Journal: Frontiers in Pharmacology

Article Title: Synergistic therapy for diabetic retinopathy via dual blockade of VEGF-A and TNF-α with a bispecific nanobody

doi: 10.3389/fphar.2025.1729606

Figure Lengend Snippet: Design, simulation and generation of bispecific nanobody Nb-TV. (A) Schematic diagram of recombinant plasmid of Nb-TV. (B) Structure diagram of Nb-TV. Green: Nb-TNFα. Blue: Nb-VEGFA. (C) Schematic diagram of Nb-TV in combination with VEGF or TNF-α. The online tool HDOCK was used for docking. (D) Schematic diagram of Nb-TV in combination with VEGF and TNF-α simultaneously. (E) Purification of TNF-α, VEGF-A, Nb-TNFα, Nb-VEGFA and Nb-TV. TNF-α: 18.5 kDa. VEGF-A: 20.4 kDa. Nb-TNFα: 15.9 kDa. Nb-VEGFA: 14.9 kDa. Nb-TV: 30.6 kDa.

Article Snippet: The Anti-TNF-alpha Rabbit pAb (Cat #GB11188, Servicebio, Wuhan, China) and recombinant Anti-VEGFA antibody (Rabbit mAb) (Cat #GB15165, Servicebio, Wuhan, China), which were diluted in PBS, were added and incubated overnight at 4 °C.

Techniques: Recombinant, Plasmid Preparation, Purification

Journal: Frontiers in Pharmacology

Article Title: Synergistic therapy for diabetic retinopathy via dual blockade of VEGF-A and TNF-α with a bispecific nanobody

doi: 10.3389/fphar.2025.1729606

Figure Lengend Snippet: The binding activity of Nb-TV to TNF-α and VEGF-A was detected by ELISA. (A) Binding activity of Nb-TV and Nb-TNFα to TNF-α. EC 50 (Nb-TNFα, TNF-α) = 0.619 μM, EC 50 (Nb-TV, TNF-α) = 0.596 μM. (B) Binding activity of Nb-TV and Nb-VEGFA to VEGF-A. EC 50 (Nb-VEGFA, VEGF-A) = 0.333 μM, EC 50 (Nb-TV, VEGF-A) = 0.283 mM.

Article Snippet: The Anti-TNF-alpha Rabbit pAb (Cat #GB11188, Servicebio, Wuhan, China) and recombinant Anti-VEGFA antibody (Rabbit mAb) (Cat #GB15165, Servicebio, Wuhan, China), which were diluted in PBS, were added and incubated overnight at 4 °C.

Techniques: Binding Assay, Activity Assay, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Pharmacology

Article Title: Synergistic therapy for diabetic retinopathy via dual blockade of VEGF-A and TNF-α with a bispecific nanobody

doi: 10.3389/fphar.2025.1729606

Figure Lengend Snippet: Nb-TV inhibits TNF-α-induced inflammation in ARPE-19 cells. (A–C) MTT assay detected the antagonism of Nb-VEGFA, Nb-TNFα and Nb-TV to TNF-α in ARPE-19 ( n = 5). (D,E) Immunofluorescence detected the inhibitory effect of Nb-TV, Nb-TNFα and Nb-VEGFA on the nuclear translocation of p65 in ARPE-19 induced by TNF-α. Green: p65; blue: DAPI ( n = 3, Scale = 50 μm). (F) qPCR detected the relative change of IL-6, IL-8 and MCP-1 mRNA expression in TNF-α-induced ARPE-19 cells treated with Nb-TV and Nb-VEGFA ( n = 5). (G,H) Western blot detected the protein expression level of IL-6, IL-8 and MCP-1 in TNF-α-induced ARPE-19 treated with Nb-TV, Nb-TNFα and Nb-VEGFA ( n = 3). (I,J) Western blot detected the protein expression level of Caspase-1 in TNF-α-induced ARPE-19 treated with Nb-TV, Nb-TNFα and Nb-VEGFA ( n = 3). Values are presented as mean ± SD; * P < 0.05, ** P < 0.01, *** P < 0.001 compared with Control group; # P < 0.05, ## P < 0.01, ### P < 0.001 compared with TNF-α group.

Article Snippet: The Anti-TNF-alpha Rabbit pAb (Cat #GB11188, Servicebio, Wuhan, China) and recombinant Anti-VEGFA antibody (Rabbit mAb) (Cat #GB15165, Servicebio, Wuhan, China), which were diluted in PBS, were added and incubated overnight at 4 °C.

Techniques: MTT Assay, Immunofluorescence, Translocation Assay, Expressing, Western Blot, Control

Journal: Frontiers in Pharmacology

Article Title: Synergistic therapy for diabetic retinopathy via dual blockade of VEGF-A and TNF-α with a bispecific nanobody

doi: 10.3389/fphar.2025.1729606

Figure Lengend Snippet: Nb-TV inhibits VEGF-A-induced HUVEC hyperproliferation and hypermigration. (A–C) MTT assay detected the antagonism of Nb-TNFα, Nb- VEGFA and Nb-TV to VEGF-A in HUVEC ( n = 5). (D,E) Western blot detected the protein expression level of p-Erk1/2 and Erk1/2 in VEGF-A -induced HUVEC treated with Nb-TV, Nb-TNFα and Nb-VEGFA ( n = 3). (F,G) Immunofluorescence detected the inhibitory effect of Nb-TV, Nb-TNFα and Nb-VEGFA on the nuclear translocation of p38α in HUVEC induced by VEGF-A. Green: p38α; blue: DAPI ( n = 3, Scale = 50 μm). (H,I) Scratch test detected the migration ability of VEGF-A -induced HUVEC treated with Nb-TV, Nb-TNFα and Nb-VEGFA ( n = 3, Scale = 100 μm). Values are presented as mean ± SD; * P < 0.05, ** P < 0.01, *** P < 0.001 compared with Control group; ## P < 0.01, ### P < 0.001, #### P < 0.0001 compared with VEGF-A group.

Article Snippet: The Anti-TNF-alpha Rabbit pAb (Cat #GB11188, Servicebio, Wuhan, China) and recombinant Anti-VEGFA antibody (Rabbit mAb) (Cat #GB15165, Servicebio, Wuhan, China), which were diluted in PBS, were added and incubated overnight at 4 °C.

Techniques: MTT Assay, Western Blot, Expressing, Immunofluorescence, Translocation Assay, Migration, Control

Journal: Frontiers in Pharmacology

Article Title: Synergistic therapy for diabetic retinopathy via dual blockade of VEGF-A and TNF-α with a bispecific nanobody

doi: 10.3389/fphar.2025.1729606

Figure Lengend Snippet: Nb-TV inhibits VEGF-A-induced angiogenesis of HUVEC in vitro and reduces vascular permeability. (A–D) Tube formation assay detected the angiogenesis ability of VEGF-A-induced HUVEC treated with Nb-TV, Nb-TNFα and Nb-VEGFA ( n = 3, Scale = 100 μm). (E,F) Immunofluorescence detected the inhibitory effect of Nb-TV, Nb-TNFα and Nb-VEGFA on the iNOS expression in HUVEC induced by VEGF-A. Green: iNOS; blue: DAPI ( n = 3, Scale = 50 μm). Values are presented as mean ± SD; * P < 0.05, ** P < 0.01, compared with Control group; ## P < 0.01, ### P < 0.001, compared with VEGF-A group.

Article Snippet: The Anti-TNF-alpha Rabbit pAb (Cat #GB11188, Servicebio, Wuhan, China) and recombinant Anti-VEGFA antibody (Rabbit mAb) (Cat #GB15165, Servicebio, Wuhan, China), which were diluted in PBS, were added and incubated overnight at 4 °C.

Techniques: In Vitro, Permeability, Tube Formation Assay, Immunofluorescence, Expressing, Control

Journal: Frontiers in Pharmacology

Article Title: Synergistic therapy for diabetic retinopathy via dual blockade of VEGF-A and TNF-α with a bispecific nanobody

doi: 10.3389/fphar.2025.1729606

Figure Lengend Snippet: Effects of Nb-TV improved retinopathy in DR mice. (A) Schematic diagram of the establishment of the STZ - induced diabetic retinopathy model in mice and the intervention of Nb-TV, Nb-TNFα and Nb-VEGFA. (B) Fasting blood glucose, diet, and body weight of mice in the control group and DR group after the injection of STZ. (C,D) Immunofluorescence detected the inhibitory effect of Nb-TV, Nb-TNFα and Nb-VEGFA on TNF-α and VEGF-A in the retinas of DR mice. Green: TNF-α; red: VEGF-A; blue: DAPI ( n = 6, Scale = 50 μm). (E,F) PAS staining of retinal vascular network flat-mounts detected the improving effect of Nb-TV, Nb-TNFα and Nb-VEGFA on retinal angiogenesis in DR mice ( n = 6, Scale = 100 μm). (G) HE staining detected the improving effect of Nb-TV, Nb-TNFα and Nb-VEGFA on retinal pathological changes in DR mice ( n = 6, Scale = 50 μm). Values are presented as mean ± SD; * P < 0.05, ** P < 0.01, *** P < 0.001 compared with Control group; # P < 0.05, ## P < 0.01, ### P < 0.001, compared with DR group.

Article Snippet: The Anti-TNF-alpha Rabbit pAb (Cat #GB11188, Servicebio, Wuhan, China) and recombinant Anti-VEGFA antibody (Rabbit mAb) (Cat #GB15165, Servicebio, Wuhan, China), which were diluted in PBS, were added and incubated overnight at 4 °C.

Techniques: Control, Injection, Immunofluorescence, Staining

Journal: Development (Cambridge, England)

Article Title: Angioblast-mesenchyme induction of early kidney development is mediated by Wt1 and Vegfa.

doi: 10.1242/dev.02095

Figure Lengend Snippet: Fig. 2. Injection of the Wt1 expression vector. (A) Effect on branching morphogenesis. (a,c,d) Injection of Wt1 cDNA. (b,e) Injection of GFP vector. In situ hybridization with Wt1 antisense probe (a,b), or sense probe (c). The strong area of hybridization is at the single injection site (a), and the endogenous Wt1 signal is observed at several induced nephrons (a,b). (d,e) Cytokeratin staining demonstrating effect of Wt1 on branching. In experiments designed to observe an effect on branching, the vector is injected at two sites, one on either side of the ureteric bud. The branching comparisons shown in each figure are between organ cultures obtained from the same embryo. (f) Overall quantitation of 12 pairs of experiments, represented by d,e. Significant differences in branching were detected after Wt1 injection (P<0.001). (B) Wt1 stimulation of Vegfa expression. Injection of Wt1 (a,c) and Gfp (b) expression vectors. In situ hybridization with Vegfa antisense (a,b) and sense (c) probes. (d) Real-time PCR with Vegfa primers in response to microinjections. As described in the text, a Gfp expression vector was co-injected, and areas of Gfp expression were separated from the remainder of the organ culture. The expression vectors used are shown at the bottom of each column. Increased Vegfa mRNA is observed in response to injection of a Wt1 expression vector. All Vegfa Ct values were normalized to the Gapdh Ct value from real-time PCR for the injected tissue.

Article Snippet: Recombinant mouse Vegfa (#493-MV), recombinant rat Gdnf (#512- GF) and goat polyclonal anti-Vegfa antibody (#AF-493-NA) were purchased from R&D Systems (Minneapolis, MN); rat polyclonal anti-Flk1 antibody (#550549) for immunostaining was purchased from BD Biosciences (San Diego, CA); rat polyclonal anti-Flk1 antibody (clone DC101) was obtained from ImClone Inc. (New York, NY); mouse polyclonal anti-pan-cytokeratin antibody was purchased from Sigma (St Louis, MO); rabbit polyclonal anti-Pax2 antibody (#71-600) was purchased from Zymed (South San Francisco, CA) or obtained from Dr Greg Dressler, (University of Michigan); rabbit polyclonal anti-Pecam antibody (H-300)), goat polyclonal anti-Pecam antibody (M-20) and rat-IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); rabbit polyclonal anti-Brush Border antibody (Miettinen and Linder, 1976) was obtained from Hannu Sariola (University of Helsinki).

Techniques: Injection, Expressing, Plasmid Preparation, In Situ Hybridization, Hybridization, Staining, Quantitation Assay, Real-time Polymerase Chain Reaction, Organ Culture

Journal: Development (Cambridge, England)

Article Title: Angioblast-mesenchyme induction of early kidney development is mediated by Wt1 and Vegfa.

doi: 10.1242/dev.02095

Figure Lengend Snippet: Fig. 3. (A) Vegfa expression in early kidney development compared to Wt1. (a,b) E12.5 kidneys, fixed immediately after dissection and sectioned. (c,d) E11.5 metanephric kidney rudiments placed in culture for 48 hours. (a,c) Vegfa antisense probe, (b) Wt1 antisense probe and (d) Vegfa sense probe. Clear Vegfa expression is present in the condensed mesenchyme of the E12.5 kidney (a) overlapping significantly with Wt1 expression at the same timepoint (b). (B) Vegfa stimulation of branching and tubulogenesis. (a) Control PBS addition, (b) Vegfa addition, Cytokeratin stain red, proximal tubule-binding lectin green. (c) Terminal branch tip (P<0.001) and (d) proximal tubule (P<0.001) quantitation showing results of four pairs of organ cultures represented by (a) and (b), which are from the same embryo. (C) Antibody inhibition of branching. (a) Control organ culture treated with rabbit IgG. (b) Organ culture from the same embryo as (a) treated with anti-Vegfa neutralizing antibody. (c,d) Quantitation of four pairs of organ cultures treated as in (a) and (b); (c) terminal branch tips (P<0.001); (d) tubules as identified by lectin staining for proximal tubules (P<0.001).

Article Snippet: Recombinant mouse Vegfa (#493-MV), recombinant rat Gdnf (#512- GF) and goat polyclonal anti-Vegfa antibody (#AF-493-NA) were purchased from R&D Systems (Minneapolis, MN); rat polyclonal anti-Flk1 antibody (#550549) for immunostaining was purchased from BD Biosciences (San Diego, CA); rat polyclonal anti-Flk1 antibody (clone DC101) was obtained from ImClone Inc. (New York, NY); mouse polyclonal anti-pan-cytokeratin antibody was purchased from Sigma (St Louis, MO); rabbit polyclonal anti-Pax2 antibody (#71-600) was purchased from Zymed (South San Francisco, CA) or obtained from Dr Greg Dressler, (University of Michigan); rabbit polyclonal anti-Pecam antibody (H-300)), goat polyclonal anti-Pecam antibody (M-20) and rat-IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); rabbit polyclonal anti-Brush Border antibody (Miettinen and Linder, 1976) was obtained from Hannu Sariola (University of Helsinki).

Techniques: Expressing, Dissection, Control, Staining, Binding Assay, Quantitation Assay, Inhibition, Organ Culture

Journal: Development (Cambridge, England)

Article Title: Angioblast-mesenchyme induction of early kidney development is mediated by Wt1 and Vegfa.

doi: 10.1242/dev.02095

Figure Lengend Snippet: Fig. 6. (A) Flk1-dependent Vegfa induction of Pax2. The injection is noted at the top of each panel, and the antibody treatment and hybridization probes are noted at the bottom of each panel. Gfp (a) or Vegfa (b-e) expression vectors were injected at the edge of the condensed mesenchyme. Rat IgG (d) or Flk1-blocking antibody (e) were added at the time of injection. Six hours after injection, cultures were analyzed by in situ hybridization with Pax2 antisense (a,b,d,e) or sense (c) probe. (B) Flk1-dependent Pax2 induction of Gdnf expression. Panels are labeled as in A. Pax2 expression (a,b.d,f) or control (c,d,g,h) vectors were injected and Gdnf antisense probe was used in all panels. (C) Real- time PCR analysis of Pax2 mRNA expression level in organ cultures after Flk1 blockade or treatment with actinomycin-D. Treatments are shown in the figure box. SU1498, actinomycin-D, SU1498 with actinomycin-D or DMSO, were added to the culture medium at the beginning of the culture period, (time 0). At 0, 1.5, 3, 4.5, 6, or 7.5 hours after beginning cultures, real-time PCR was performed to measure levels of Pax2 mRNA. All Pax2 Ct values were normalized to the Gapdh Ct values and converted to relative mRNA levels as described in the methods. Relative RNA levels are shown on a log scale. (D) Flk1 dependency of Pax2 expression. Panels a-h were injected with the Pax2 expression vector. The hybridization probes are noted at the bottom of each panel (a-d), and the antibody treatment is noted at the top (a-d) or bottom (e-h) of each panel. (a,e) Rat IgG; (b,f) Flk1-blocking antibody; (c,g) DMSO 1:1000; (d,h) SU1498 treatment. (a-d) In situ hybridization with Pax2 antisense probe; (e-h) staining with anti-Pax2 antibody. Pax2 mRNA or protein expression in response to injection of the Pax2 expression vector could be blocked by antibody or pharmacological inhibition of signaling through Flk1. (i-l) Injections of the GFP expression vector, detecting direct GFP fluorescence.

Article Snippet: Recombinant mouse Vegfa (#493-MV), recombinant rat Gdnf (#512- GF) and goat polyclonal anti-Vegfa antibody (#AF-493-NA) were purchased from R&D Systems (Minneapolis, MN); rat polyclonal anti-Flk1 antibody (#550549) for immunostaining was purchased from BD Biosciences (San Diego, CA); rat polyclonal anti-Flk1 antibody (clone DC101) was obtained from ImClone Inc. (New York, NY); mouse polyclonal anti-pan-cytokeratin antibody was purchased from Sigma (St Louis, MO); rabbit polyclonal anti-Pax2 antibody (#71-600) was purchased from Zymed (South San Francisco, CA) or obtained from Dr Greg Dressler, (University of Michigan); rabbit polyclonal anti-Pecam antibody (H-300)), goat polyclonal anti-Pecam antibody (M-20) and rat-IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); rabbit polyclonal anti-Brush Border antibody (Miettinen and Linder, 1976) was obtained from Hannu Sariola (University of Helsinki).

Techniques: Injection, Hybridization, Expressing, Blocking Assay, In Situ Hybridization, Labeling, Control, Real-time Polymerase Chain Reaction, Plasmid Preparation, Staining, Inhibition

Journal: iScience

Article Title: Revisiting the effects of exercise on cerebral neurovascular functions in rats using multimodal assessment techniques

doi: 10.1016/j.isci.2023.106354

Figure Lengend Snippet: Key resources table

Article Snippet: Rabbit polyclonal anti-VEGFA , Elabscience , Cat# E-AB-64001; RRID:N/A.

Techniques: Recombinant, Protease Inhibitor, Bicinchoninic Acid Protein Assay, Western Blot, Software