Journal: Angiogenesis

Article Title: Angiopoietin-2 binds to FGFR2, inhibits FGF-FGFR2 signaling, and delays cutaneous wound healing by inhibiting wound angiogenesis

doi: 10.1007/s10456-025-09988-2

Figure Lengend Snippet: Recombinant Ang2 binds to recombinant FGFR2-Fc. A Lysates prepared from HUVEC in starvation medium for 1 hour and then incubated with FGF1 (5 nM, 5 min) or/and Ang2 (5 nM, 5 min) in the same medium were analyzed by Western blotting; antibodies to p-FGFR Y653/654 and FGFR2 recognize a band at ~130 kDa. Representative of 3 experiments. B Schematic of the pull-down experiment. FGFR2: FGFR2ß (IIIb)-Fc. C Ang2 (0.1 μM) specifically binds to recombinant FGFR2ß (IIIb)-Fc (0.1-0.5 μM) but not to human IgG-Fc (Fc, 0.5 μM). The precipitated proteins were immunoblotted with antibodies to Ang2 (left) or to FGFR2ß (IIIb) (right). The asterisks point to bands specifically identifying Ang2 (left) and FGFR2 (right). Representative of 3 experiments. D Schematic of the pull-down experiment. FGFR2: FGFR2α (IIIc)-Fc. E Ang2 (0.1 μM) does not compete with the binding of FGF1 (0.1-0.4 μM) to FGFR2α (IIIc)-Fc (0.2 μM). IgG-Fc used at 0.2 mM. The precipitates were immunoblotted with antibodies to Ang2, FGF1, or anti-human Fc (hFc). The asterisks point to Ang2 (left), FGF1 (middle), and to FGFR2-Fc or Fc (right). Representative of 3 experiments. F Structural modeling of the Ang2, FGF1 and FGFR2α (IIIb) trimeric complex from amino acid sequences by AlphaFold2-Multimer. Red: predicted structure of Ang2 in the predicted trimeric complex. Pink: Ang2 structure from the crystal structure of Ang2 alone (PDB 1z3s). Blue: predicted structure of FGFR2α (IIIb). Light blue: FGFR2ß (IIIb) structure from the crystal structure of FGFR2ß (IIIb)-FGF1 dimeric complex (PDB 1djs). Yellow: predicted structure of FGF1. Green: FGF1 structure from the crystal structure of FGFR2ß (IIIb)-FGF1 complex (PDB 1djs). The three predicted protein structures are largely consistent when superimposed on the respective crystal structures. The predicted structure suggests that Ang2 and FGF1 interact with FGFR2 at distinct sites. G Structural models of the Ang2, FGF1 and FGFR2α (IIIc) trimeric complex (left) and the Ang2, FGF2 and FGFR2α (IIIc) trimeric complex (right) from amino acid sequences by AlphaFold3

Article Snippet: Cells were then incubated with 100–1000 ng/ml recombinant Ang2 (expressed with a C-terminal His-tag in CHO cells; Biolegend, No. 753106) or/and 3 or 5 nM FGF1 (R&D Systems, No. 232-FA) for 5 min, and/or 314.8 nM (10 μg/ml) AMG386 (obtained under material CRADA from Amgen).

Techniques: Recombinant, Incubation, Western Blot, Binding Assay

Journal: Angiogenesis

Article Title: Angiopoietin-2 binds to FGFR2, inhibits FGF-FGFR2 signaling, and delays cutaneous wound healing by inhibiting wound angiogenesis

doi: 10.1007/s10456-025-09988-2

Figure Lengend Snippet: The Ang2 inhibitor AMG386 reduces Ang2 binding to FGFR2-Fc. A Schematic of the pull-down experiment. FGFR2α (IIIc)-Fc. B AMG386 (AMG, 0.2 or 0.05 μM) inhibits the binding of Ang2 (0.1 μM) to FGFR2-Fc (0.2 μM). IgG-Fc (Fc, 0.2 μM). The precipitates were immunoblotted with antibodies to Fc and Ang2. The black asterisk points to FGFR2-Fc (left) and Ang2 (right); the red asterisk points to AMG386. Representative experiment (of three performed). C Overall complex structure of AMG386 (yellow) bound to Ang2 (blue), FGF1 (green), and FGFR2α (IIIb) (pink) is shown as a surface representation. AMG386 occupies the main binding cavity of the complex, highlighting its central role in coordinating interactions with the other proteins. D and E Trp 280 from AMG386 (yellow) forms hydrophobic interactions with nearby residues, including Tyr 135 and His 108 from FGFR2 (pink) and Phe 190 from Ang2 (blue). These interactions include π-stacking between Trp 280 (AMG386) and Phe 190 (Ang2), contributing to the stabilization of the complex. Additional π-π stacking interactions involving His 108 (FGFR2) and Phe 190 (Ang2) underline the importance of aromatic side chains in maintaining the structural integrity of the complex. F A hydrogen bond between Lys 176 (FGFR2, pink) and Glu 274 (AMG386, yellow) reinforces the binding interface. G Another hydrogen bond is observed between Glu 283 (AMG386, yellow) and Gly 200 (Ang2, blue), further enhancing the specificity and stability of the interaction

Article Snippet: Cells were then incubated with 100–1000 ng/ml recombinant Ang2 (expressed with a C-terminal His-tag in CHO cells; Biolegend, No. 753106) or/and 3 or 5 nM FGF1 (R&D Systems, No. 232-FA) for 5 min, and/or 314.8 nM (10 μg/ml) AMG386 (obtained under material CRADA from Amgen).

Techniques: Binding Assay

Journal: Angiogenesis

Article Title: Angiopoietin-2 binds to FGFR2, inhibits FGF-FGFR2 signaling, and delays cutaneous wound healing by inhibiting wound angiogenesis

doi: 10.1007/s10456-025-09988-2

Figure Lengend Snippet: Ang2 reduces FGF1-induced activation of FGFR, Erk1/2, STAT3 and AKT in HUVEC . A HUVEC were incubated in starvation medium only (None), with FGF1 (3 nM), with Ang2 (3 nM) or with Ang2 (3 nM) + FGF1 (3 nM) for the indicated times (min). Cell lysates were immunoblotted with the indicated antibodies. When FGF1 + Ang2 were added to the cells, the incubation time of Ang2 was 5 min and the incubation time of FGF1 was either 1, 2 or 5 minutes (displayed as 5/1, 5/2, 5/5). Representative results of 3 experiments. B HUVEC were incubated in starvation medium only (None), with FGF1 (3 nM), with Ang2 (3 nM) or with Ang2 (3 nM) + FGF1 (3 nM) for the indicated times (min). When Ang2 + FGF1 were added to the cells, the incubation time of Ang2 was 10 min and the incubation time of FGF1 was either 1, 2 or 5 minutes (displayed as 10/1, 10/2, 10/5). Representative of 3 experiments. C HUVEC were incubated in starvation medium only (None), with FGF1 (3 nM), FGF2 (3 nM), Ang2 (3 nM), Ang2 (3 nM) + FGF1 (3 nM) or with Ang2 (3 nM) + FGF2 (3 nM). When Ang2+FGF1 or Ang2+FGF2 were added together to the cells, the incubation time was either 2- or 5-min. Representative of 3 experiments. D Effects of AMG386 on p-Erk1/2 levels in HUVEC activated by FGF1 alone, Ang2 alone, or FGF1 + Ang2. HUVEC were cultured in starvation medium only (none), FGF1 only (5 nM; 5 min), Ang2 only (5 nM; 10 min) or FGF1 (5 nM; 5 min) + Ang2 (5 nM; 10 min). Where indicated, AMG386 (AMG, 315 nM) was added to HUVEC 60 min prior to the addition of FGF1 alone, Ang2 alone or Ang2 + FGF1. Relative band intensity (p-Erk/total Erk) is shown in the bar graph. Representative of 3 experiments

Article Snippet: Cells were then incubated with 100–1000 ng/ml recombinant Ang2 (expressed with a C-terminal His-tag in CHO cells; Biolegend, No. 753106) or/and 3 or 5 nM FGF1 (R&D Systems, No. 232-FA) for 5 min, and/or 314.8 nM (10 μg/ml) AMG386 (obtained under material CRADA from Amgen).

Techniques: Activation Assay, Incubation, Cell Culture

Journal: Angiogenesis

Article Title: Angiopoietin-2 binds to FGFR2, inhibits FGF-FGFR2 signaling, and delays cutaneous wound healing by inhibiting wound angiogenesis

doi: 10.1007/s10456-025-09988-2

Figure Lengend Snippet: Ang2 reduces p-FGFR, p-Erk1/2, p-STAT3 and p-AKT in HEK293T cells. A, B HEK293T cells were incubated in starvation medium only (None), with Ang2 (3 nM), with FGF1 (3 nM), or with Ang2 (3 nM) + FGF1 (3 nM) for the indicated times (min). Cell lysates were immunoblotted with the indicated antibodies. Representative results from 3 experiments. When FGF1 + Ang2 were added to the cells, the incubation time of FGF1 was 5 min and the incubation time of Ang2 is either 1, 2 or 5 minutes (displayed as 5/1, 5/2, 5/5). C HEK293T cells were incubated in starvation medium with FGF2 (3 nM) alone (2 or 5 min), or with Ang2 (3 nM), 5 min. The results show Ang2 + FGF2 for 5 min reduce FGF2-induced Erk1/2 activity (5 min). D Left panel: HEK293T cells were incubated in starvation medium only (None), FGF1 only (5 nM; 5 min), or with Ang2 (5 nM; 5 min) without or with AMG386 (AMG, 5 ng/ml; 1, 2, or 5 min). Right panel: HEK293T cells were incubated with medium only (None), FGF1 only (5 nM; 5 min), or FGF1 (5 nM; 5 min) + Ang2 (5 nM; 5 min), with or without AMG386 (5 ng/ml; 1, 2, or 5 min). Relative band intensity (p-Erk/total Erk) is shown in the bar graph. Representative of 3 experiments

Article Snippet: Cells were then incubated with 100–1000 ng/ml recombinant Ang2 (expressed with a C-terminal His-tag in CHO cells; Biolegend, No. 753106) or/and 3 or 5 nM FGF1 (R&D Systems, No. 232-FA) for 5 min, and/or 314.8 nM (10 μg/ml) AMG386 (obtained under material CRADA from Amgen).

Techniques: Incubation, Activity Assay

Journal: Angiogenesis

Article Title: Angiopoietin-2 binds to FGFR2, inhibits FGF-FGFR2 signaling, and delays cutaneous wound healing by inhibiting wound angiogenesis

doi: 10.1007/s10456-025-09988-2

Figure Lengend Snippet: Ang2 impairs endothelial cell migration induced by FGF1. A Effects of Ang2 (100 ng/ml) and FGF1 (3 ng/ml) individually or together on HUVEC proliferation after 72 hours incubation. Results from 3 H thymidine incorporation are expressed as cpm/culture. Dots reflects results of individual experiments performed in triplicate cultures; experimental means (±SD) are reflected by the bar graphs and error bars. B Ang2 reduces FGF1-induced wound healing in vitro. Images from a representative wound healing assay (of 5 assays) evaluated at 0, 12 and 16 hours (h) after HUVEC wounding. Ang2 (100 ng/ml), FGF1 (10 ng/ml) were added individually or together to the wounded HUVEC monolayers. Quantification of the results from triplicate cultures. The results of % wound closure from individual values (shown as dots) are expressed as mean (±SD), reflected by the error bars. Representative of 5 experiments. C The Ang2 inhibitor, AMG386 (AMG, 5 ng/ml) mitigates inhibition of wound healing by Ang2 (100 ng/ml) in the presence of FGF1 (10 ng/ml). Results of HUVEC wound closure from 4 experiments (evaluated at 12 hours after wounding) are presented as individual dots and means (±SD), reflected by the error bars. D, E Ang2 reduces FGF1-induced HUVEC transmigration. HUVEC (5x10 5 ) were tested in transmigration assays using Transwells (8.0 µm pore size) with or without Ang2 (100 ng/ml) and FGF1 (50 ng/ml). The number of cells migrated to lower surface of the membrane separating the upper from the lower chamber was counted after staining 0.5% crystal violet. Representative images (D) and quantification of results from 3 experiments, each performed in triplicate (E). Significant differences:*P<0.05; ** P<0.01; ***P<0.001 by two-way ANOVA for multiple comparisons with Tukey’s correction

Article Snippet: Cells were then incubated with 100–1000 ng/ml recombinant Ang2 (expressed with a C-terminal His-tag in CHO cells; Biolegend, No. 753106) or/and 3 or 5 nM FGF1 (R&D Systems, No. 232-FA) for 5 min, and/or 314.8 nM (10 μg/ml) AMG386 (obtained under material CRADA from Amgen).

Techniques: Migration, Incubation, In Vitro, Wound Healing Assay, Inhibition, Transmigration Assay, Pore Size, Membrane, Staining

Journal: JCI insight

Article Title: Myocardial protection by nanomaterials formulated with CHIR99021 and FGF1.

doi: 10.1172/jci.insight.132796

Figure Lengend Snippet: Figure 1. CHIR99021 and/or FGF1 NP treatment: assessment of cardiac function and infarct size in a mouse model of MI. Mice that were treated with intramyocardial injections of different NPs, including CHIR + FGF1-NPs, CHIR- NPs, FGF1-NPs, and empty NPs (nonloaded); MI-only control mice; and sham-operated control mice were subjected to echocardiographic assessments of left ventricular (LV) function (A). Ejection fraction (EF) (B), fractional shorten- ing (FS) (C), end-systolic diameter of the left ventricle (D), and end-diastolic diameter of the left ventricle (E) were assessed before MI induction (pre-S) and on post-MI day 28 (post-S). On day 28 after MI, CHIR + FGF1-NP treatment groups presented significantly greater EF and FS compared with other treatment as well as control groups (B and C) and significantly lower values of systolic/diastolic diameters of the left ventricle. Data are given as means ± SEM. There were 10–12 animals per group. Statistical analysis: 2-way ANOVA with Dunn’s multiple comparisons test. *P < 0.01 vs. sham; †P < 0.01 vs. MI; ‡P < 0.05 vs. empty NPs; §P < 0.05 vs. CHIR-NPs, ||P < 0.01 vs. FGF1-NPs. Sirius Red/Fast Green histochemical staining revealing areas of infarcted (red, nonviable) and noninfarcted (green, viable) zones in post-MI day 28 ventricular tissue sections (F). The infarct size was quantified as the ratio of the scar area to the total surface area of the left ventricle and expressed as a percentage, for day 28 samples (G). At day 28, the CHIR + FGF1-NP treatment group showed significant reduction in infarct size compared with other NP treatment groups or the untreated control MI animals. Scale bar: 1 mm (panels in F). Data are given as means ± SEM. There were 10–12 animals per group. Statistical analysis: 1-way ANOVA with Dunn’s multiple comparisons test. *P < 0.01 vs. MI; †P < 0.01 vs. empty NP; ‡P < 0.01 vs. CHIR-NP; §P < 0.01 vs. FGF1-NP.

Article Snippet: A double-emulsion (water/oil/water phase) technique for recombinant human FGF1 acidic protein (rhFGF1, aa 16-155 (Novus Biologicals, Inc.) and a single-emulsion (oil/water phase) technique for CHIR99021 were used.

Techniques: Control, Staining

Journal: JCI insight

Article Title: Myocardial protection by nanomaterials formulated with CHIR99021 and FGF1.

doi: 10.1172/jci.insight.132796

Figure Lengend Snippet: Figure 2. Direct intramyocardial injection of free CHIR99021 and/or FGF1: assessment of cardiac function and infarct size in a mouse model of MI. Mice were treated with direct intramyocardial injections of free CHIR, FGF1, and CHIR + FGF1 while MI-only mice served as controls. (A) Echocardiographic assessment of LV function. EF (B) and FS (C) were determined before MI (pre-S) and after 28 days (post-S). On day 28 after MI, no difference was identified among different treatment groups (means ± SEM, 5 animals per group). (D) Fibrosis, as assessed by Sirius Red/Fast Green histochemical stain- ing (scale bar: 1 mm). At day 28, fibrosis, expressed as the ratio of the scar area to the total surface area of the left ventricle, did not differ between groups (E). There were 5 animals per group. Statistical analysis: 2-way ANOVA (B and C) and 1-way ANOVA (E) with Dunn’s multiple comparisons test. *P < 0.01 vs. sham (B and C, post-S).

Article Snippet: A double-emulsion (water/oil/water phase) technique for recombinant human FGF1 acidic protein (rhFGF1, aa 16-155 (Novus Biologicals, Inc.) and a single-emulsion (oil/water phase) technique for CHIR99021 were used.

Techniques: Injection, Staining

Journal: JCI insight

Article Title: Myocardial protection by nanomaterials formulated with CHIR99021 and FGF1.

doi: 10.1172/jci.insight.132796

Figure Lengend Snippet: Figure 3. Detection and quantification of apoptosis by TUNEL assay in the mouse model of MI. (A) Seventy-two hours posttreatment, the CHIR + FGF1- NP treatment group showed significantly fewer numbers of TUNEL+ cells in the border zone compared with the other treatment groups (scale bar: 50 μm) (B). Cells were also counterstained for nuclei (blue, DAPI). Data are given as means ± SEM. There were 5 animals per group. Statistical analysis: 1-way ANOVA with Dunn’s multiple comparisons test. *P < 0.01 vs. sham; †P < 0.01 vs. MI; ‡P < 0.01 vs. empty NP; §P < 0.01 vs. CHIR-NPs; ||P < 0.01 vs. FGF1-NPs.

Article Snippet: A double-emulsion (water/oil/water phase) technique for recombinant human FGF1 acidic protein (rhFGF1, aa 16-155 (Novus Biologicals, Inc.) and a single-emulsion (oil/water phase) technique for CHIR99021 were used.

Techniques: TUNEL Assay

Journal: JCI insight

Article Title: Myocardial protection by nanomaterials formulated with CHIR99021 and FGF1.

doi: 10.1172/jci.insight.132796

Figure Lengend Snippet: Figure 4. Evaluation of NPs mediated neo-angiogenesis in the mouse model of MI. (A) Expression of the endothelium-specific lectin, isolectin B4 (red, IB4) and smooth muscle protein, SM22-α (green, SM22α), in the periinfarct border zone sections 28 days after MI (scale bar: 50 μm). (B and C) Graphical expression of angiogenesis data. The CHIR + FGF1-NP–treated group showed significantly elevated vascular densities compared with other NP treatment groups and the untreated MI group, but no significant difference in vascular densities was observed between the CHIR + FGF1-NP treat- ment group and the sham-operated controls. Data are given as means ± SEM. There were 10–12 animals per group. Statistical analysis: 1-way ANOVA with Dunn’s multiple comparisons test. *P < 0.01 vs. sham; †P < 0.01 vs. MI; ‡P < 0.01 vs. empty NPs; §P < 0.01 vs. CHIR-NPs; ||P < 0.01 vs. FGF1-NPs. (D–G) Cell cycle activity of endothelial cells assessed by a dual staining for Ki-67 (green) and IB4 (red; D and F) and phospho–histone H3 (PH3) (green) and IB4 (red; E and G). Scale bar: 20 μm. Data are given as means ± SEM. There were 10–12 animals per group. Statistical analysis: 1-way ANOVA with Dunn’s multiple comparisons test. *P < 0.01 vs. sham; †P < 0.01 vs. MI; ‡P < 0.01 vs. empty NPs; §P < 0.01 vs. CHIR-NPs; ||P < 0.01 vs. FGF1-NPs.

Article Snippet: A double-emulsion (water/oil/water phase) technique for recombinant human FGF1 acidic protein (rhFGF1, aa 16-155 (Novus Biologicals, Inc.) and a single-emulsion (oil/water phase) technique for CHIR99021 were used.

Techniques: Expressing, Activity Assay, Staining

Journal: JCI insight

Article Title: Myocardial protection by nanomaterials formulated with CHIR99021 and FGF1.

doi: 10.1172/jci.insight.132796

Figure Lengend Snippet: Figure 5. Assessment of LV morphology and function in a pig model of IR injury. Cardiac MRI recordings in the experimental groups at end-diastole (ED) and end-systole (ES). At day 28, the CHIR + FGF1-NP-treated group revealed significant reduction in infarct size compared with the untreated IR group (B). On the contrary, EF (C), CO (D), and SV (E) were significantly greater in CHIR + FGF1-NP–treated groups than in untreated IR groups while LV end-diastolic volume and LV end-systolic volume were significantly lower (F and G). Data are given as means ± SEM. There were 4 animals per group. Statistical analysis: 2-way ANOVA with Dunn’s multiple comparisons test. *P < 0.01 vs. pre-IR; †P < 0.05 vs. week 1; ‡P < 0.05 vs. IR. Macroscopic areas of infarction/fibrosis/scar (H) at after-IR day 28 in serial transverse sections of fresh (scale bar: 1 cm) representative micrographs of Sirius Red/Fast Green histochemical staining, revealing areas of infarcted (red, nonviable) and noninfarcted (green, viable) zones (I) (scale bar: 1 mm) and quantification of left anterior wall thickness (J). Data are given as means ± SEM. There were 4 animals per group. Statistical analysis: 1-way ANOVA with Dunn’s multiple comparisons test. *P < 0.01 vs. control; †P < 0.05 vs. IR.

Article Snippet: A double-emulsion (water/oil/water phase) technique for recombinant human FGF1 acidic protein (rhFGF1, aa 16-155 (Novus Biologicals, Inc.) and a single-emulsion (oil/water phase) technique for CHIR99021 were used.

Techniques: Staining, Control

Journal: Oncotarget

Article Title: Human papillomavirus 16L1-58L2 chimeric virus-like particles elicit durable neutralizing antibody responses against a broad-spectrum of human papillomavirus types

doi: 10.18632/oncotarget.19327

Figure Lengend Snippet: Neutralizing antibody response of rabbits vaccinated 4 times with KLH-58RG1 or KLH-6RG1

Article Snippet: Synthetic peptides that mimic HPV58 L2 aa.15-37 (100% identity with HPV52) or HPV6 L2 aa.16-39 were synthesized by GL Biochem LTD (Shanghai).

Techniques: