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R&D Systems vegf neutralizing antibody

Continuous intraosseous administration of SCS prevents glucocorticoid-induced bone degeneration. ( A ) Schematic illustration of the glucocorticoid (GC; MPS)-induced bone deterioration and intraosseous SCS treatment. ( B-D ) Representative H&E staining images of the femur at 6 weeks (B). Magnified views of the cortical bone and trabecular bone in the marrow cavity are shown on the right. Solid arrows indicate normal osteocytes, while hollow arrows indicate empty osteocyte lacunae. Quantification of empty lacunae ratios in cortical bone (C) and trabecular bone (D). n = 6 biological replicates. (Scale bars, 500 μm and 25 μm) ( E-H ) Representative immunofluorescence staining of OPN + mature osteoblasts, osteolectin + osteoprogenitors, and VE-cadherin + endothelial cells (ECs) in femur at 6 weeks (E), and corresponding quantifications (F–H). n = 6 biological replicates. (Scale bars, 100 μm and 20 μm) ( I and J ) Representative flow cytometry plots of capillary subtypes in the femur (I), with quantification of CD45 − Ter119 − CD31 hi Emcn hi ECs (J). n = 6 biological replicates. ( K and L ) Flow cytometry plots showing Sca-1 hi CD31 hi arteriolar ECs (K), and corresponding quantification (L). n = 6 biological replicates. ( M and N ) Representative micro-CT 3D images of the femur (M). Quantitative analysis of percent bone volume (BV/TV) (N). n = 6 biological replicates. (Scale bars, 1.5 mm, 600 μm and 545 μm) ( O and P ) ELISA analysis of <t>VEGF</t> (O) and PDGF-BB (P) levels in bone marrow supernatant and peripheral serum from PBS- and SCS-treated groups at week 6. n = 6 biological replicates. ( Q ) ELISA quantification of the osteogenic factor osteocalcin in peripheral serum at week 6. n = 6 biological replicates. Data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant. Statistical significance was determined using one-way ANOVA with Tukey's post hoc test ( C, D, F, G, H, J, L, N, O, P and Q ).

Vegf Neutralizing Antibody, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 154 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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vegf neutralizing antibody - by Bioz Stars, 2026-09

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Images

1) Product Images from "Sulfated polysaccharide prevents senescent adipocyte-driven osteonecrosis by stem cell fate reprogramming"

Article Title: Sulfated polysaccharide prevents senescent adipocyte-driven osteonecrosis by stem cell fate reprogramming

Journal: Bioactive Materials

doi: 10.1016/j.bioactmat.2025.11.039

Figure Legend Snippet: Continuous intraosseous administration of SCS prevents glucocorticoid-induced bone degeneration. ( A ) Schematic illustration of the glucocorticoid (GC; MPS)-induced bone deterioration and intraosseous SCS treatment. ( B-D ) Representative H&E staining images of the femur at 6 weeks (B). Magnified views of the cortical bone and trabecular bone in the marrow cavity are shown on the right. Solid arrows indicate normal osteocytes, while hollow arrows indicate empty osteocyte lacunae. Quantification of empty lacunae ratios in cortical bone (C) and trabecular bone (D). n = 6 biological replicates. (Scale bars, 500 μm and 25 μm) ( E-H ) Representative immunofluorescence staining of OPN + mature osteoblasts, osteolectin + osteoprogenitors, and VE-cadherin + endothelial cells (ECs) in femur at 6 weeks (E), and corresponding quantifications (F–H). n = 6 biological replicates. (Scale bars, 100 μm and 20 μm) ( I and J ) Representative flow cytometry plots of capillary subtypes in the femur (I), with quantification of CD45 − Ter119 − CD31 hi Emcn hi ECs (J). n = 6 biological replicates. ( K and L ) Flow cytometry plots showing Sca-1 hi CD31 hi arteriolar ECs (K), and corresponding quantification (L). n = 6 biological replicates. ( M and N ) Representative micro-CT 3D images of the femur (M). Quantitative analysis of percent bone volume (BV/TV) (N). n = 6 biological replicates. (Scale bars, 1.5 mm, 600 μm and 545 μm) ( O and P ) ELISA analysis of VEGF (O) and PDGF-BB (P) levels in bone marrow supernatant and peripheral serum from PBS- and SCS-treated groups at week 6. n = 6 biological replicates. ( Q ) ELISA quantification of the osteogenic factor osteocalcin in peripheral serum at week 6. n = 6 biological replicates. Data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant. Statistical significance was determined using one-way ANOVA with Tukey's post hoc test ( C, D, F, G, H, J, L, N, O, P and Q ).

Techniques Used: Staining, Immunofluorescence, Flow Cytometry, Micro-CT, Enzyme-linked Immunosorbent Assay

SCS targets downstream senescent lineage commitment of bone marrow MSCs to mitigate GC-induced bone deterioration. ( A ) Schematic diagram illustrating the experimental design: CD45 − Ter119 − CD31 − LepR + MSCs isolated from mice co-treated with SCS and MPS for 7 days were subjected to in vitro lineage-competitive differentiation, followed by DEX-induced senescence in lineage-mixed cells. These cells were then adoptively transplanted into healthy bone marrow cavity to assess bone deterioration development. ( B ) Representative H&E-stained images of the femur 12 weeks after adoptive transfer. PBS-DEX group: LepR + MSCs from PBS and MPS co-treated mice subjected to in vitro lineage differentiation and DEX-induced senescence, followed by transplantation. SCS-DEX group: LepR + MSCs from SCS and MPS co-treated mice processed similarly. PBS group: solvent control without cell transplantation. Solid arrows indicate intact osteocytes; hollow arrows indicate empty lacunae. (Scale bars, 250 μm and 25 μm) ( C – E ) Quantitative analysis of marrow hypertrophic adipocyte diameter (C), proportion of empty osteocyte lacunae in trabecular bone (D), and adipocyte number (E) in the metaphysis 12 weeks post-transplantation. n = 19 biological replicates (C), n = 6 biological replicates (D), n = 8 biological replicates (E). ( F ) Quantification of empty lacunae in epiphysis at 12 weeks post-transplantation. n = 6 biological replicates. ( G – I ) Representative flow cytometry plots of capillary ECs subtypes in the femur at 12 weeks (G), with quantification of CD45 − Ter119 − CD31 hi Emcn hi ECs (H) and CD45 − Ter119 − CD31 lo Emcn lo ECs (I). n = 6 biological replicates. ( J and K ) Representative flow cytometry plots (J) and corresponding quantification (K) of CD45 − Ter119 − Sca-1 hi CD31 hi arteriolar ECs in the femur at 12 weeks post-transplantation. n = 6 biological replicates. ( L ) Representative micro-CT images of the femur at 12 weeks post-transplantation across different treatment groups. (Scale bars, 1.5 mm and 500 μm) ( M – P ) Quantitative analysis of bone parameters in the metaphysis: bone mineral density (BMD) (M), percent bone volume (BV/TV) (N), trabecular separation (Tb.Sp) (O), and trabecular number (Tb.N) (P). n = 6 biological replicates. ( Q ) Serum ELISA analysis of the osteogenic marker osteocalcin at 12 weeks post-transplantation. n = 6 biological replicates. ( R and S ) ELISA analysis of PDGF-BB (R) and VEGF (S) in both bone marrow supernatant and peripheral serum at 12 weeks post-transplantation. n = 6 biological replicates. Data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant. Statistical significance was determined using one-way ANOVA with Tukey's post hoc test ( C, D, E, F, H, I, K, M, N, O, P, Q, R and S ).

Figure Legend Snippet: SCS targets downstream senescent lineage commitment of bone marrow MSCs to mitigate GC-induced bone deterioration. ( A ) Schematic diagram illustrating the experimental design: CD45 − Ter119 − CD31 − LepR + MSCs isolated from mice co-treated with SCS and MPS for 7 days were subjected to in vitro lineage-competitive differentiation, followed by DEX-induced senescence in lineage-mixed cells. These cells were then adoptively transplanted into healthy bone marrow cavity to assess bone deterioration development. ( B ) Representative H&E-stained images of the femur 12 weeks after adoptive transfer. PBS-DEX group: LepR + MSCs from PBS and MPS co-treated mice subjected to in vitro lineage differentiation and DEX-induced senescence, followed by transplantation. SCS-DEX group: LepR + MSCs from SCS and MPS co-treated mice processed similarly. PBS group: solvent control without cell transplantation. Solid arrows indicate intact osteocytes; hollow arrows indicate empty lacunae. (Scale bars, 250 μm and 25 μm) ( C – E ) Quantitative analysis of marrow hypertrophic adipocyte diameter (C), proportion of empty osteocyte lacunae in trabecular bone (D), and adipocyte number (E) in the metaphysis 12 weeks post-transplantation. n = 19 biological replicates (C), n = 6 biological replicates (D), n = 8 biological replicates (E). ( F ) Quantification of empty lacunae in epiphysis at 12 weeks post-transplantation. n = 6 biological replicates. ( G – I ) Representative flow cytometry plots of capillary ECs subtypes in the femur at 12 weeks (G), with quantification of CD45 − Ter119 − CD31 hi Emcn hi ECs (H) and CD45 − Ter119 − CD31 lo Emcn lo ECs (I). n = 6 biological replicates. ( J and K ) Representative flow cytometry plots (J) and corresponding quantification (K) of CD45 − Ter119 − Sca-1 hi CD31 hi arteriolar ECs in the femur at 12 weeks post-transplantation. n = 6 biological replicates. ( L ) Representative micro-CT images of the femur at 12 weeks post-transplantation across different treatment groups. (Scale bars, 1.5 mm and 500 μm) ( M – P ) Quantitative analysis of bone parameters in the metaphysis: bone mineral density (BMD) (M), percent bone volume (BV/TV) (N), trabecular separation (Tb.Sp) (O), and trabecular number (Tb.N) (P). n = 6 biological replicates. ( Q ) Serum ELISA analysis of the osteogenic marker osteocalcin at 12 weeks post-transplantation. n = 6 biological replicates. ( R and S ) ELISA analysis of PDGF-BB (R) and VEGF (S) in both bone marrow supernatant and peripheral serum at 12 weeks post-transplantation. n = 6 biological replicates. Data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant. Statistical significance was determined using one-way ANOVA with Tukey's post hoc test ( C, D, E, F, H, I, K, M, N, O, P, Q, R and S ).

Techniques Used: Isolation, In Vitro, Staining, Adoptive Transfer Assay, Transplantation Assay, Solvent, Control, Flow Cytometry, Micro-CT, Enzyme-linked Immunosorbent Assay, Marker

Incubation:

Article Title: Mesenchymal stem cell-derived extracellular vesicles exert pro-angiogenic and pro-lymphangiogenic effects in ischemic tissues by transferring various microRNAs and proteins including ITGa5 and NRP1
Article Snippet: Briefly, homogenized tissue samples were separated by SDS-PAGE and transferred onto PVDF membranes (Merck). .. The blocked PVDF membranes were incubated overnight at 4 °C with one of the following primary monoclonal antibodies: anti-endothelial NOS (eNOS; 1:2500, BD Biosciences), anti-caveolin-1 (1000, BD Biosciences), anti-phospho-eNOS (Ser 1177, 1:1000, Cell Signaling Technology), anti-Akt (1:1000, Cell Signaling Technology), anti-phospho-Akt (Ser 473, 1:2000, Cell Signaling Technology), and anti-VEGF (1:1000, R&D Systems). .. A rabbit polyclonal anti-actin antibody (1:5000, Merck) was used to visualize protein loading on the gel.

Article Title: Mc LTP1, a lipid transfer protein isolated from noni seeds induces effective healing of superficial burns
Article Snippet: .. A microtiter plates with 96 well were incubated with anti-TNF-α or anti-IL-1β primary antibodies (R&D Systems) diluted in PBS (1:1000), followed by incubation with biotinylated monoclonal anti-TNF-α, anti-IL-1β, anti-IL6, anti-IL10, anti-VEGF and anti-TGF-β detection antibodies (R&D Systems) diluted in BSA 1% (1:1000). .. Plates were washed and HRP-streptavidin 1000 (R&D Systems) diluted and were added in each well.

Article Title: Inhibition of triple negative breast cancer-associated inflammation and progression by N- acylethanolamine acid amide hydrolase (NAAA)
Article Snippet: .. The sections were incubated with the primary antibody Anti-IL6 (GB11117, 1:100) and anti-VEGF (R&D systems AF293, 1–100) overnight at 4 °C. .. Sections were rinsed with PBS and incubated with Donkey anti-Goat AF488 (Invitrogen, A32814, 1:500) for 1 h at room temperature followed by 3 times of wash.

Bioprocessing:

Staining:

Article Title: Digital spatial profiling reveals additive effects of triple therapy on tumor microenvironment: anti-PD-L1, anti-VEGF, and PARP inhibition in mouse models
Article Snippet: .. Sections were stained with the following primary antibodies: anti-Granzyme B (rabbit anti-mouse; Abcam, #ab4059), anti-CD8 (rabbit anti-mouse; Cell Signaling, #98,941), anti-PD-L1 (rabbit monoclonal clone D5V3B; Cell Signaling, #64,988), anti-VEGF (goat polyclonal; R&D SYSTEMS, #AF-493-NA), anti-PARP1 (rabbit polyclonal; Abcam, #ab227244), anti-CD31 (rabbit monoclonal clone EPR17259 ; Abcam, #ab182981), and anti-MECA79 (rat monoclonal clone MECA79; BioLegend, #120,801). ..

SDS Page:

Article Title: Vascular endothelial growth factor antagonists
Article Snippet: Purified recombinant NRP1-Fc (Sino Biologics) was added as indicated (−NRP1 or +NRP1) to 24 h conditioned media from vector transfected 293/KDR cells(left lanes), 293/KDR/VEGF 3S transfectants (middle lanes), or 293/KDR/VEGF WT transfectants (right lanes) in the absence (−) or presence (+) of the crosslinking reagent BS3 (Thermo Fisher Pierce). .. Samples were resolved by SDS-PAGE under reducing conditions, transferred to PVDF and immunodetected with anti-Fc antibody (top panel; Thermo Fisher Pierce) or anti-VEGF (lower panels; R&D Systems). ..

Image Search Results

Positive and negative control cells for both marker panels. ( A ) HeLa (white arrows): in the first panel positive for cytokeratin (CK), Vimentin (Vim), VEGF and in the second panel positive for p16INK4A, negative for PD-L1 and CD45. ( B ) T98G (red arrows): positive for Vim. ( C ) CaSki (yellow arrows): positive for CK, Vim, VEGF, PD-L1 and p16INK4A. ( D ) MCF-7 (green arrows): positive for CK. All cell lines were negative for CD45. Only hematopoietic cells showed CD45 positive staining. Scale bar = 10 μm. Images were processed using Zeiss ZEN 3.7 software and the figure was assembled using Microsoft PowerPoint.

Journal: International Journal of Molecular Sciences

Article Title: Disseminated Tumor Cells (DTCs) in Patients with Cervical Cancer Reveal Mesenchymal Properties and Potential Therapeutic Targets—A New Perspective?

doi: 10.3390/ijms27114875

Figure Lengend Snippet: Positive and negative control cells for both marker panels. ( A ) HeLa (white arrows): in the first panel positive for cytokeratin (CK), Vimentin (Vim), VEGF and in the second panel positive for p16INK4A, negative for PD-L1 and CD45. ( B ) T98G (red arrows): positive for Vim. ( C ) CaSki (yellow arrows): positive for CK, Vim, VEGF, PD-L1 and p16INK4A. ( D ) MCF-7 (green arrows): positive for CK. All cell lines were negative for CD45. Only hematopoietic cells showed CD45 positive staining. Scale bar = 10 μm. Images were processed using Zeiss ZEN 3.7 software and the figure was assembled using Microsoft PowerPoint.

Article Snippet: In the first round of staining, anti-human Pan-Cytokeratin APC-conjugated REAdye_lease antibody (Cat. No. 130-123-091, Miltenyi Biotech, Bergisch Gladbach, Germany), anti-human vimentin FITC-conjugated REAdye_lease antibody (Cat. No. 130-127-022, Miltenyi Biotech, Bergisch Gladbach, Germany) and anti-human VEGF PE-conjugated REAdye_lease antibody (Cat. No. 130-118-061, Miltenyi Biotech, Bergisch Gladbach, Germany) were applied.

Techniques: Negative Control, Marker, Staining, Software

Representative images of patient-derived DTCs with three different profiles. ( A ) DTC positive for Vim, VEGF and p16, negative for CK, PD-L1 and CD45. ( B ) CK and Vim positive while negative for VEGF, PD-L1, p16 and CD45. ( C ) DTC positive for Vim and PD-L1, negative for all other markers. Scale bar = 10 μm. Images were processed using Zeiss ZEN 3.7 software and figure was assembled using Microsoft PowerPoint.

Journal: International Journal of Molecular Sciences

Article Title: Disseminated Tumor Cells (DTCs) in Patients with Cervical Cancer Reveal Mesenchymal Properties and Potential Therapeutic Targets—A New Perspective?

doi: 10.3390/ijms27114875

Figure Lengend Snippet: Representative images of patient-derived DTCs with three different profiles. ( A ) DTC positive for Vim, VEGF and p16, negative for CK, PD-L1 and CD45. ( B ) CK and Vim positive while negative for VEGF, PD-L1, p16 and CD45. ( C ) DTC positive for Vim and PD-L1, negative for all other markers. Scale bar = 10 μm. Images were processed using Zeiss ZEN 3.7 software and figure was assembled using Microsoft PowerPoint.

Techniques: Derivative Assay, Software

Comparison of p16, VEGF and PD-L1 in matching tumor tissue. In four cases we found VEGF on DTCs but not in matching tumor tissue, indicating a potential therapeutic target. Further, we detected PD-L1 on DTCs in 11 patients that had PD-L1 negative tissue at diagnosis, suggesting a potential discordance between tumor tissue and DTC phenotype. Chart generated using Microsoft Excel.

Journal: International Journal of Molecular Sciences

Article Title: Disseminated Tumor Cells (DTCs) in Patients with Cervical Cancer Reveal Mesenchymal Properties and Potential Therapeutic Targets—A New Perspective?

doi: 10.3390/ijms27114875

Figure Lengend Snippet: Comparison of p16, VEGF and PD-L1 in matching tumor tissue. In four cases we found VEGF on DTCs but not in matching tumor tissue, indicating a potential therapeutic target. Further, we detected PD-L1 on DTCs in 11 patients that had PD-L1 negative tissue at diagnosis, suggesting a potential discordance between tumor tissue and DTC phenotype. Chart generated using Microsoft Excel.

Techniques: Comparison, Biomarker Discovery, Generated

Study design. At diagnosis a tissue biopsy was obtained to histologically confirm cervical cancer cases. The FFPE tissue samples were stained against p16, PD-L1 and VEGF for comparison with DTC profiles. One day prior to oncologic surgery, blood samples were collected for CTC detection using the standardized CK based brightfield method. Bone marrow aspirates were sampled during surgery and DTCs were analyzed using the CK based and the multi-parameter IF staining method. Post-operative follow-up blood samples were collected within two years after surgery during routine visits if applicable. Clinical Outcome was assessed up to February 2025. Created with BioRender.com.

Journal: International Journal of Molecular Sciences

Article Title: Disseminated Tumor Cells (DTCs) in Patients with Cervical Cancer Reveal Mesenchymal Properties and Potential Therapeutic Targets—A New Perspective?

doi: 10.3390/ijms27114875

Figure Lengend Snippet: Study design. At diagnosis a tissue biopsy was obtained to histologically confirm cervical cancer cases. The FFPE tissue samples were stained against p16, PD-L1 and VEGF for comparison with DTC profiles. One day prior to oncologic surgery, blood samples were collected for CTC detection using the standardized CK based brightfield method. Bone marrow aspirates were sampled during surgery and DTCs were analyzed using the CK based and the multi-parameter IF staining method. Post-operative follow-up blood samples were collected within two years after surgery during routine visits if applicable. Clinical Outcome was assessed up to February 2025. Created with BioRender.com.

Techniques: Biomarker Discovery, Staining, Comparison

Sequential multi-parameter immunofluorescent staining. After applying the first antibody panel (CK, Vim and VEGF), the slides were scanned and DTC profiles were assessed. Subsequently, releasable fluorochrome-conjugates were digested and the slides were subjected to the second antibody panel (PD-L1, p16, CD45) followed by scanning and DTC detection. Created with BioRender.com.

Journal: International Journal of Molecular Sciences

Article Title: Disseminated Tumor Cells (DTCs) in Patients with Cervical Cancer Reveal Mesenchymal Properties and Potential Therapeutic Targets—A New Perspective?

doi: 10.3390/ijms27114875

Figure Lengend Snippet: Sequential multi-parameter immunofluorescent staining. After applying the first antibody panel (CK, Vim and VEGF), the slides were scanned and DTC profiles were assessed. Subsequently, releasable fluorochrome-conjugates were digested and the slides were subjected to the second antibody panel (PD-L1, p16, CD45) followed by scanning and DTC detection. Created with BioRender.com.

Techniques: Staining

Box plot showing the residual fluorescence signal after the release step (%) in 295 CaSki cells following treatment with a VEGF-PE conjugated antibody. The release efficiency was evaluated by signal quantification in the cells before and after treatment with the release reagent. The median fluorescence signal was 24.5% (mean 26.7%), corresponding to a signal reduction of 75.5%. The range was 1.75% to 87.78%. Statistical graph was generated using IBM SPSS Statistics Version 29.0.

Journal: International Journal of Molecular Sciences

Article Title: Disseminated Tumor Cells (DTCs) in Patients with Cervical Cancer Reveal Mesenchymal Properties and Potential Therapeutic Targets—A New Perspective?

doi: 10.3390/ijms27114875

Figure Lengend Snippet: Box plot showing the residual fluorescence signal after the release step (%) in 295 CaSki cells following treatment with a VEGF-PE conjugated antibody. The release efficiency was evaluated by signal quantification in the cells before and after treatment with the release reagent. The median fluorescence signal was 24.5% (mean 26.7%), corresponding to a signal reduction of 75.5%. The range was 1.75% to 87.78%. Statistical graph was generated using IBM SPSS Statistics Version 29.0.

Techniques: Fluorescence, Generated

CaSki cells before and after the release step. Representative immunofluorescence image of two CaSki cells spiked into bone marrow cells ( A ) before and ( B ) after the release step. The PE channel shows VEGF staining before staining with CD45, while nuclei were stained with DAPI (blue). Images were processed using Zeiss ZEN software and figure was assembled using Microsoft PowerPoint.

Journal: International Journal of Molecular Sciences

Article Title: Disseminated Tumor Cells (DTCs) in Patients with Cervical Cancer Reveal Mesenchymal Properties and Potential Therapeutic Targets—A New Perspective?

doi: 10.3390/ijms27114875

Figure Lengend Snippet: CaSki cells before and after the release step. Representative immunofluorescence image of two CaSki cells spiked into bone marrow cells ( A ) before and ( B ) after the release step. The PE channel shows VEGF staining before staining with CD45, while nuclei were stained with DAPI (blue). Images were processed using Zeiss ZEN software and figure was assembled using Microsoft PowerPoint.

Techniques: Immunofluorescence, Staining, Software

Journal: Bioactive Materials

Article Title: Sulfated polysaccharide prevents senescent adipocyte-driven osteonecrosis by stem cell fate reprogramming

doi: 10.1016/j.bioactmat.2025.11.039

Figure Lengend Snippet: Continuous intraosseous administration of SCS prevents glucocorticoid-induced bone degeneration. ( A ) Schematic illustration of the glucocorticoid (GC; MPS)-induced bone deterioration and intraosseous SCS treatment. ( B-D ) Representative H&E staining images of the femur at 6 weeks (B). Magnified views of the cortical bone and trabecular bone in the marrow cavity are shown on the right. Solid arrows indicate normal osteocytes, while hollow arrows indicate empty osteocyte lacunae. Quantification of empty lacunae ratios in cortical bone (C) and trabecular bone (D). n = 6 biological replicates. (Scale bars, 500 μm and 25 μm) ( E-H ) Representative immunofluorescence staining of OPN + mature osteoblasts, osteolectin + osteoprogenitors, and VE-cadherin + endothelial cells (ECs) in femur at 6 weeks (E), and corresponding quantifications (F–H). n = 6 biological replicates. (Scale bars, 100 μm and 20 μm) ( I and J ) Representative flow cytometry plots of capillary subtypes in the femur (I), with quantification of CD45 − Ter119 − CD31 hi Emcn hi ECs (J). n = 6 biological replicates. ( K and L ) Flow cytometry plots showing Sca-1 hi CD31 hi arteriolar ECs (K), and corresponding quantification (L). n = 6 biological replicates. ( M and N ) Representative micro-CT 3D images of the femur (M). Quantitative analysis of percent bone volume (BV/TV) (N). n = 6 biological replicates. (Scale bars, 1.5 mm, 600 μm and 545 μm) ( O and P ) ELISA analysis of VEGF (O) and PDGF-BB (P) levels in bone marrow supernatant and peripheral serum from PBS- and SCS-treated groups at week 6. n = 6 biological replicates. ( Q ) ELISA quantification of the osteogenic factor osteocalcin in peripheral serum at week 6. n = 6 biological replicates. Data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant. Statistical significance was determined using one-way ANOVA with Tukey's post hoc test ( C, D, F, G, H, J, L, N, O, P and Q ).

Article Snippet: Other drugs and compounds used in this study included: GW9662 (MCE, HY-16578; intraperitoneal injection, 1 mg/kg body weight/day, administered continuously for 4 weeks), T0070907 (Selleck, S2871; intraperitoneal injection, 2 mg/kg body weight/day, administered continuously for 4 weeks), rapamycin (MCE, HY-10219; subcutaneous injection, 3 mg/kg body weight/day, administered continuously for 4 weeks), Rosiglitazone (MCE, HY-17386; oral gavage, 3 mg/kg body weight/day, administered continuously for 2 weeks), LY294002 (Selleck, S1105; intraosseous injection, 10 μM, 5 μL per dose per week, administered for 1 or 4 weeks), DMH1 (Selleck, S7146; intraperitoneal injection, 5 mg/kg body weight/day, administered continuously for 4 weeks), Noggin (PeproTech, 250-38; intraosseous injection, 50 ng per dose, twice per week, administered for 2 or 4 weeks), LDN-193189 (Selleck, S2618; intraperitoneal injection, 3 mg/kg body weight/day, administered for 1 or 4 weeks), IGF-1 (PeproTech, 250-19; intraosseous injection, 4 μg per dose per week, administered for 2 weeks), IGF-1 neutralizing antibody (R&D Systems, AF-791; intraosseous injection, 2 μg per dose, twice per week, administered for 2 or 4 weeks), VEGF neutralizing antibody (R&D Systems, AF-493-NA; intraosseous injection, 2 μg per dose, twice per week, administered for 4 weeks), PDGF-AA neutralizing antibody (R&D Systems, AF-221-NA; intraosseous injection, 2 μg per dose, twice per week, administered for 4 weeks), PDGF-BB neutralizing antibody (R&D Systems, AF-220-NA; intraosseous injection, 2 μg per dose, twice per week, administered for 4 weeks), TGF-β1 neutralizing antibody (R&D Systems, MAB2401; intraosseous injection, 2 μg per dose, twice per week, administered for 4 weeks), TGF-β2 neutralizing antibody (R&D Systems, AB-112-NA; intraosseous injection, 2 μg per dose, twice per week, administered for 4 weeks).

Techniques: Staining, Immunofluorescence, Flow Cytometry, Micro-CT, Enzyme-linked Immunosorbent Assay

Journal: Bioactive Materials

Article Title: Sulfated polysaccharide prevents senescent adipocyte-driven osteonecrosis by stem cell fate reprogramming

doi: 10.1016/j.bioactmat.2025.11.039

Figure Lengend Snippet: SCS targets downstream senescent lineage commitment of bone marrow MSCs to mitigate GC-induced bone deterioration. ( A ) Schematic diagram illustrating the experimental design: CD45 − Ter119 − CD31 − LepR + MSCs isolated from mice co-treated with SCS and MPS for 7 days were subjected to in vitro lineage-competitive differentiation, followed by DEX-induced senescence in lineage-mixed cells. These cells were then adoptively transplanted into healthy bone marrow cavity to assess bone deterioration development. ( B ) Representative H&E-stained images of the femur 12 weeks after adoptive transfer. PBS-DEX group: LepR + MSCs from PBS and MPS co-treated mice subjected to in vitro lineage differentiation and DEX-induced senescence, followed by transplantation. SCS-DEX group: LepR + MSCs from SCS and MPS co-treated mice processed similarly. PBS group: solvent control without cell transplantation. Solid arrows indicate intact osteocytes; hollow arrows indicate empty lacunae. (Scale bars, 250 μm and 25 μm) ( C – E ) Quantitative analysis of marrow hypertrophic adipocyte diameter (C), proportion of empty osteocyte lacunae in trabecular bone (D), and adipocyte number (E) in the metaphysis 12 weeks post-transplantation. n = 19 biological replicates (C), n = 6 biological replicates (D), n = 8 biological replicates (E). ( F ) Quantification of empty lacunae in epiphysis at 12 weeks post-transplantation. n = 6 biological replicates. ( G – I ) Representative flow cytometry plots of capillary ECs subtypes in the femur at 12 weeks (G), with quantification of CD45 − Ter119 − CD31 hi Emcn hi ECs (H) and CD45 − Ter119 − CD31 lo Emcn lo ECs (I). n = 6 biological replicates. ( J and K ) Representative flow cytometry plots (J) and corresponding quantification (K) of CD45 − Ter119 − Sca-1 hi CD31 hi arteriolar ECs in the femur at 12 weeks post-transplantation. n = 6 biological replicates. ( L ) Representative micro-CT images of the femur at 12 weeks post-transplantation across different treatment groups. (Scale bars, 1.5 mm and 500 μm) ( M – P ) Quantitative analysis of bone parameters in the metaphysis: bone mineral density (BMD) (M), percent bone volume (BV/TV) (N), trabecular separation (Tb.Sp) (O), and trabecular number (Tb.N) (P). n = 6 biological replicates. ( Q ) Serum ELISA analysis of the osteogenic marker osteocalcin at 12 weeks post-transplantation. n = 6 biological replicates. ( R and S ) ELISA analysis of PDGF-BB (R) and VEGF (S) in both bone marrow supernatant and peripheral serum at 12 weeks post-transplantation. n = 6 biological replicates. Data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant. Statistical significance was determined using one-way ANOVA with Tukey's post hoc test ( C, D, E, F, H, I, K, M, N, O, P, Q, R and S ).

Techniques: Isolation, In Vitro, Staining, Adoptive Transfer Assay, Transplantation Assay, Solvent, Control, Flow Cytometry, Micro-CT, Enzyme-linked Immunosorbent Assay, Marker

Schematic illustration of the a) preparation and application of Ti-OH-ePV; b) VEGF release of Ti-OH-ePV under different pH conditions; c) anastomotic healing performance with Ti and Ti-OH-ePV; d) healing-promotion mechanism of Ti-OH-ePV.

Journal: Bioactive Materials

Article Title: Bioactive-coated porous anastomotic staples enhance anastomotic healing

doi: 10.1016/j.bioactmat.2026.01.005

Figure Lengend Snippet: Schematic illustration of the a) preparation and application of Ti-OH-ePV; b) VEGF release of Ti-OH-ePV under different pH conditions; c) anastomotic healing performance with Ti and Ti-OH-ePV; d) healing-promotion mechanism of Ti-OH-ePV.

Article Snippet: Vascular Endothelial Growth Factor (VEGF) protein was supplied by MedChemExpress LLC (USA).

Techniques:

Structure characterization of Ti-OH-ePV. a) SEM images of Ti, Ti-OH, and Ti-OH-ePV (scale bars: 1 μm); b) Elemental mapping of Ti-OH-ePV; c) CV test of DA and VEGF solution under a nitrogen atmosphere; d) AFM height images of Ti, Ti-OH, and Ti-OH-ePV; e) Surface Sa (arithmetic mean height) via AFM of Ti, Ti-OH, and Ti-OH-ePV; f) Water contact angle of Ti, Ti-OH, and Ti-OH-ePV; g) FTIR spectra of Ti, Ti-OH, and Ti-OH-ePV; h) Tensile testing of the Ti, Ti-OH, and Ti-OH-ePV; i) Single anastomotic staple tensile strength testing of the Ti, Ti-OH, and Ti-OH-ePV; j) VEGF release profiles of the Ti-OH-ePV, Ti-ePV, and Ti-OH-PV in buffer solutions at pH = 7.4; k) VEGF release profiles of the Ti-OH-ePV, Ti-ePV, and Ti-OH-PV in buffer solutions at pH = 6.5; n = 3; ns = not significant, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Journal: Bioactive Materials

Article Title: Bioactive-coated porous anastomotic staples enhance anastomotic healing

doi: 10.1016/j.bioactmat.2026.01.005

Figure Lengend Snippet: Structure characterization of Ti-OH-ePV. a) SEM images of Ti, Ti-OH, and Ti-OH-ePV (scale bars: 1 μm); b) Elemental mapping of Ti-OH-ePV; c) CV test of DA and VEGF solution under a nitrogen atmosphere; d) AFM height images of Ti, Ti-OH, and Ti-OH-ePV; e) Surface Sa (arithmetic mean height) via AFM of Ti, Ti-OH, and Ti-OH-ePV; f) Water contact angle of Ti, Ti-OH, and Ti-OH-ePV; g) FTIR spectra of Ti, Ti-OH, and Ti-OH-ePV; h) Tensile testing of the Ti, Ti-OH, and Ti-OH-ePV; i) Single anastomotic staple tensile strength testing of the Ti, Ti-OH, and Ti-OH-ePV; j) VEGF release profiles of the Ti-OH-ePV, Ti-ePV, and Ti-OH-PV in buffer solutions at pH = 7.4; k) VEGF release profiles of the Ti-OH-ePV, Ti-ePV, and Ti-OH-PV in buffer solutions at pH = 6.5; n = 3; ns = not significant, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: Vascular Endothelial Growth Factor (VEGF) protein was supplied by MedChemExpress LLC (USA).

Techniques:

a) The procedure of gastrointestinal anastomosis in New Zealand rabbit; b) Immumohistochemical staining images of IL-6, TNF-α, TGF-β, and IL-10 at the anastomotic stoma on day 3 for Ti, Ti-OH, Ti-OH-ePDA (PDA-only), and Ti-OH-ePV (VEGF-loaded PDA) groups (scale bar: 25 μm); c) Statistical analysis of IL-6 expression in different groups; d) Statistical analysis of TNF-α expression in different groups; e) Statistical analysis of TGF-β expression in different groups; f) Statistical analysis of IL-10 expression in different groups; n = 3; ns = not significant, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Journal: Bioactive Materials

Article Title: Bioactive-coated porous anastomotic staples enhance anastomotic healing

doi: 10.1016/j.bioactmat.2026.01.005

Figure Lengend Snippet: a) The procedure of gastrointestinal anastomosis in New Zealand rabbit; b) Immumohistochemical staining images of IL-6, TNF-α, TGF-β, and IL-10 at the anastomotic stoma on day 3 for Ti, Ti-OH, Ti-OH-ePDA (PDA-only), and Ti-OH-ePV (VEGF-loaded PDA) groups (scale bar: 25 μm); c) Statistical analysis of IL-6 expression in different groups; d) Statistical analysis of TNF-α expression in different groups; e) Statistical analysis of TGF-β expression in different groups; f) Statistical analysis of IL-10 expression in different groups; n = 3; ns = not significant, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: Vascular Endothelial Growth Factor (VEGF) protein was supplied by MedChemExpress LLC (USA).

Techniques: Staining, Expressing

a) Immumohistochemical staining images of CD31 on day 7 at the anastomotic stoma for Ti, Ti-OH, Ti-OH-ePDA (PDA-only), and Ti-OH-ePV (VEGF-loaded PDA) groups (scale bar: 25 μm); b) Masson staining images on day 14 at the anastomotic stoma for Ti, Ti-OH, Ti-OH-ePDA (PDA-only), and Ti-OH-ePV (VEGF-loaded PDA) groups (scale bar: 20 μm); c) H&E staining images on day 14 at the anastomotic stoma for Ti, Ti-OH, Ti-OH-ePDA (PDA-only), and Ti-OH-ePV (VEGF-loaded PDA) groups; d) Statistical analysis for the number of blood vessels in different groups; e) Statistical analysis of collagen expression in different groups; f) Statistical analysis of bursting pressure on days 7, and 14 in different groups; g) Statistical analysis of WBC on days pre-1, 3, 7, and 14 in different groups; h) Statistical analysis of APTT on days pre-1, 3, 7, and 14 in different groups; i) Statistical analysis of ALT on days pre-1, 3, 7, and 14 in different groups; n = 3; ns = not significant, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Journal: Bioactive Materials

Article Title: Bioactive-coated porous anastomotic staples enhance anastomotic healing

doi: 10.1016/j.bioactmat.2026.01.005

Figure Lengend Snippet: a) Immumohistochemical staining images of CD31 on day 7 at the anastomotic stoma for Ti, Ti-OH, Ti-OH-ePDA (PDA-only), and Ti-OH-ePV (VEGF-loaded PDA) groups (scale bar: 25 μm); b) Masson staining images on day 14 at the anastomotic stoma for Ti, Ti-OH, Ti-OH-ePDA (PDA-only), and Ti-OH-ePV (VEGF-loaded PDA) groups (scale bar: 20 μm); c) H&E staining images on day 14 at the anastomotic stoma for Ti, Ti-OH, Ti-OH-ePDA (PDA-only), and Ti-OH-ePV (VEGF-loaded PDA) groups; d) Statistical analysis for the number of blood vessels in different groups; e) Statistical analysis of collagen expression in different groups; f) Statistical analysis of bursting pressure on days 7, and 14 in different groups; g) Statistical analysis of WBC on days pre-1, 3, 7, and 14 in different groups; h) Statistical analysis of APTT on days pre-1, 3, 7, and 14 in different groups; i) Statistical analysis of ALT on days pre-1, 3, 7, and 14 in different groups; n = 3; ns = not significant, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: Vascular Endothelial Growth Factor (VEGF) protein was supplied by MedChemExpress LLC (USA).

Techniques: Staining, Expressing

(A) , Wild-type (WT) and KI mice HTRZ for either PS1 FAD mutant M146V (WT/M146V) or I213T (WT/I213T) were infused with vehicle (0.2% BSA in PBS) or VEGF-A (a total of 3.5μg in 100μl of vehicle) through the carotid artery for 15 days using a mini osmotic pump as in Methods. Left: Brain coronal sections (40μm thick) were prepared and immunostained with anti-Col IV antibodies to visualize brain vessels. Enhanced visualization surfaces were generated using Imaris software from representative confocal images of ipsilateral hemispheres. Scale bar: 50μm. Right: Graph shows total vessel length density in WT and PS1 FAD brains quantified using Imaris 9.9 software as in Methods. (B) , WT and HTRZ for PS1 FAD mutants M146V or I213T mice were injected through the carotid artery for 20 minutes with either vehicle or 100ng of VEGF-A in vehicle prepared as in 1A using a catheter as described in Methods. Brain microvessels (MV) were isolated as in Methods, lysed in Triton X-100 buffer, and subjected to immunoprecipitation (IP) with anti-VEGFR2 antibody or control IgG. Left: IPs were analyzed on Western blots (WBs) using anti-endoglin or anti-VEGFR2 antibodies (upper panel). Input samples are shown in lower panel. β-actin: loading control. Right: Graph shows quantification of endoglin co-IPed with VEGFR2, normalized to IPed VEGFR2. (C) , WT mice were infused for 15 days through the carotid artery with vehicle or VEGF-A in vehicle as in 1A using a mini osmotic pump (as in 1A). For RO injection, mice were treated with vehicle (2% DMSO, 30% PEG 300, 5% Tween-80 in ddH2O) or RO in vehicle (5mg/kg body weight) via five injections in tail vein one injection every three days, with first injection administered 1 hour before osmotic pump implantation. Brain coronal sections (40μm) were prepared and immunostained with anti-Col IV antibodies as in 1A. Left: Representative confocal images of ipsilateral hemispheres are shown prepared as in 1A. Scale bar: 50μm. Right: Graph shows total vessel length density quantified using Imaris software as in 1A. (D) , WT adult mice were treated with either 50μl vehicle as in 1C or 1mg/kg RO in vehicle via carotid artery as in Methods. 15-16 hrs later, 50 μl vehicle prepared as in 1A or 100ng VEGF-A in vehicle was administered via carotid artery for 10-20 minutes using a catheter as in 1B. Brain MVs were isolated and extracted as in 1B. Left: p-VEGFR2 (Tyr1054/Tyr1059), VEGFR2, p-ERK1/2 and ERK1/2 are detected on WBs of extracts with specific antibodies in MV extracts. Vinculin: loading control. Right: Graphs show fold change of phosphorylated to total protein ratio. (E) , WT mice and mice HTRZ for PS1 FAD mutant M146V (WT/M146V) or I213T (WT/I213T) were treated with vehicle or VEGF-A via carotid artery for 10-20 minutes using a catheter as in 1D. Brain MVs were isolated and extracted as in 1B. Left: p-VEGFR2 (Tyr1054/Tyr1059), VEGFR2, p-ERK1/2 and ERK1/2 are detected on WBs of extracts with specific antibodies in MV extracts. β-actin: loading control. Right: Graphs show fold change of phosphorylated to total protein ratio. (F) , WT pCECs were prepared and treated as in Methods with vehicle (DMSO) or RO (200nM in DMSO) and then stimulated with either vehicle (PBS) or VEGF-A (20ng in PBS) for 15min. Upper: Cells were co-immunostained with either anti-VEGFR2 antibodies (green) or early endosome marker Rab5 (red) and cell nuclei were stained with Hoechst (blue) as in Methods. Yellow fluorescence in merged images indicates co-localization of VEGFR2 with Rab5. Scale bar 0.5μm. Lower: Graph shows percent of VEGFR2 co-localized with Rab5 in RO-treated WT cells compared to vehicle-treated cells measured with Imaris software. (G) , pCECs from either WT or mice HTRZ for PS1 FAD mutant M146V (WT/M146V) or I213T (WT/I213T), were stimulated with vehicle or VEGF-A in vehicle as in 1F. Upper: Cells were co-stained with anti-VEGFR2 antibodies and early endosome marker Rab5 as in 1F. Cell nuclei were stained with Hoechst (blue) as in 1F. Yellow fluorescence in merged images indicates co-localization of VEGFR2 with Rab5. Scale bar 0.5μm. Lower: Graph shows percent of VEGFR2 co-localized with Rab5 in PS1 FAD WT/M146V or WT/I213T HTRZ mice compared to WT measured with Imaris software. For Figs A-G, data are shown as Mean ± S.E. from at least three independent experiments or as indicated in the dot plots. Statistical analysis was performed using two-way ANOVA followed by Tukey post-hoc test. ns = not significant, *p<0.05, **p<0.01, ***p<0.001.

Journal: bioRxiv

Article Title: PS FAD mutants and γ-secretase inhibition accumulate VEGFR2-derived peptide VCTF1 suppressing brain VEGFR2 dimerization, angiogenesis and neuroprotection

doi: 10.64898/2026.05.12.724648

Figure Lengend Snippet: (A) , Wild-type (WT) and KI mice HTRZ for either PS1 FAD mutant M146V (WT/M146V) or I213T (WT/I213T) were infused with vehicle (0.2% BSA in PBS) or VEGF-A (a total of 3.5μg in 100μl of vehicle) through the carotid artery for 15 days using a mini osmotic pump as in Methods. Left: Brain coronal sections (40μm thick) were prepared and immunostained with anti-Col IV antibodies to visualize brain vessels. Enhanced visualization surfaces were generated using Imaris software from representative confocal images of ipsilateral hemispheres. Scale bar: 50μm. Right: Graph shows total vessel length density in WT and PS1 FAD brains quantified using Imaris 9.9 software as in Methods. (B) , WT and HTRZ for PS1 FAD mutants M146V or I213T mice were injected through the carotid artery for 20 minutes with either vehicle or 100ng of VEGF-A in vehicle prepared as in 1A using a catheter as described in Methods. Brain microvessels (MV) were isolated as in Methods, lysed in Triton X-100 buffer, and subjected to immunoprecipitation (IP) with anti-VEGFR2 antibody or control IgG. Left: IPs were analyzed on Western blots (WBs) using anti-endoglin or anti-VEGFR2 antibodies (upper panel). Input samples are shown in lower panel. β-actin: loading control. Right: Graph shows quantification of endoglin co-IPed with VEGFR2, normalized to IPed VEGFR2. (C) , WT mice were infused for 15 days through the carotid artery with vehicle or VEGF-A in vehicle as in 1A using a mini osmotic pump (as in 1A). For RO injection, mice were treated with vehicle (2% DMSO, 30% PEG 300, 5% Tween-80 in ddH2O) or RO in vehicle (5mg/kg body weight) via five injections in tail vein one injection every three days, with first injection administered 1 hour before osmotic pump implantation. Brain coronal sections (40μm) were prepared and immunostained with anti-Col IV antibodies as in 1A. Left: Representative confocal images of ipsilateral hemispheres are shown prepared as in 1A. Scale bar: 50μm. Right: Graph shows total vessel length density quantified using Imaris software as in 1A. (D) , WT adult mice were treated with either 50μl vehicle as in 1C or 1mg/kg RO in vehicle via carotid artery as in Methods. 15-16 hrs later, 50 μl vehicle prepared as in 1A or 100ng VEGF-A in vehicle was administered via carotid artery for 10-20 minutes using a catheter as in 1B. Brain MVs were isolated and extracted as in 1B. Left: p-VEGFR2 (Tyr1054/Tyr1059), VEGFR2, p-ERK1/2 and ERK1/2 are detected on WBs of extracts with specific antibodies in MV extracts. Vinculin: loading control. Right: Graphs show fold change of phosphorylated to total protein ratio. (E) , WT mice and mice HTRZ for PS1 FAD mutant M146V (WT/M146V) or I213T (WT/I213T) were treated with vehicle or VEGF-A via carotid artery for 10-20 minutes using a catheter as in 1D. Brain MVs were isolated and extracted as in 1B. Left: p-VEGFR2 (Tyr1054/Tyr1059), VEGFR2, p-ERK1/2 and ERK1/2 are detected on WBs of extracts with specific antibodies in MV extracts. β-actin: loading control. Right: Graphs show fold change of phosphorylated to total protein ratio. (F) , WT pCECs were prepared and treated as in Methods with vehicle (DMSO) or RO (200nM in DMSO) and then stimulated with either vehicle (PBS) or VEGF-A (20ng in PBS) for 15min. Upper: Cells were co-immunostained with either anti-VEGFR2 antibodies (green) or early endosome marker Rab5 (red) and cell nuclei were stained with Hoechst (blue) as in Methods. Yellow fluorescence in merged images indicates co-localization of VEGFR2 with Rab5. Scale bar 0.5μm. Lower: Graph shows percent of VEGFR2 co-localized with Rab5 in RO-treated WT cells compared to vehicle-treated cells measured with Imaris software. (G) , pCECs from either WT or mice HTRZ for PS1 FAD mutant M146V (WT/M146V) or I213T (WT/I213T), were stimulated with vehicle or VEGF-A in vehicle as in 1F. Upper: Cells were co-stained with anti-VEGFR2 antibodies and early endosome marker Rab5 as in 1F. Cell nuclei were stained with Hoechst (blue) as in 1F. Yellow fluorescence in merged images indicates co-localization of VEGFR2 with Rab5. Scale bar 0.5μm. Lower: Graph shows percent of VEGFR2 co-localized with Rab5 in PS1 FAD WT/M146V or WT/I213T HTRZ mice compared to WT measured with Imaris software. For Figs A-G, data are shown as Mean ± S.E. from at least three independent experiments or as indicated in the dot plots. Statistical analysis was performed using two-way ANOVA followed by Tukey post-hoc test. ns = not significant, *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: Mouse monoclonal anti-Flag tag (M2; F1804) was from Millipore Sigma, anti-GAPDH (2118S) from Cell Signaling Technologies (Beverly, MA), anti-VEGFR2 (OTI12C1) from Origene, anti-endoglin (CD-105; NBP2-22122) and anti-LAMP2 (NBP2-22217) from Novus Biologicals, Inc, anti-Rab5 (D-11) and anti-Rab7 (B-3) from Santa Cruz Biotechnology, Inc. Chicken polyclonal anti-GFAP (ab4674) was from Abcam.

Techniques: Mutagenesis, Generated, Software, Injection, Isolation, Immunoprecipitation, Control, Western Blot, Marker, Staining, Fluorescence

$(A), HEK293T cells transfected with either pCMV3 vector or VEGFR2-Myc-expressing vector were treated with either vehicle or RO overnight as in 1F. Membrane fractions were prepared as in Methods and incubated with vehicle (-) or VEGF-A as in 1F for 30 minutes in the presence of lactacystin. VEGFR2-Myc, VCTF1-Myc, and VCTF2-Myc, were then detected on WB using anti-Myc antibodies. Representative blot shows Myc-labelled fragments as indicated in Figure. (B), Left: HEK293T cells were transfected with VEGFR2-Myc and treated with vehicle or RO as in 3A and extracted in SDS buffer as in methods. VEGFR2-Myc and VCTF1-Myc were detected in cell extracts on WBs with antibodies recognizing the cytoplasmic sequence of VEGFR2 (ab39256). β-actin: loading control. Right: HEK293T cells were transfected as in 3A. Cells were pretreated with 200nm ADAM17 inhibitor D1 (A12: ADAM17 inh) for 1 hour and then stimulated with vehicle or VEGF-A as in 3A for 1h. Cells were extracted as in 3B Left. VEGFR2-Myc and its proteolytic product VCTF1-Myc are detected with anti-Myc antibody on WB as in 3A. Vinculin: loading control. (C), Left: WT pCECs were treated with either vehicle (DMSO; Veh) or RO as in 1F for 15-16 h and extracted in SDS buffer. VEGFR2 and VCTF1 were detected in cell extracts on WB with anti-VEGFR2 antibody as in 3B Left. β-actin: loading control. Right: Brain MVs were isolated from adult WT mice as in 1B following 15 hours of treatment with either vehicle or RO as in 1D and extracted in SDS buffer as in Methods. VEGFR2 and VCTF1 were detected in MV extracts on WB with anti-VEGFR2 antibody as in 3B Left. β-actin: loading control. (D), Left: pCECs isolated from WT or PS1 knockout (PS1 KO) mouse embryos were extracted as in 3C. VEGFR2 and VCTF1 were detected in cell extracts on WB with anti-VEGFR2 antibody as in 3B Left. β-actin: loading control. Right: HEK293T cells expressing VEGFR2-Myc were transfected with anti-PS1 siRNA or non-targeting control siRNA (NT siRNA) as in Methods and extracted as in 3B. VEGFR2-Myc and VCTF1-Myc were detected in cell extracts with anti-Myc antibody as in 3A. PS1 N-terminal fragment (PS1/NTF) was detected in cell extracts with anti-PS1 antibody (R222; 41). β-actin: loading control. (E), Left: Extracts from WT and either WT/M146V- or WT/I213T-expressing pCECs were prepared as in 3C. VEGFR2 and VCTF1 were detected in cell extracts on WB with anti-VEGFR2 antibody as in 3B Left. β-actin: loading control. Right: Brain MVs were isolated from WT and either WT/M146V- or WT/I213T-expressing mice as in 1B and extracted as in 3C Right. VEGFR2 and VCTF1 were detected in MV extracts on WB with anti-VEGFR2 antibody as in 3B Left. β-actin: loading control. Each WB is representative of at least three independent experiments.$

Journal: bioRxiv

Article Title: PS FAD mutants and γ-secretase inhibition accumulate VEGFR2-derived peptide VCTF1 suppressing brain VEGFR2 dimerization, angiogenesis and neuroprotection

doi: 10.64898/2026.05.12.724648

Figure Lengend Snippet: (A), HEK293T cells transfected with either pCMV3 vector or VEGFR2-Myc-expressing vector were treated with either vehicle or RO overnight as in 1F. Membrane fractions were prepared as in Methods and incubated with vehicle (-) or VEGF-A as in 1F for 30 minutes in the presence of lactacystin. VEGFR2-Myc, VCTF1-Myc, and VCTF2-Myc, were then detected on WB using anti-Myc antibodies. Representative blot shows Myc-labelled fragments as indicated in Figure. (B), Left: HEK293T cells were transfected with VEGFR2-Myc and treated with vehicle or RO as in 3A and extracted in SDS buffer as in methods. VEGFR2-Myc and VCTF1-Myc were detected in cell extracts on WBs with antibodies recognizing the cytoplasmic sequence of VEGFR2 (ab39256). β-actin: loading control. Right: HEK293T cells were transfected as in 3A. Cells were pretreated with 200nm ADAM17 inhibitor D1 (A12: ADAM17 inh) for 1 hour and then stimulated with vehicle or VEGF-A as in 3A for 1h. Cells were extracted as in 3B Left. VEGFR2-Myc and its proteolytic product VCTF1-Myc are detected with anti-Myc antibody on WB as in 3A. Vinculin: loading control. (C), Left: WT pCECs were treated with either vehicle (DMSO; Veh) or RO as in 1F for 15-16 h and extracted in SDS buffer. VEGFR2 and VCTF1 were detected in cell extracts on WB with anti-VEGFR2 antibody as in 3B Left. β-actin: loading control. Right: Brain MVs were isolated from adult WT mice as in 1B following 15 hours of treatment with either vehicle or RO as in 1D and extracted in SDS buffer as in Methods. VEGFR2 and VCTF1 were detected in MV extracts on WB with anti-VEGFR2 antibody as in 3B Left. β-actin: loading control. (D), Left: pCECs isolated from WT or PS1 knockout (PS1 KO) mouse embryos were extracted as in 3C. VEGFR2 and VCTF1 were detected in cell extracts on WB with anti-VEGFR2 antibody as in 3B Left. β-actin: loading control. Right: HEK293T cells expressing VEGFR2-Myc were transfected with anti-PS1 siRNA or non-targeting control siRNA (NT siRNA) as in Methods and extracted as in 3B. VEGFR2-Myc and VCTF1-Myc were detected in cell extracts with anti-Myc antibody as in 3A. PS1 N-terminal fragment (PS1/NTF) was detected in cell extracts with anti-PS1 antibody (R222; 41). β-actin: loading control. (E), Left: Extracts from WT and either WT/M146V- or WT/I213T-expressing pCECs were prepared as in 3C. VEGFR2 and VCTF1 were detected in cell extracts on WB with anti-VEGFR2 antibody as in 3B Left. β-actin: loading control. Right: Brain MVs were isolated from WT and either WT/M146V- or WT/I213T-expressing mice as in 1B and extracted as in 3C Right. VEGFR2 and VCTF1 were detected in MV extracts on WB with anti-VEGFR2 antibody as in 3B Left. β-actin: loading control. Each WB is representative of at least three independent experiments.

Techniques: Transfection, Plasmid Preparation, Expressing, Membrane, Incubation, Sequencing, Control, Isolation, Knock-Out

(A), HEK293T cells were transfected with VEGFR2-Myc as in 3A and treated with vehicle or VEGF-A for the indicated times, in the presence or absence of RO as in 1F and extracted in SDS buffer. Left: VEGFR2 dimer and monomer were detected on WB with anti-Myc antibodies. β-actin: loading control. Right: Graph shows the fold change in the VEGFR2 dimer to monomer ratio. (B), WT mice were treated with vehicle or RO as in 1D. Mice were treated with VEGF-A, and brain MVs were isolated and extracted as in 1B. Left: VEGFR2 dimers and monomers were detected on WB with anti-VEGFR2 antibody D5B1. Vinculin: loading control. Right: Graph shows the fold change in the VEGFR2 dimer to monomer ratio. (C), WT and WT/M146V or WT/I213T mice were injected with either vehicle or VEGF-A via the carotid artery as in 1D, and brain MVs were prepared as in 1B and extracted as in 3C Right. Left: VEGFR2 dimers and monomers were detected on WB with anti-VEGFR2 antibody as in 4B Left. β-actin: loading control. Right: graph shows the fold change in the VEGFR2 dimer to monomer ratio. (D), HEK293T cells were co-transfected with VEGFR2-Myc as in 3A and either WT PS1 or PS1 mutant M146V or I213T in FCbAIGW vector as indicated in Figure. Cells were treated with VEGF-A as in 1G for the indicated times and extracted in SDS buffer. Left: VEGFR2 dimers and monomers were detected in cell extract on WB with anti-Myc antibody as in 4A. Full length PS1 (FL-PS1) and PS1/NTF were detected with R222 (middle). Vinculin: loading control. Right: Graph shows the fold change in the VEGFR2 dimer to monomer ratio. (E), HEK293 cells were co-transfected with VEGFR2-Myc and either non-targeting or anti-PS1 siRNA as in 3D. Cells were treated with vehicle (0 lanes) or VEGF-A as in 4D above for the indicated times and extracted in SDS buffer. Left: VEGFR2 dimers and monomers and VCTF1 were detected on WB with anti-Myc antibody as in 4A. PS1/NTF was detected in cell extracts with R222. β-actin: loading control. Right: graphs show fold change in PS1/NTF levels (upper) and fold change in the VEGFR2 dimer/monomer ratio (lower) following treatment with anti-PS1 siRNA. PS1 downregulation resulted in decreased VEGFR2 dimerization and increased VCTF1-Myc (upper panel). (F), Embryonic brain (E15.5) extract from WT or PS1 knockout (PS1 KO) mice were prepared as described . Representative WB of extracts shows VEGFR2 dimers, detected with anti-VEGFR2 antibody D5B1, and PS1-NTF detected with R222 antibody. Vinculin: loading control. A–E: data are presented as mean ± SE from at least three independent experiments. Statistical analysis was performed by two-way ANOVA followed by Tukey post-hoc test. ns = not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Journal: bioRxiv

Article Title: PS FAD mutants and γ-secretase inhibition accumulate VEGFR2-derived peptide VCTF1 suppressing brain VEGFR2 dimerization, angiogenesis and neuroprotection

doi: 10.64898/2026.05.12.724648

Figure Lengend Snippet: (A), HEK293T cells were transfected with VEGFR2-Myc as in 3A and treated with vehicle or VEGF-A for the indicated times, in the presence or absence of RO as in 1F and extracted in SDS buffer. Left: VEGFR2 dimer and monomer were detected on WB with anti-Myc antibodies. β-actin: loading control. Right: Graph shows the fold change in the VEGFR2 dimer to monomer ratio. (B), WT mice were treated with vehicle or RO as in 1D. Mice were treated with VEGF-A, and brain MVs were isolated and extracted as in 1B. Left: VEGFR2 dimers and monomers were detected on WB with anti-VEGFR2 antibody D5B1. Vinculin: loading control. Right: Graph shows the fold change in the VEGFR2 dimer to monomer ratio. (C), WT and WT/M146V or WT/I213T mice were injected with either vehicle or VEGF-A via the carotid artery as in 1D, and brain MVs were prepared as in 1B and extracted as in 3C Right. Left: VEGFR2 dimers and monomers were detected on WB with anti-VEGFR2 antibody as in 4B Left. β-actin: loading control. Right: graph shows the fold change in the VEGFR2 dimer to monomer ratio. (D), HEK293T cells were co-transfected with VEGFR2-Myc as in 3A and either WT PS1 or PS1 mutant M146V or I213T in FCbAIGW vector as indicated in Figure. Cells were treated with VEGF-A as in 1G for the indicated times and extracted in SDS buffer. Left: VEGFR2 dimers and monomers were detected in cell extract on WB with anti-Myc antibody as in 4A. Full length PS1 (FL-PS1) and PS1/NTF were detected with R222 (middle). Vinculin: loading control. Right: Graph shows the fold change in the VEGFR2 dimer to monomer ratio. (E), HEK293 cells were co-transfected with VEGFR2-Myc and either non-targeting or anti-PS1 siRNA as in 3D. Cells were treated with vehicle (0 lanes) or VEGF-A as in 4D above for the indicated times and extracted in SDS buffer. Left: VEGFR2 dimers and monomers and VCTF1 were detected on WB with anti-Myc antibody as in 4A. PS1/NTF was detected in cell extracts with R222. β-actin: loading control. Right: graphs show fold change in PS1/NTF levels (upper) and fold change in the VEGFR2 dimer/monomer ratio (lower) following treatment with anti-PS1 siRNA. PS1 downregulation resulted in decreased VEGFR2 dimerization and increased VCTF1-Myc (upper panel). (F), Embryonic brain (E15.5) extract from WT or PS1 knockout (PS1 KO) mice were prepared as described . Representative WB of extracts shows VEGFR2 dimers, detected with anti-VEGFR2 antibody D5B1, and PS1-NTF detected with R222 antibody. Vinculin: loading control. A–E: data are presented as mean ± SE from at least three independent experiments. Statistical analysis was performed by two-way ANOVA followed by Tukey post-hoc test. ns = not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Techniques: Transfection, Control, Isolation, Injection, Mutagenesis, Plasmid Preparation, Knock-Out

(A), HEK293T cells were co-transfected with both constructs expressing complementary VEGFR2-Venus fusion proteins as in Methods. Cells were treated with vehicle or VEGF-A for 30 minutes in the presence or absence of RO as in 1F and imaged as in Methods. Left upper panel: Green fluorescence indicates VEGFR2 homodimer formation. Scale bar: 200μm. Left lower panel: Corresponding phase contrast images of cell cultures. Right: Graph shows fold induction of VEGFR2 homodimer formation based on fluorescent intensity measured using ImageJ as in Methods. (B), HEK293T cells were co-transfected with VEGFR2-Myc in pCMV3 vector and either WT PS1 or one of the PS1 FAD mutants indicated in Figure in FCbAIGW. Cells were treated with vehicle or VEGF-A for 0, 15 or 30 mins and then extracted in SDS buffer as in Methods. Left: VEGFR2 dimers and monomers were detected on WB with anti-VEGFR2 antibody (OTI12C1). PS1 (FL-PS1) and PS1/NTF were detected with R222. Vinculin: loading control. Right: Graph shows fold change in the VEGFR2 dimer to monomer ratio. For statistical analysis, two-way ANOVA followed by Tukey post-hoc test was used. ns = not significant, **p<0.01, ***p<0.001.

Journal: bioRxiv

Article Title: PS FAD mutants and γ-secretase inhibition accumulate VEGFR2-derived peptide VCTF1 suppressing brain VEGFR2 dimerization, angiogenesis and neuroprotection

doi: 10.64898/2026.05.12.724648

Figure Lengend Snippet: (A), HEK293T cells were co-transfected with both constructs expressing complementary VEGFR2-Venus fusion proteins as in Methods. Cells were treated with vehicle or VEGF-A for 30 minutes in the presence or absence of RO as in 1F and imaged as in Methods. Left upper panel: Green fluorescence indicates VEGFR2 homodimer formation. Scale bar: 200μm. Left lower panel: Corresponding phase contrast images of cell cultures. Right: Graph shows fold induction of VEGFR2 homodimer formation based on fluorescent intensity measured using ImageJ as in Methods. (B), HEK293T cells were co-transfected with VEGFR2-Myc in pCMV3 vector and either WT PS1 or one of the PS1 FAD mutants indicated in Figure in FCbAIGW. Cells were treated with vehicle or VEGF-A for 0, 15 or 30 mins and then extracted in SDS buffer as in Methods. Left: VEGFR2 dimers and monomers were detected on WB with anti-VEGFR2 antibody (OTI12C1). PS1 (FL-PS1) and PS1/NTF were detected with R222. Vinculin: loading control. Right: Graph shows fold change in the VEGFR2 dimer to monomer ratio. For statistical analysis, two-way ANOVA followed by Tukey post-hoc test was used. ns = not significant, **p<0.01, ***p<0.001.

Techniques: Transfection, Construct, Expressing, Fluorescence, Plasmid Preparation, Control

(A), pCECs from WT or HTRZ (WT/M146V or WT/I213T) or HMZG (M146V/M146V or I213T/I213T) mice were extracted in SDS buffer as in Methods. VEGFR2 and VCTF1 were detected on WBs of cell extracts as in 3B with the anti-VEGFR2 antibody (ab39256). β-actin: loading control. (B), Brain MVs were isolated from adult WT mice or mice HTRZ or HMZG for PS1 FAD mutants M146V or I213T (see Suppl. 5A). VEGFR2 and VCTF1 were detected in MV extracts on WB as in Suppl. 5A with anti-VEGFR2 antibody (ab39256). β-actin: loading control.

Journal: bioRxiv

Article Title: PS FAD mutants and γ-secretase inhibition accumulate VEGFR2-derived peptide VCTF1 suppressing brain VEGFR2 dimerization, angiogenesis and neuroprotection

doi: 10.64898/2026.05.12.724648

Figure Lengend Snippet: (A), pCECs from WT or HTRZ (WT/M146V or WT/I213T) or HMZG (M146V/M146V or I213T/I213T) mice were extracted in SDS buffer as in Methods. VEGFR2 and VCTF1 were detected on WBs of cell extracts as in 3B with the anti-VEGFR2 antibody (ab39256). β-actin: loading control. (B), Brain MVs were isolated from adult WT mice or mice HTRZ or HMZG for PS1 FAD mutants M146V or I213T (see Suppl. 5A). VEGFR2 and VCTF1 were detected in MV extracts on WB as in Suppl. 5A with anti-VEGFR2 antibody (ab39256). β-actin: loading control.

Techniques: Control, Isolation

(A), HEK293T cells were co-transfected with vector expressing VEGFR2-Myc and plasmids expressing EphB2/CTF1-Flag, VCTF1-Flag, or vector alone. Cells were stimulated with vehicle (-) or VEGF-A (+) as in 1F and then extracted in SDS buffer. Upper panel: VEGFR2 monomers and dimers are detected on WBs using anti-VEGFR2 antibody OTI12C1. Middle panel: VCTF1-Flag and EphB2/CTF1-Flag expression was detected using anti-Flag antibody. Lower panel: GAPDH, loading control. Graph shows fold change of the VEGFR2 dimer to monomer ratio. (B), HEK293T cells were co-transfected with vector expressing VEGFR2-Myc and either vector alone or increasing amounts of vector expressing VCTF1-Flag (200ng, 400ng, 800ng, or 1000ng). Forty-eight hours post-transfection cells were stimulated with either vehicle or VEGF-A as in 1F for 20 min and extracted in SDS buffer. Upper panel: VEGFR2 dimers and monomers (VEGFR2-Myc) are detected in cell lysates on WBs using anti-VEGFR2 antibody OTI12C1. Middle panel: VCTF1-Flag is detected with anti-Flag antibody. Lower panel: GAPDH: loading control. Bottom: Non-linear regression analysis (inhibitor vs. response, three-parameter model) showed a good fit to the curve (R² = 0.7410), indicating that increase of VCTF1-Flag expression inhibits VEGF-A-induced VEGFR2 dimerization. The red line (squares) represents VEGF-A-treated cells, whereas the blue line (circles) represents vehicle-treated cells. (C), Cells described in 5A were lysed in Triton X-100 buffer as in Methods and lysates were IPed with anti-Flag antibody. Upper panel: VEGFR2-Myc co-IPed with VCTF1-Flag is detected on WB using anti-Myc antibody as in 3A. Second panel: IPed VCTF1-Flag and EphB2/CTF1-Flag are detected on WBs with anti-Flag antibody. Third panel: Input of VEGFR2-Myc is detected with anti-Myc antibody as in 3A. Fourth panel: Input VCTF1-Flag and EphB2/CTF1-Flag are detected with Flag antibody. GAPDH: loading control. (D), bEnd3 cells were transduced with lentiviral vector FCbAIGW expressing VCTF1-Flag or empty vector as in Methods. Cells were treated with vehicle (-) or VEGF-A (+) as in Suppl. 4A for 7 minutes and extracted in SDS buffer as in Methods. Left: VEGFR2 dimers, monomers, p-VEGFR2 (Tyr1175), p-ERK1/2, ERK1/2 and VCTF1-Flag are detected on WBs with specific antibodies. Right: Graphs show fold change of VEGFR2 dimer to monomer ratio or p-VEGFR2/VEGFR2 and p-ERK1/2/ERK1 protein ratios. (E), bend3 cells expressing either empty vector (FCbAIGW) or VCTF1-Flag as in 5D were seeded as in Methods and treated with vehicle (-) or VEGF-A (+) as in Suppl. 4A for 6 hours. Upper: Representative photomicrographs show tube-like (loop/mesh) structures. Fluorescent images (EGFP, green) are shown. Scale bar 200μm. Lower: Graph shows quantification of tube formation as average number of loops/meshes per field. For statistical analysis, two-way ANOVA followed by Tukey post-hoc test was performed. ns = not significant, *p<0.05, **p<0.01, ***p<0.001.

Journal: bioRxiv

Article Title: PS FAD mutants and γ-secretase inhibition accumulate VEGFR2-derived peptide VCTF1 suppressing brain VEGFR2 dimerization, angiogenesis and neuroprotection

doi: 10.64898/2026.05.12.724648

Figure Lengend Snippet: (A), HEK293T cells were co-transfected with vector expressing VEGFR2-Myc and plasmids expressing EphB2/CTF1-Flag, VCTF1-Flag, or vector alone. Cells were stimulated with vehicle (-) or VEGF-A (+) as in 1F and then extracted in SDS buffer. Upper panel: VEGFR2 monomers and dimers are detected on WBs using anti-VEGFR2 antibody OTI12C1. Middle panel: VCTF1-Flag and EphB2/CTF1-Flag expression was detected using anti-Flag antibody. Lower panel: GAPDH, loading control. Graph shows fold change of the VEGFR2 dimer to monomer ratio. (B), HEK293T cells were co-transfected with vector expressing VEGFR2-Myc and either vector alone or increasing amounts of vector expressing VCTF1-Flag (200ng, 400ng, 800ng, or 1000ng). Forty-eight hours post-transfection cells were stimulated with either vehicle or VEGF-A as in 1F for 20 min and extracted in SDS buffer. Upper panel: VEGFR2 dimers and monomers (VEGFR2-Myc) are detected in cell lysates on WBs using anti-VEGFR2 antibody OTI12C1. Middle panel: VCTF1-Flag is detected with anti-Flag antibody. Lower panel: GAPDH: loading control. Bottom: Non-linear regression analysis (inhibitor vs. response, three-parameter model) showed a good fit to the curve (R² = 0.7410), indicating that increase of VCTF1-Flag expression inhibits VEGF-A-induced VEGFR2 dimerization. The red line (squares) represents VEGF-A-treated cells, whereas the blue line (circles) represents vehicle-treated cells. (C), Cells described in 5A were lysed in Triton X-100 buffer as in Methods and lysates were IPed with anti-Flag antibody. Upper panel: VEGFR2-Myc co-IPed with VCTF1-Flag is detected on WB using anti-Myc antibody as in 3A. Second panel: IPed VCTF1-Flag and EphB2/CTF1-Flag are detected on WBs with anti-Flag antibody. Third panel: Input of VEGFR2-Myc is detected with anti-Myc antibody as in 3A. Fourth panel: Input VCTF1-Flag and EphB2/CTF1-Flag are detected with Flag antibody. GAPDH: loading control. (D), bEnd3 cells were transduced with lentiviral vector FCbAIGW expressing VCTF1-Flag or empty vector as in Methods. Cells were treated with vehicle (-) or VEGF-A (+) as in Suppl. 4A for 7 minutes and extracted in SDS buffer as in Methods. Left: VEGFR2 dimers, monomers, p-VEGFR2 (Tyr1175), p-ERK1/2, ERK1/2 and VCTF1-Flag are detected on WBs with specific antibodies. Right: Graphs show fold change of VEGFR2 dimer to monomer ratio or p-VEGFR2/VEGFR2 and p-ERK1/2/ERK1 protein ratios. (E), bend3 cells expressing either empty vector (FCbAIGW) or VCTF1-Flag as in 5D were seeded as in Methods and treated with vehicle (-) or VEGF-A (+) as in Suppl. 4A for 6 hours. Upper: Representative photomicrographs show tube-like (loop/mesh) structures. Fluorescent images (EGFP, green) are shown. Scale bar 200μm. Lower: Graph shows quantification of tube formation as average number of loops/meshes per field. For statistical analysis, two-way ANOVA followed by Tukey post-hoc test was performed. ns = not significant, *p<0.05, **p<0.01, ***p<0.001.

Techniques: Transfection, Plasmid Preparation, Expressing, Control, Transduction

(A), Brain tissue extracts were prepared as in Methods from twelve PS1 FAD patients each carrying a different PS1 mutation, and twelve non-demented controls described in Methods. Left: VEGFR2 dimers and monomers were detected in brain extracts on WBs using anti-VEGFR2 antibody D5B1. Representative gels with control (C1-4) or FAD samples (FAD1-4) expressing mutants P264L, A260V, N135S and P242H respectively are shown. Vinculin: loading control. Right: Graph shows the fold change in VEGFR2 dimer to monomer ratio of FAD and control samples. (B), Brain tissue extract from control and PS1 FAD patient brains described in 7A were prepared and IPed with anti-endoglin antibody (ab252345) or IgG as in Methods. Upper panel: VEGFR2 co-IPed with endoglin was detected on WBs using an anti-VEGFR2 antibody as in 7A. Lower panel: Input samples are shown. Representative gel with control samples (C1, C2) and FAD samples (FAD1, FAD2) expressing mutants A260V and P264L respectively is shown. β-actin: loading control. Right: Graph shows relative levels of VEGFR2 co-precipitated with endoglin. (C), Brain sections from control and PS1 FAD patients were prepared as in Methods and stained for Col IV as in 1A. Upper: Representative images show brain vessels in either PS1 FAD or control (CT) brain sections. Scale bar: 80μm. Lower: Graph shows total vessel length density in PS1 FAD and CT brains quantified with Imaris software as in 1A. A-C , bars represent Mean ± S.E. For statistical analysis, unpaired t-test was performed. *p < 0.05, **p<0.01 and ***p<0.001.

Journal: bioRxiv

Article Title: PS FAD mutants and γ-secretase inhibition accumulate VEGFR2-derived peptide VCTF1 suppressing brain VEGFR2 dimerization, angiogenesis and neuroprotection

doi: 10.64898/2026.05.12.724648

Figure Lengend Snippet: (A), Brain tissue extracts were prepared as in Methods from twelve PS1 FAD patients each carrying a different PS1 mutation, and twelve non-demented controls described in Methods. Left: VEGFR2 dimers and monomers were detected in brain extracts on WBs using anti-VEGFR2 antibody D5B1. Representative gels with control (C1-4) or FAD samples (FAD1-4) expressing mutants P264L, A260V, N135S and P242H respectively are shown. Vinculin: loading control. Right: Graph shows the fold change in VEGFR2 dimer to monomer ratio of FAD and control samples. (B), Brain tissue extract from control and PS1 FAD patient brains described in 7A were prepared and IPed with anti-endoglin antibody (ab252345) or IgG as in Methods. Upper panel: VEGFR2 co-IPed with endoglin was detected on WBs using an anti-VEGFR2 antibody as in 7A. Lower panel: Input samples are shown. Representative gel with control samples (C1, C2) and FAD samples (FAD1, FAD2) expressing mutants A260V and P264L respectively is shown. β-actin: loading control. Right: Graph shows relative levels of VEGFR2 co-precipitated with endoglin. (C), Brain sections from control and PS1 FAD patients were prepared as in Methods and stained for Col IV as in 1A. Upper: Representative images show brain vessels in either PS1 FAD or control (CT) brain sections. Scale bar: 80μm. Lower: Graph shows total vessel length density in PS1 FAD and CT brains quantified with Imaris software as in 1A. A-C , bars represent Mean ± S.E. For statistical analysis, unpaired t-test was performed. *p < 0.05, **p<0.01 and ***p<0.001.

Techniques: Mutagenesis, Control, Expressing, Staining, Software

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