Journal: Nature Communications

Article Title: Endothelial AGGF1 promotes retinal angiogenesis by coordinating TNFSF12/FN14 signalling

doi: 10.1038/s41467-025-55970-3

Figure Lengend Snippet: a Heat maps from proteomic array analysis with ranked enriched genes in 3 db/m retina compared with 3 db/db retina (fold-change >1.2 or <0.83, p < 0.05). High and low expression are denoted by red and navy, respectively. b , c Western blot analysis and quantification of AGGF1 protein levels in db/m retina and db/db retina ( n = 6 mice per group). d Immunohistochemical detection of AGGF1 expression in db/m and db/db mice. Scale bar: 50 μm. e Representative fundus fluorescein angiography (FFA) and SD-OCT pictures obtained from patients with PDR. n = 3 individuals. Scale bars: 2000 μm (top), 500 μm (centre). f – h Western blot analysis and quantification of AGGF1 and VEGF protein levels in equal volumes of vitreous fluid from patients. n = 3 individuals. i – k Western blot analysis and quantification of AGGF1 and VEGF protein levels in equal volumes of aqueous humour from patients. n = 12 individuals. l , m The ELISA analysis of AGGF1 and VEGF levels in aqueous humour from patients. n = 12 individuals. Error bars represent mean ± SEM. 2-tailed unpaired Student’s t test ( a , c , h ) and one-way ANOVA with Tukey’s multiple comparisons test ( k , l , m ). GCL ganglion cell layer, INL inner nuclear layer, ONL outer nuclear layer. Source data are provided as a Source Data file.

Article Snippet: Then, isolectin B4 (1:200, Invitrogen, I21411) staining with different primary antibodies against AGGF1 (1:200, Novus, NB100-455), GFAP (1:200, Proteintech, 60190-1-Ig), Iba-1 (1:200, Abcam, ab283319), TER119 (1:100, Invitrogen, 14-5921-81), and F4/80 (1:200, Proteintech, 28463-1-AP) was performed overnight at 4 °C.

Techniques: Expressing, Western Blot, Immunohistochemical staining, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: Endothelial AGGF1 promotes retinal angiogenesis by coordinating TNFSF12/FN14 signalling

doi: 10.1038/s41467-025-55970-3

Figure Lengend Snippet: a Schematic illustration of the mouse oxygen-induced retinopathy (OIR) model. b FITC-dextran staining of whole-mount retinas from normoxic and OIR mice at P12, P17, and P21 ( n = 3 mice per group). In the inserts, the red line represents the edge of the retina, the blue area represents the avascular area, and the red area represents neovascular tufts (NVTs). c , d Western blot analysis and quantification of AGGF1 and VEGF protein levels in the OIR retinas at P12, P17, P21 ( n = 4 mice per group). e Immunofluorescence staining for AGGF1 on retinal sections at P12, P17, and P21 of OIR mice ( n = 4 mice per group). AGGF1 is expressed by blood vessels in the superficial, intermediate, and deep layers. f Schematic illustration of the structure of retinal layers and the distribution of vessels in retinal sections. g Double immunofluorescence staining for AGGF1 (red) and CD31 (green) in P17 OIR retinas ( n = 4 mice per group). h Localization and quantification of AGGF1 protein in whole-mount retinas upon normoxia and OIR at P17 ( n = 5 mice per group). i Schematic illustration of the vascular network in flat-mounted retinas. j Immunofluorescence for AGGF1 and isolectin B4 (IB4) in whole-mounted retinas of OIR mice at P17 ( n = 5 mice per group). AGGF1 co-localized with IB4 in microvessels and large blood vessels. k Immunofluorescence for AGGF1 and Iba-1 (a microglial marker) and GFAP (an astroglial marker) in whole-mount retinas of OIR mice at P17 ( n = 5 mice per group). Error bars represent mean ± SEM. One-way ANOVA with Tukey’s multiple comparisons test ( d ). Scale bars: 1000 μm ( b ), 50 μm ( e , g ) and 100 μm ( h , j , k ); magnified images: 50 μm ( g ). GCL ganglion cell layer, INL inner nuclear layer, ONL outer nuclear layer. Source data are provided as a Source Data file.

Article Snippet: Then, isolectin B4 (1:200, Invitrogen, I21411) staining with different primary antibodies against AGGF1 (1:200, Novus, NB100-455), GFAP (1:200, Proteintech, 60190-1-Ig), Iba-1 (1:200, Abcam, ab283319), TER119 (1:100, Invitrogen, 14-5921-81), and F4/80 (1:200, Proteintech, 28463-1-AP) was performed overnight at 4 °C.

Techniques: Staining, Western Blot, Immunofluorescence, Double Immunofluorescence Staining, Marker

Journal: Nature Communications

Article Title: Endothelial AGGF1 promotes retinal angiogenesis by coordinating TNFSF12/FN14 signalling

doi: 10.1038/s41467-025-55970-3

Figure Lengend Snippet: a – i FITC-dextran staining of whole-mount retinas and quantification of the avascular area and NVTs area from Aggf1 fl/fl and Cdh5-Cre Aggf1 fl/fl normoxic and OIR mice at P12 ( a , d , g ) ( n = 10 Aggf1 fl/fl and 8 Cdh5-Cre Aggf1 fl/fl OIR mice), P17 ( b , e , h ) ( n = 9 Aggf1 fl/fl and 12 Cdh5-Cre Aggf1 fl/fl OIR mice) and P21 ( c , f , i ) ( n = 5 Aggf1 fl/fl and 4 Cdh5-Cre Aggf1 fl/fl OIR mice). In the inserts, the red line represents the edge of the retina, the blue area represents the avascular area, and the red area represents NVTs. j TER119-positive RBC leakage in Aggf1 fl/fl and Cdh5-Cre Aggf1 fl/fl OIR mice are shown ( n = 5 mice per group). k F4/80-positive macrophage infiltration in Aggf1 fl/fl and Cdh5-Cre Aggf1 fl/fl OIR mice are shown ( n = 5 mice per group). Error bars represent mean ± SEM. 2-tailed unpaired Student’s t test ( g , h , i ). Scale bars: 1000 μm ( a – f ) and 100 μm ( j , k ). Source data are provided as a Source Data file.

Article Snippet: Then, isolectin B4 (1:200, Invitrogen, I21411) staining with different primary antibodies against AGGF1 (1:200, Novus, NB100-455), GFAP (1:200, Proteintech, 60190-1-Ig), Iba-1 (1:200, Abcam, ab283319), TER119 (1:100, Invitrogen, 14-5921-81), and F4/80 (1:200, Proteintech, 28463-1-AP) was performed overnight at 4 °C.

Techniques: Staining

Journal: Nature Communications

Article Title: Endothelial AGGF1 promotes retinal angiogenesis by coordinating TNFSF12/FN14 signalling

doi: 10.1038/s41467-025-55970-3

Figure Lengend Snippet: a Heat maps from proteomic array analysis with ranked enriched genes between control and AGGF1 -silenced HRMECs treated with HG (fold-change >1.2 or <0.83, p < 0.05). High and low expression are denoted by red and navy, respectively ( n = 3 independent cultures). b , c GO terms and KEGG pathway analysis of the differentially expressed genes between control and AGGF1 -silenced HRMECs treated with normal glucose and HG. Statistical analysis was performed using Fisher exact test. d , e Western blot analysis and quantification of AGGF1, CyclinA2, CyclinD1 and CDK1 protein levels in control and AGGF1 -silenced HRMECs treated with HG ( n = 6 independent cultures). f Cell proliferation was performed by EdU staining of control and AGGF1 -silenced HRMECs treated with HG ( n = 3 independent cultures). g , h Representative images and quantification of control and AGGF1 -silenced HRMECs treated with HG in wound healing assay at 0 and 24 h ( n = 3 independent cultures). i Representative images of angiogenesis in control and AGGF1 -silenced HRMECs treated with HG, measured using in vitro tube formation assays ( n = 3 independent cultures). Error bars represent mean ± SEM. 2-tailed unpaired Student’s t test ( a , e , h ). Scale bars: 100 μm ( f , i ), 1000 μm ( g ). HG high glucose (33.3 mmol/L). Source data are provided as a Source Data file.

Article Snippet: Then, isolectin B4 (1:200, Invitrogen, I21411) staining with different primary antibodies against AGGF1 (1:200, Novus, NB100-455), GFAP (1:200, Proteintech, 60190-1-Ig), Iba-1 (1:200, Abcam, ab283319), TER119 (1:100, Invitrogen, 14-5921-81), and F4/80 (1:200, Proteintech, 28463-1-AP) was performed overnight at 4 °C.

Techniques: Control, Expressing, Western Blot, Staining, Wound Healing Assay, In Vitro

Journal: Nature Communications

Article Title: Endothelial AGGF1 promotes retinal angiogenesis by coordinating TNFSF12/FN14 signalling

doi: 10.1038/s41467-025-55970-3

Figure Lengend Snippet: a AGGF1 protein interaction network analysed by STRING (string-db.org). b Immunoprecipitated (IP) AGGF1 was immunoblotted (IB) with AGGF1 or TNFSF12 antibody in HRMECs. c , d Western blot analysis and quantification of TNFSF12, CyclinA2, CyclinD1, and CDK1 protein levels in control and TNFSF12 -silenced HRMECs treated with HG ( n = 4 independent cultures). e , f Representative images and quantification of control and TNFSF12 -silenced HRMECs treated with HG (Scratching wound healing assay) ( n = 6 independent cultures). g Representative images of control and TNFSF12 -silenced HRMECs treated with HG (EdU staining) ( n = 3 independent cultures). h Western blot analysis of CyclinA2, CyclinD1, CDK1, and TNFSF12 protein levels in blank control and TNFSF12 -plasmid HRMECs ( n = 6 independent cultures). i Western blot analysis of CyclinA2, CyclinD1, CDK1, AGGF1, and TNFSF12 protein levels in blank control and AGGF1 + TNFSF12 -plasmid HRMECs ( n = 4 independent cultures). j Representative images of blank control and AGGF1 + TNFSF12 -plasmid HRMECs (EdU staining) ( n = 3 independent cultures). k TNFSF12 protein interaction network analysed by STRING (string-db.org). l Immunoprecipitated (IP) FN14 immunoblotted (IB) with AGGF1 or TNFSF12 antibody in HRMECs. m Immunoprecipitated (IP) FN14 immunoblotted (IB) with TNFSF12 or FN14 antibody in HRMECs treated with HG. n Schematic illustration of AGGF1 promoting the binding of TNFSF12 to Fn14 to increase angiogenesis. Error bars represent mean ± SEM. one-way ANOVA with Tukey’s multiple comparisons test ( d , f ). Scale bars: 1000 μm ( e ) and 100 μm ( g , j ). HG, high glucose (33.3 mmol/L). Source data are provided as a Source Data file.

Article Snippet: Then, isolectin B4 (1:200, Invitrogen, I21411) staining with different primary antibodies against AGGF1 (1:200, Novus, NB100-455), GFAP (1:200, Proteintech, 60190-1-Ig), Iba-1 (1:200, Abcam, ab283319), TER119 (1:100, Invitrogen, 14-5921-81), and F4/80 (1:200, Proteintech, 28463-1-AP) was performed overnight at 4 °C.

Techniques: Immunoprecipitation, Western Blot, Control, Wound Healing Assay, Staining, Plasmid Preparation, Binding Assay

Journal: Nature Communications

Article Title: Endothelial AGGF1 promotes retinal angiogenesis by coordinating TNFSF12/FN14 signalling

doi: 10.1038/s41467-025-55970-3

Figure Lengend Snippet: a Schematic illustration of the OIR mice treated with intravitreal injections. At P13, mouse pups were intravitreally injected with 2.5 μL anti-IgG, anti-AGGF1, anti-TNFSF12, anti-AGGF1 + TNFSF12, anti-VEGF, or anti-AGGF1 + VEGF antibodies. Retinas were analysed at P17. b – d FITC-dextran staining of whole-mount retinas from OIR mice injected with anti-IgG ( n = 6 mice), or anti-AGGF1 antibody ( n = 5 mice), or anti-TNFSF12 antibody ( n = 4 mice), or anti-AGGF1 + TNFSF12 antibody ( n = 7 mice), or anti-VEGF antibody ( n = 6 mice), or anti-AGGF1 + VEGF antibody ( n = 11 mice). In the inserts, the red line represents the edge of the retina, the blue area represents the avascular area, and the red area represents NVTs. c , d Quantification of the avascular and NVT areas at P18 in OIR mice, related to B. Error bars represent mean ± SEM. one-way ANOVA with Tukey’s multiple comparisons test ( c , d ). Scale bars: 1000 μm ( b ). Source data are provided as a Source Data file.

Article Snippet: Then, isolectin B4 (1:200, Invitrogen, I21411) staining with different primary antibodies against AGGF1 (1:200, Novus, NB100-455), GFAP (1:200, Proteintech, 60190-1-Ig), Iba-1 (1:200, Abcam, ab283319), TER119 (1:100, Invitrogen, 14-5921-81), and F4/80 (1:200, Proteintech, 28463-1-AP) was performed overnight at 4 °C.

Techniques: Injection, Staining

Journal: Nature Communications

Article Title: Endothelial AGGF1 promotes retinal angiogenesis by coordinating TNFSF12/FN14 signalling

doi: 10.1038/s41467-025-55970-3

Figure Lengend Snippet: a , b Fundus and OCT cross-sectional structural images and quantification of the retinal thickness measured by OCT in db/db mice compared to those in age-matched db/db+SGLT2i (Dapagliflozin, 1.5 mg/kg/d) mice ( n = 6 mice per group). c Paraffin HE staining for retinal thickness in db/db mice and db/db+SGLT2i mice ( n = 4 mice per group). d PAS staining of retinal trypsin digestion visualizing retinal vasculature and acellular capillaries (white arrowheads) in db/db mice and db/db+SGLT2i mice ( n = 5 mice per group). e Heat maps from proteomic array analysis with ranked enriched genes in 3 db/db mice and 3 db/db+SGLT2i mice retinas (fold-change >1.2 or <0.83, p < 0.05). High and low expression are denoted by red and navy, respectively. f Western blot analysis and quantification of AGGF1 protein levels in the retinas of db/db and db/db+SGLT2i mice ( n = 6 mice per group). g Immunohistochemical detection of AGGF1 expression in db/db and db/db+SGLT2i mice ( n = 3 mice per group). h , i Western blot analysis and quant i fication of AGGF1, CyclinA2, CyclinD1 and CDK1 protein levels in the retinas of db/db and db/db+SGLT2i mice ( n = 6 mice per group). j , k Western blot analysis and quantification of AGGF1, CyclinA2, CyclinD1 and CDK1 protein levels in NG, MA, HG and HG+SGLT2i HRMECs ( n = 3 independent cultures). l Representative images of HRMECs treated with HG and HG+SGLT2i was performed by EdU staining to reflect cell proliferation ( n = 3 mice per group). m , n Representative images and qua n tification of HRMECs treated with HG and HG+SGLT2i in wound healing assay at 0 and 24 h ( n = 6 independent cultures). Error bars represent mean ± SEM. 2-tailed unpaired Student’s t tests ( b , f , i , k ). Scale bars: 50 μm ( c , d and g ); 100 μm ( l ), 1000 μm ( m ). NG normal glucose (5.5 mmol/L glucose), MA mannitol (5.5mmol/L glucose+ 27.8 mmol/L mannitol), HG high glucose (33.3 mmol/L glucose), SGLT2i SGLT2 inhibitors (Dapagliflozin), GCL ganglion cell layer, INL inner nuclear layer, ONL outer nuclear layer. Source data are provided as a Source Data file.

Article Snippet: Then, isolectin B4 (1:200, Invitrogen, I21411) staining with different primary antibodies against AGGF1 (1:200, Novus, NB100-455), GFAP (1:200, Proteintech, 60190-1-Ig), Iba-1 (1:200, Abcam, ab283319), TER119 (1:100, Invitrogen, 14-5921-81), and F4/80 (1:200, Proteintech, 28463-1-AP) was performed overnight at 4 °C.

Techniques: Staining, Expressing, Western Blot, Immunohistochemical staining, Wound Healing Assay

Journal: Nature Communications

Article Title: Endothelial AGGF1 promotes retinal angiogenesis by coordinating TNFSF12/FN14 signalling

doi: 10.1038/s41467-025-55970-3

Figure Lengend Snippet: Endothelial AGGF1 deficiency ameliorates pathological angiogenesis in an OIR model. Mechanistically, AGGF1 expression in HRMECs is regulated by retinal hypoxia caused by pathological changes. Our study also suggests an action model through which AGGF1 promotes angiogenesis by activating the cell cycle via the AGGF1/TNFSF12/FN14 signalling pathway. Thus, we speculate that targeting the AGGF1/TNFSF12/FN14 signalling pathway could provide a therapy for the treatment of ischaemic retinopathy.

Article Snippet: Then, isolectin B4 (1:200, Invitrogen, I21411) staining with different primary antibodies against AGGF1 (1:200, Novus, NB100-455), GFAP (1:200, Proteintech, 60190-1-Ig), Iba-1 (1:200, Abcam, ab283319), TER119 (1:100, Invitrogen, 14-5921-81), and F4/80 (1:200, Proteintech, 28463-1-AP) was performed overnight at 4 °C.

Techniques: Expressing

Journal:

Article Title: Identification of an angiogenic factor that when mutated causes susceptibility to Klippel-Trenaunay syndrome

doi: 10.1038/nature02320.

Figure Lengend Snippet: Positional cloning of the VG5Q gene. a, Definition of chromosome breakpoints involved in translocation t(5;11)(q13.3;q15.1) associated with KTS. Chr 5/Chr 11, normal chromosomes; Der 5/Der 11, derivative chromosomes. b, The amino acid sequences of human VG5Q protein. The FHA domain (435–508) and a G-patch domain (619–663) are indicated. The estimated pI of VG5Q is 5.2. c, d, Identification of VG5Q mutation E133K in five independent KTS patients. c, DNA sequence analysis (G to A substitution at codon 133). d, Schematic representation of mutation E113K (substitution of a glutamic acid residue by a lysine residue).

Article Snippet: HUVECs were plated on Lab-Tek II chamber slides coated with or without matrigel ( in vitro angiogenesis) for 4 h. The media were collected, incubated for 30 min with an optimum concentration (200 ng ml −1 ) of anti-VG5Q antibody (determined experimentally with antigen), and transferred to wells coated with peptide immunogen (1μg ml −1 , 6 replicates).

Techniques: Cloning, Translocation Assay, Mutagenesis, Sequencing, Residue

Journal:

Article Title: Identification of an angiogenic factor that when mutated causes susceptibility to Klippel-Trenaunay syndrome

doi: 10.1038/nature02320.

Figure Lengend Snippet: Expression profile of VG5Q and dynamic redistribution and secretion of VG5Q protein during angiogenesis. A, B, VG5Q expression in HMVECs and HUVECs determined by northern (A) and western blot analyses (B). C, Tissue expression pattern of VG5Q determined by northern blot analysis. D, Expression of VG5Q in different cell lines determined by RT–PCR. Tubulin, internal control. E, Immunostaining of mouse kidney for VG5Q protein expression. The nucleus was stained with DAPI (blue signal). CD31, endothelial cell marker; α-actin, VSMC marker. F, HMVECs were cultured on plastic (a, b, g) and matrigel (c–f, h). VG5Q protein, red; nucleus, blue (DAPI). c, d, f, One hour on matrigel; e, 4 h on matrigel; h, 24 h on matrigel. G, Competitive ELISA analysis to show that VG5Q is secreted. Relative HRP activity, absorbance reading of the wells. An asterisk indicates statistical significance (P < 0.05). H, Detection of VG5Q in the media by metabolic labelling and immunoprecipitation, indicating that cells secrete VG5Q into the media. Control, HUVECs transfected with pcDNA3.1; plastic and matrigel, HUVECs transfected with pcDNA3.1-VG5Q and plated on either plastic or matrigel plates, respectively. The 84-kDa band indicated by an arrow represents VG5Q. The nature of the small 45-kDa band is unknown, but may be a cleaved VG5Q product. Faint signal was also observed in the media from cells cultured on plastic dishes. This may suggest weak secretion of VG5Q under this condition.

Article Snippet: HUVECs were plated on Lab-Tek II chamber slides coated with or without matrigel ( in vitro angiogenesis) for 4 h. The media were collected, incubated for 30 min with an optimum concentration (200 ng ml −1 ) of anti-VG5Q antibody (determined experimentally with antigen), and transferred to wells coated with peptide immunogen (1μg ml −1 , 6 replicates).

Techniques: Expressing, Northern Blot, Western Blot, Reverse Transcription Polymerase Chain Reaction, Control, Immunostaining, Staining, Marker, Cell Culture, Competitive ELISA, Activity Assay, Metabolic Labelling, Immunoprecipitation, Transfection

Journal:

Article Title: Identification of an angiogenic factor that when mutated causes susceptibility to Klippel-Trenaunay syndrome

doi: 10.1038/nature02320.

Figure Lengend Snippet: VG5Q is an angiogenic factor and both VG5Q E133K and KTS translocation t(5;11) are functional mutations. A, Angiogenesis promoted by VG5Q protein was determined by CAM assays. EK, VG5Q with mutation E133K. B, Effect of siRNA against VG5Q on endothelial tube formation. Scramble, control siRNA. Suppression of VG5Q expression by siRNAs was determined by RT–PCR (ribosomal RNA as control) and western blot analyses (tubulin as control). C, Binding of VG5Q to endothelial cells by cell adhesion assays. D, The t(5;11)(q13.3;p15.1) translocation associated with KTS increases expression of VG5Q. Results represent mean of triplicate cultures ± standard deviation from three independent experiments. EC, endothelial cell.

Article Snippet: HUVECs were plated on Lab-Tek II chamber slides coated with or without matrigel ( in vitro angiogenesis) for 4 h. The media were collected, incubated for 30 min with an optimum concentration (200 ng ml −1 ) of anti-VG5Q antibody (determined experimentally with antigen), and transferred to wells coated with peptide immunogen (1μg ml −1 , 6 replicates).

Techniques: Translocation Assay, Functional Assay, Mutagenesis, Control, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Binding Assay, Standard Deviation

Journal:

Article Title: Identification of an angiogenic factor that when mutated causes susceptibility to Klippel-Trenaunay syndrome

doi: 10.1038/nature02320.

Figure Lengend Snippet: VG5Q interacts with TWEAK. a, Pull-down assays using GST–TWEAK. Input, 35S-labelled luciferase (Luc, about 61 kDa) and VG5Q (about 84 kDa). Right panel, binding of VG5Q with GST–TWEAK, but not with GST; middle panel, no interaction between GST–TWEAK with luciferase. b, Co-immunoprecipitation of TWEAK with VG5Q from HVSMC protein extract by a rabbit anti-VG5Q antibody. Bound materials were analysed by western blot using a goat anti-TWEAK antibody. NRS, normal rabbit serum as a negative control. 50-kDa band, IgG cross-reaction. c, Co-localization of VG5Q and TWEAK in HUVECs. VG5Q, red signal; TWEAK, green; nuclei, blue (DAPI).

Article Snippet: HUVECs were plated on Lab-Tek II chamber slides coated with or without matrigel ( in vitro angiogenesis) for 4 h. The media were collected, incubated for 30 min with an optimum concentration (200 ng ml −1 ) of anti-VG5Q antibody (determined experimentally with antigen), and transferred to wells coated with peptide immunogen (1μg ml −1 , 6 replicates).

Techniques: Luciferase, Binding Assay, Immunoprecipitation, Western Blot, Negative Control