon stimulation with inflammatory cytokines IL-9, IL-23, and IFN-γ in HIMECs (n = 3 in each group). (B, C) The results of WB demonstrated that stimulation with IL-9, IL-23, and IFN-γ upregulated the protein expression of TGM2 in HIMEC. (D) Stimulation with inflammatory cytokines IL-9, IL-23, and IFN-γ significantly augmented the in vitro angiogenic capacity of HIMECs, while knockdown of TGM2 effectively reversed this effect. (E, F) IL-9, IL-23 and IFN-γ stimulation significantly enhanced the phosphorylation of VEGFR2 at Tyr1059, Tyr1214 and activated the downstream pathways such as FAK, PLC-γ and MAPK pathways in HIMEC, which were reversed by TGM2 knockdown. (G) IF results showed VEGFR2 co-localizes with TGM2 in HIMEC, mainly on the cell membrane. (H) Co-IP results substantiated the interaction between TGM2 and VEGFR2. (I) The CHIP assay confirmed the binding of STAT1 to the upstream promoter region of TGM2, thereby facilitating transcription following IFN-γ stimulation. (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: Plasma TGM2 levels were quantified by ELISA (ELH-TGM2-1, RayBiotech, USA).

Techniques: Expressing, In Vitro, Knockdown, Phospho-proteomics, Membrane, Co-Immunoprecipitation Assay, Binding Assay

Journal: Journal of Advanced Research

Article Title: Transglutaminase 2 modulates inflammatory angiogenesis via vascular endothelial growth factor receptor 2 pathway in inflammatory bowel disease

doi: 10.1016/j.jare.2025.07.002

Figure Lengend Snippet: Tgm2 knockout relieve tissue inflammation and inhibit angiogenesis in DSS colitis model. (A) Body weight lost of Tgm2 KO mice and control mice after DSS treatment (n = 5 in WT-Water and KO-Water group, n = 10 in WT-DSS and KO-DSS group). (B) Representative colonic images of DSS-treated Tgm2 KO mice and control mice. (C)Appearance of colon length of DSS-treated Tgm2 KO mice and control mice (n = 5 in water group, n = 10 in DSS group,). (D, E) DAI and Histological score of DSS-treated Tgm2 KO mice and control mice. (n = 5 in water group, n = 10 in DSS group). (F, H) Representative HE staining and Tgm2, CD31 immunohistochemical images of Tgm2 KO mice and control mice show decreased MVD in Tgm2 KO mice after DSS treatment. (G)WB result display that Tgm2 KO mice significantly reduce the phosphorylation of VEGFR2 at Tyr1059 and Tyr1214 after DSS treatment. (I) Tgm2 KO mice exhibit decrease in the number of Cd31 (green), Tgm2 (red), Ki-67 (cyan) positive proliferative vascular endothelial cells comparing to control mice. (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Plasma TGM2 levels were quantified by ELISA (ELH-TGM2-1, RayBiotech, USA).

Techniques: Knock-Out, Control, Staining, Immunohistochemical staining, Phospho-proteomics

Journal: Journal of Advanced Research

Article Title: Transglutaminase 2 modulates inflammatory angiogenesis via vascular endothelial growth factor receptor 2 pathway in inflammatory bowel disease

doi: 10.1016/j.jare.2025.07.002

Figure Lengend Snippet: TGM2 specific inhibitor significantly attenuates colitis and angiogenesis in DSS colitis model. (A) Body lost of GK921 treatment group and solvent treatment group after DSS colitis model establishment (n = 9 in Con group, n = 7 in DSS-DMSO, n = 8 in DSS-GK921 treatment group). (B) Representative colonic images of GK921 treatment group and solvent treatment group after DSS colitis model establishment. (C) Appearance of colon length of GK921 treatment group and solvent treatment group after DSS establishment (n = 9 in Con group, n = 7 in DSS-DMSO, n = 8 in DSS-GK921 treatment group). (D, E) DAI and CDMI score of GK921 treatment group and solvent treatment group after DSS colitis model establishment (n = 9 in Con group, n = 7 in DSS-DMSO, n = 8 in DSS-GK921 treatment group). (F, H) Representative HE staining and TGM2, CD31 immunohistochemical images of GK921 treatment group and solvent treatment group after DSS colitis model establishment show decreased MVD in DSS mice after GK921 treatment. (G) WB result display that GK921 treatment significantly reduce the phosphorylation of VEGFR2 at Tyr1059 and Tyr1214 after DSS treatment. (I) GK921 treatment exhibit decrease in the number of CD31 (green), TGM2 (red), Ki-67 (cyan) positive proliferative vascular endothelial cells comparing to controls. (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Plasma TGM2 levels were quantified by ELISA (ELH-TGM2-1, RayBiotech, USA).

Techniques: Solvent, Staining, Immunohistochemical staining, Phospho-proteomics

Journal: Journal of Advanced Research

Article Title: Transglutaminase 2 modulates inflammatory angiogenesis via vascular endothelial growth factor receptor 2 pathway in inflammatory bowel disease

doi: 10.1016/j.jare.2025.07.002

Figure Lengend Snippet: Serum TGM2 is a potential biomarker for monitoring endoscopic disease activity in CD. (A) Serum TGM2 concentrations were significantly increased in CD patients. (B-E) Levels of serum TGM2, CRP, PLT and ESR were markedly elevated in the endoscopically active cohort. (F-I) Serum TGM2, CRP, PLT and ESR exhibited positive correlations with CDEIS. (J) ROC curves of serum TGM2, CRP and PLT in assessing endoscopic disease activity show serum TGM2 is a reliable biomarker for monitoring endoscopic disease activity in CD. (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: Plasma TGM2 levels were quantified by ELISA (ELH-TGM2-1, RayBiotech, USA).

Techniques: Biomarker Discovery, Activity Assay