Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Nrf2 acts cell-autonomously in endothelium to regulate tip cell formation and vascular branching

doi: 10.1073/pnas.1309276110

Figure Lengend Snippet: Deletion of endothelial Nrf2 leads to increased Dll4/Notch signaling. (A–C) VEGF protein levels at P5 (n = 5). (D) Quantitative RT-PCR analysis of Dll4 and Notch target genes in the retinas of Nrf2−/− and WT mice at P5 (n = 4). (E) Quantitative RT-PCR analysis of Dll4 and Notch target genes in the retinas of Nrf2fl/fl;Six3-Cre and control mice at P5 (n = 6). (F, Upper) Laser-capture microdissection of blood vessels. (Scale bar, 100 µm.) (F, Lower) Quantitative RT-PCR analysis of Dll4 and Notch target genes in laser-capture microdissected blood vessels from Nrf2fl/fl;Cdh5-Cre and control retinas at P5 (n = 5). (G) Increased Dll4 expression was observed in the angiogenic front (arrowheads) in Nrf2fl/fl;Cdh5-Cre retinas compared with control at P5. (Scale bar, 25 µm.) Data are presented as mean ± SEM (*P < 0.05 and **P < 0.01; NS, not significant).

Article Snippet: The following primary antibodies were used: monoclonal mouse anti-Nrf2 (5 ng/μL; R&D Systems), monoclonal rat anti-mouse PECAM-1(1:50; BD Pharmingen), and polyclonal goat anti-mouse Dll4 (15 ng/μL; R&D Systems).

Techniques: Quantitative RT-PCR, Control, Laser Capture Microdissection, Expressing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Nrf2 acts cell-autonomously in endothelium to regulate tip cell formation and vascular branching

doi: 10.1073/pnas.1309276110

Figure Lengend Snippet: Inhibition of Dll4/Notch signaling abrogates the restrained sprouting angiogenesis in Nrf2-deficient mice. (A and C) PECAM-1–stained P5 retina from Nrf2−/− and WT mice after 24 h treatment (P4–P5) with Dll4 antibody (intravitreous injection) (A) or DAPT (i.p. injection) (C). Dll4 antibody or DAPT administration results in comparable vascular hyperplasia in the affected region (between dashed lines) in Nrf2−/− and WT retinas. (B and D) Quantification of vascular density in P5 retinas after 24 h treatment (P4–P5) with Dll4 antibody (intravitreous injection; B) or DAPT (i.p. injection; D); n = 6 per group for Dll4 antibody injection; n = 5 per group for DAPT injection. (E) Aortic ring explants from Nrf2fl/fl;Cdh5-Cre and control mice treated with Dll4 antibody or DAPT, respectively. (F) Quantification of outgrowth area of aortic explants shown in E (n = 7). (Scale bar, 200 µm.) Data are presented as mean ± SEM (*P < 0.05 and **P < 0.01; NS, not significant).

Article Snippet: The following primary antibodies were used: monoclonal mouse anti-Nrf2 (5 ng/μL; R&D Systems), monoclonal rat anti-mouse PECAM-1(1:50; BD Pharmingen), and polyclonal goat anti-mouse Dll4 (15 ng/μL; R&D Systems).

Techniques: Inhibition, Staining, Injection, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Nrf2 acts cell-autonomously in endothelium to regulate tip cell formation and vascular branching

doi: 10.1073/pnas.1309276110

Figure Lengend Snippet: Deletion of the Nrf2 repressor Keap1 in ECs promotes sprouting angiogenesis via suppression of Dll4/Notch signaling. (A) Visualization of blood vessels by PECAM-1 staining of control (Keap1fl/fl) and Keap1fl/fl;Cdh5-Cre retinas at P5. Enhanced vascular density (B), branch points (C), and hypersprouting characterized by increased tip cell numbers (D) and filopodia (E) were observed in Keap1fl/fl;Cdh5-Cre retina (n = 6). (F and G) Isolectin B4 (green) and BrdU labeling (red) of control and Keap1fl/fl;Cdh5-Cre retinas at P5 (n = 4). (Scale bar, 50 µm.) (H and I) Increased area of the deep vascular plexus was observed in Keap1fl/fl;Cdh5-Cre retina at P9 (n = 6). (Scale bar, 500 µm.) (J and K) Keap1 knockdown enhanced HREC tube formation. (Scale bar, 100 µm.) (L) Increased Dll4 expression was observed at the angiogenic front (arrowheads) in Nrf2fl/fl;Cdh5-Cre retinas at P5. (Scale bar, 25 µm.) (M) Quantitative RT-PCR analysis of Dll4 and Notch target genes in HRECs cultured in a collagen-sandwich gel. (N) Immunoblot analysis of NICD and quantitative RT-PCR analysis of Dll4 in HRECs stimulated with rDll4. GAPDH was detected as a loading control. (O and P) Keap1 knockdown enhanced HREC spheroid sprouting with lower or higher level of VEGF. DAPT corrected the differential sprouting between control siRNA and Keap1 siRNA-transfected HRECs. (Scale bar, 50 µm.) (Q) Immunoblot analysis of Nrf2, HIF-1α, HIF-2α, Dll4, and NICD in HRECs under normoxia or hypoxia. (R and S) Quantitative RT-PCR analysis of HIF-2α in HRECs under hypoxia (R) or in blood vessels (S) from Keap1fl/fl;Cdh5-Cre mice at P5 (n = 4). Data are presented as mean ± SEM (*P < 0.05 and **P < 0.01; NS, not significant).

Article Snippet: The following primary antibodies were used: monoclonal mouse anti-Nrf2 (5 ng/μL; R&D Systems), monoclonal rat anti-mouse PECAM-1(1:50; BD Pharmingen), and polyclonal goat anti-mouse Dll4 (15 ng/μL; R&D Systems).

Techniques: Staining, Control, Labeling, Knockdown, Expressing, Quantitative RT-PCR, Cell Culture, Western Blot, Transfection

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Nrf2 acts cell-autonomously in endothelium to regulate tip cell formation and vascular branching

doi: 10.1073/pnas.1309276110

Figure Lengend Snippet: Nrf2 activation by Keap1 knockdown inhibits PI3K/Akt-dependent Notch signaling in ECs. (A) PI3K activity in HRECs. Knockdown of Keap1 blocked VEGF-induction of PI3K activity. (B) Immunoblot analysis of VEGFR2, Akt, and Erk in HRECs transfected with control siRNA or Keap1 siRNA in the presence or absence of VEGF. Knockdown of Keap1 inhibited VEGF-induced phosphorylation of VEGFR2 and Akt, but not Erk. (C) Immunoblot analysis of Dll4, NICD, Akt in lung or retina homogenates from Keap1fl/fl;Cdh5-Cre mice at P5. (D and E) Quantitative RT-PCR analysis of Dll4 (D) and Hey1 (E) in HRECs treated with PI3K inhibitor LY294002. (F) Immunoblot analysis of Dll4 and NICD in HRECs expressing constitutively active Akt. Suppression of Dll4 and NICD by Keap1 knockdown was reversed by adenovirus-mediated enhancement of Akt activity. GAPDH was detected as a loading control. (G) Proposed schematic of Nrf2 in developmental angiogenesis. Nrf2 activation by release from Keap1 suppresses Dll4/Notch signaling via inhibition of VEGF-induced VEGFR2-PI3K/Akt and down-regulation of HIF-2α, leading to the enhanced sprouting angiogenesis. Data are presented as mean ± SEM (*P < 0.05 and **P < 0.01; NS, not significant).

Article Snippet: The following primary antibodies were used: monoclonal mouse anti-Nrf2 (5 ng/μL; R&D Systems), monoclonal rat anti-mouse PECAM-1(1:50; BD Pharmingen), and polyclonal goat anti-mouse Dll4 (15 ng/μL; R&D Systems).

Techniques: Activation Assay, Knockdown, Activity Assay, Western Blot, Transfection, Control, Phospho-proteomics, Quantitative RT-PCR, Expressing, Inhibition

Journal: The Journal of Physiology

Article Title: Delta like 4 regulates cerebrovascular development and endothelial integrity via DLL4‐NOTCH‐CLDN5 pathway and is vulnerable to neonatal hyperoxia

doi: 10.1113/jp285716

Figure Lengend Snippet: Figure 1. Haploinsufficiency of Dll4 decreased brain weights at P10 compared to Dll4+/+ littermate controls Data are presented as the mean ± SD. A, brain weights at P10 (n = 8–13 mice/sex/group). B, body weights at P10 (n = 8–13 mice/sex/group). C, brain weights at P21 (n = 7–13 mice/sex/group). D, body weights at P21 (n = 5–20 mice/sex/group). E, brain weights at P100 (n = 3–4 mice/sex/group). F, body weights at P100 (n = 8–15 mice/sex/group). ∗P < 0.05, ∗∗P < 0.01.

Article Snippet: Antibodies against mouse DLL4 (#MAB1389-SP; R&D Systems Minneapolis, MN, USA), NICD (#4147; Cell Signaling, Danvers, MA, USA), HEY1 (#19929-1-AP; Thermo Fisher, Rockford, IL, USA), VEGFA (#ab46154; Abcam, Waltham, MA, USA), VEGFR2 (KDR) (#9698S; Cell Signaling), JAG1(#70109; Cell Signaling), HES1 (#11988; Cell Signaling), albumin (#16475-1-AP; Thermo Fisher), CLDN5 (#PA5-99415; Thermo Fisher) and ZO1 (TJP1, #ab276131; Abcam) were used to determine the protein abundances.

Techniques:

Journal: The Journal of Physiology

Article Title: Delta like 4 regulates cerebrovascular development and endothelial integrity via DLL4‐NOTCH‐CLDN5 pathway and is vulnerable to neonatal hyperoxia

doi: 10.1113/jp285716

Figure Lengend Snippet: Figure 2. DLL4 expression pattern in brain A–F, representative images of X-gal and CD31 double staining in the brains of Dll4+/+ (A, C and E) and Dll4+/LacZ

Article Snippet: Antibodies against mouse DLL4 (#MAB1389-SP; R&D Systems Minneapolis, MN, USA), NICD (#4147; Cell Signaling, Danvers, MA, USA), HEY1 (#19929-1-AP; Thermo Fisher, Rockford, IL, USA), VEGFA (#ab46154; Abcam, Waltham, MA, USA), VEGFR2 (KDR) (#9698S; Cell Signaling), JAG1(#70109; Cell Signaling), HES1 (#11988; Cell Signaling), albumin (#16475-1-AP; Thermo Fisher), CLDN5 (#PA5-99415; Thermo Fisher) and ZO1 (TJP1, #ab276131; Abcam) were used to determine the protein abundances.

Techniques: Expressing, Double Staining

Journal: The Journal of Physiology

Article Title: Delta like 4 regulates cerebrovascular development and endothelial integrity via DLL4‐NOTCH‐CLDN5 pathway and is vulnerable to neonatal hyperoxia

doi: 10.1113/jp285716

Figure Lengend Snippet: Figure 3. DLL4 was differentially expressed in different cell types in mouse brain A–R, immunofluorescence double staining with antibodies against DLL4 and different cell type markers in P21 Dll4+/+ mouse brain. Colocalization of DLL4 (green) and cell type marker (red) makes yellow. A–C, DLL4 double staining with CD31 (endothelial cell marker). D–F, DLL4 double staining with GFAP (astrocyte marker). G–I, DLL4 double staining with NeuN (neuronal marker). J–L, DLL4 double staining with CD11b (microglia marker). M–O, DLL4 double staining with Nestin (stem cell marker). P–R, DLL4 double staining with SOX10 (oligodendrocyte marker). S, X-gal (blue) double staining with venous marker NR2F2 (brown). T, X-gal (blue) double staining with venous marker EPHB$ (brown). Images were taken using a Keyence microscope at 20× magnification. Scale bar = 100 μm. U, Dll4 mRNA levels in different cell types of P21 Dll4+/+ female (F) and male (M) mouse brain. Data are presented as the mean ± SD. N = 3 mice/sex/group.

Article Snippet: Antibodies against mouse DLL4 (#MAB1389-SP; R&D Systems Minneapolis, MN, USA), NICD (#4147; Cell Signaling, Danvers, MA, USA), HEY1 (#19929-1-AP; Thermo Fisher, Rockford, IL, USA), VEGFA (#ab46154; Abcam, Waltham, MA, USA), VEGFR2 (KDR) (#9698S; Cell Signaling), JAG1(#70109; Cell Signaling), HES1 (#11988; Cell Signaling), albumin (#16475-1-AP; Thermo Fisher), CLDN5 (#PA5-99415; Thermo Fisher) and ZO1 (TJP1, #ab276131; Abcam) were used to determine the protein abundances.

Techniques: Immunofluorescence, Double Staining, Marker, Microscopy

Journal: The Journal of Physiology

Article Title: Delta like 4 regulates cerebrovascular development and endothelial integrity via DLL4‐NOTCH‐CLDN5 pathway and is vulnerable to neonatal hyperoxia

doi: 10.1113/jp285716

Figure Lengend Snippet: Figure 4. Haploinsufficiency of Dll4 decreased capillary diameter and increased vessel densities in P7, P21, and P100 mouse brains compared to Dll4+/+ littermate controls A–J, representative images of CD31 staining in female cortex (CTX, A and B), hippocampus (HP, C and D), mid brain (MB, E and F), cerebellum (CB, G and H) and brain stem (BS, I and J) regions of Dll4+/+ (A, C, E, G and I)

Article Snippet: Antibodies against mouse DLL4 (#MAB1389-SP; R&D Systems Minneapolis, MN, USA), NICD (#4147; Cell Signaling, Danvers, MA, USA), HEY1 (#19929-1-AP; Thermo Fisher, Rockford, IL, USA), VEGFA (#ab46154; Abcam, Waltham, MA, USA), VEGFR2 (KDR) (#9698S; Cell Signaling), JAG1(#70109; Cell Signaling), HES1 (#11988; Cell Signaling), albumin (#16475-1-AP; Thermo Fisher), CLDN5 (#PA5-99415; Thermo Fisher) and ZO1 (TJP1, #ab276131; Abcam) were used to determine the protein abundances.

Techniques: Staining

Journal: The Journal of Physiology

Article Title: Delta like 4 regulates cerebrovascular development and endothelial integrity via DLL4‐NOTCH‐CLDN5 pathway and is vulnerable to neonatal hyperoxia

doi: 10.1113/jp285716

Figure Lengend Snippet: Figure 5. Haploinsufficiency of Dll4 altered gene expression in the EC of Dll4+/Lacz mouse brain at P10 and impaired BBB integrity at P21 compared to Dll4+/+ littermate controls Data are presented as the mean ± SD. A, mRNA levels of Dll4, Hey1, Jag1, Hes1, Vegfa, Vegfr1-3, Nrp1-2 and Ephb4 in the EC of Dll4+/+ vs. Dll4+/Lacz female (F) and male (M) mouse brains. N = 5 mice/sex/group, ∗P < 0.05. B, protein levels of DLL4, NICD, VEGFA, VEGFR2, JAG1 and HES1 in the EC of Dll4+/+ vs. Dll4+/Lacz mouse brains. N = 4 mice/sex/group. C, Albumin levels in whole brain lysates of Dll4+/+ vs. Dll4+/Lacz mice at P7. N = 4–5 mice/sex/group, female P = 0.413, male P = 0.73. D, Albumin levels in whole brain lysates of Dll4+/+ vs. Dll4+/Lacz

Article Snippet: Antibodies against mouse DLL4 (#MAB1389-SP; R&D Systems Minneapolis, MN, USA), NICD (#4147; Cell Signaling, Danvers, MA, USA), HEY1 (#19929-1-AP; Thermo Fisher, Rockford, IL, USA), VEGFA (#ab46154; Abcam, Waltham, MA, USA), VEGFR2 (KDR) (#9698S; Cell Signaling), JAG1(#70109; Cell Signaling), HES1 (#11988; Cell Signaling), albumin (#16475-1-AP; Thermo Fisher), CLDN5 (#PA5-99415; Thermo Fisher) and ZO1 (TJP1, #ab276131; Abcam) were used to determine the protein abundances.

Techniques: Gene Expression

Journal: The Journal of Physiology

Article Title: Delta like 4 regulates cerebrovascular development and endothelial integrity via DLL4‐NOTCH‐CLDN5 pathway and is vulnerable to neonatal hyperoxia

doi: 10.1113/jp285716

Figure Lengend Snippet: Figure 6. DLL4 insufficiency led to a hypersprouting angiogenic phenotype in HBMEC and increased HBMEC permeability in vitro Data are presented as the mean ± SD. A–F, EC phenotype confirmation in HBMEC-Im cells by LDL uptake (J and K), CDH5 (L and M), and ERG (N and O) staining. Images were taken using a Keyence microscope at a 60× magnification. Scale bar = 100 μm. G and H, DLL4 insufficiency caused hyperbranching network in 3-D cell culture (H) compared to the controls, red arrow heads point to branches (G). Images were taken using a Keyence microscope at a 10× magnification. Scale bar = 100 μm. I, quantitative analysis of cells per bead in a 3-D angiogenesis assay. N = 17–19 beads/group across three different experiments. ∗∗∗∗P < 0.0001. J, quantitative analysis of length of tube formation. N = 13–14 beads/group across three different experiments. ∗∗∗∗P < 0.0001. K, mRNA levels of DLL4 signalling genes in DLL4 sh-RNA transduced cells relative to scRNA transduced cells. Data were collected from four different experiments. L, protein levels of DLL4 signalling genes in DLL4 sh-RNA trans- duced cells relative to scRNA transduced cells. Data were collected from four different experiments. ∗P < 0.05, ∗∗P < 0.01. M, normalized TEER values in scRNA transduced HBMEC (circles, top) vs. DLL4-shRNA transduced cells (triangles, bottom). Changes in the TEER were normalized to the initial values. Each plotted data point is presented as the mean ± SD. Data were collected from three different experiments. ∗P < 0.05. ∗∗P < 0.01, ∗∗∗P < 0.001. N, HMBEC-Im cells transduced with either scRNA or DLL4-shRNA were seeded and cultured in growth medium until a monolayer was formed and confirmed by cell membrane marker wheat germ agglutinin (WGA, green) and nuclei marker Hoechst (blue) double staining. O, non-monolayer controls. Images were taken using a Keyence microscope at 20× magnification. Scale bar = 100 μm. P, quantitative analysis of fluorescence value (485/535 nm excitation/emission). Data were collected from three different experiments, ∗P = 0.0256.

Article Snippet: Antibodies against mouse DLL4 (#MAB1389-SP; R&D Systems Minneapolis, MN, USA), NICD (#4147; Cell Signaling, Danvers, MA, USA), HEY1 (#19929-1-AP; Thermo Fisher, Rockford, IL, USA), VEGFA (#ab46154; Abcam, Waltham, MA, USA), VEGFR2 (KDR) (#9698S; Cell Signaling), JAG1(#70109; Cell Signaling), HES1 (#11988; Cell Signaling), albumin (#16475-1-AP; Thermo Fisher), CLDN5 (#PA5-99415; Thermo Fisher) and ZO1 (TJP1, #ab276131; Abcam) were used to determine the protein abundances.

Techniques: Permeability, In Vitro, Staining, Microscopy, Cell Culture, Angiogenesis Assay, shRNA, Transduction, Membrane, Marker, Double Staining, Fluorescence

Journal: The Journal of Physiology

Article Title: Delta like 4 regulates cerebrovascular development and endothelial integrity via DLL4‐NOTCH‐CLDN5 pathway and is vulnerable to neonatal hyperoxia

doi: 10.1113/jp285716

Figure Lengend Snippet: Figure 7. DLL4 insufficiency decreased tight junction protein CLDN5 expression in HBMEC through NOTCH-NICD-RBPJ-CLDN5 signalling Data are presented as the mean ± SD. A, mRNA levels of CLDN5 and TJP1 in HBMEC-im treated with scRNA vs. DLL4-shRNA. Data were collected from four different experiments, CLDN5, ∗P = 0.0104; TJP, P = 0.697. B, protein levels of CLDN5 and TJP1 in HBMEC-im treated with scRNA vs. DLL4-shRNA. Data were collected from four different experiments, CLDN5, ∗P = 0.0255; TJP, P = 0.113. C, mRNA levels of DLL4 and CLDN5 in primary HBMEC. Data were collected from four different experiments. DLL4, ∗∗P = 0.00202; CLDN5, ∗P = 0.0312. D, schematic representation of DLL4-NOTCH-NICD-CLDN5 pathway. E, schematic representation of human CLDN5 promoter with RBPJ site at –2168 bp upstream of transcription start site set as +1. F, sequences around RBPJ binding site of CLDN5 promoter among different species with the sequence for RBPJ bonding site capitalized and bold. G, NOTCH density at RBPJ site of CLDN5 promoter in HBMEC-im. Data were collected from three different experiments. ∗P = 0.0141. H, CLDN5 promoter reporter activity assay in HEK293T cells. Data were collected from three different experiments. ∗∗P = 0.0058 or 0.0059 compared to either empty vector pGL4.10-pcDNA 3.1 or vector + NICD only or vector + CLDN5 only. I, CLDN5 and HEY1 mRNA fold change in HBMEC-im treated with y-secretase inhibitors DAPT and DBZ. Data were collected from three different experiments. ∗∗∗∗P < 0.0001.

Article Snippet: Antibodies against mouse DLL4 (#MAB1389-SP; R&D Systems Minneapolis, MN, USA), NICD (#4147; Cell Signaling, Danvers, MA, USA), HEY1 (#19929-1-AP; Thermo Fisher, Rockford, IL, USA), VEGFA (#ab46154; Abcam, Waltham, MA, USA), VEGFR2 (KDR) (#9698S; Cell Signaling), JAG1(#70109; Cell Signaling), HES1 (#11988; Cell Signaling), albumin (#16475-1-AP; Thermo Fisher), CLDN5 (#PA5-99415; Thermo Fisher) and ZO1 (TJP1, #ab276131; Abcam) were used to determine the protein abundances.

Techniques: Expressing, shRNA, Binding Assay, Sequencing, Activity Assay, Plasmid Preparation

Journal: The Journal of Physiology

Article Title: Delta like 4 regulates cerebrovascular development and endothelial integrity via DLL4‐NOTCH‐CLDN5 pathway and is vulnerable to neonatal hyperoxia

doi: 10.1113/jp285716

Figure Lengend Snippet: Figure 8. Neonatal hyperoxia exposure decreased cerebral vessel density and DLL4 expression in the EC of mouse brain Data are presented as the mean ± SD. A–J, representative images of CD31 staining in male cortex (CTX, A and B), hippocampus (HP, C and D), mid brain (MB, E and F), cerebellum (CB, G and H) and brain stem (BS, I and J) regions of RA (A, C, E, G and I) and HOX (B, D, F, H and J). Images were taken using a Keyence light microscope at 20× magnification. Scale bar = 200 μm. K, quantitative analysis of vessel densities in different brain regions, female (F) and male (M). N = 4 mice/sex/group. ∗P < 0.05. L, Dll4 mRNA fold change in brain EC of room air (RA) vs. hyperoxia exposure (HOX) relative to Tubb5. N = 6 mice/sex/group; female, ∗∗P = 0.0079; male, ∗∗P = 0.0098. M, DLL4 protein levels in brain EC of RA vs. HOX relative vinculin. N = 4–5 mice/sex/group, female P = 0.382, male ∗P = 0.0317. N, representative immunofluorescence double staining of CD31 (green) and DLL4 (red) images in the CTX of female mice exposed to RA (top) and HOX (bottom) in females. Images were taken using a Keyence microscope at 20× magnification. Scale bar = 100 μm. O, quantitative analysis of DLL4 densities in cortex. N = 4 mice/sex/group; female, ∗P = 0.0286; male, ∗P = 0.0286.

Article Snippet: Antibodies against mouse DLL4 (#MAB1389-SP; R&D Systems Minneapolis, MN, USA), NICD (#4147; Cell Signaling, Danvers, MA, USA), HEY1 (#19929-1-AP; Thermo Fisher, Rockford, IL, USA), VEGFA (#ab46154; Abcam, Waltham, MA, USA), VEGFR2 (KDR) (#9698S; Cell Signaling), JAG1(#70109; Cell Signaling), HES1 (#11988; Cell Signaling), albumin (#16475-1-AP; Thermo Fisher), CLDN5 (#PA5-99415; Thermo Fisher) and ZO1 (TJP1, #ab276131; Abcam) were used to determine the protein abundances.

Techniques: Expressing, Staining, Light Microscopy, Immunofluorescence, Double Staining, Microscopy

Journal: The Journal of Physiology

Article Title: Delta like 4 regulates cerebrovascular development and endothelial integrity via DLL4‐NOTCH‐CLDN5 pathway and is vulnerable to neonatal hyperoxia

doi: 10.1113/jp285716

Figure Lengend Snippet: Figure 9. Neonatal hyperoxia exposure decreased Cldn5 mRNA levels in the EC and protein abundance in CTX region of mouse brain Data are presented as the mean ± SD. A, Cldn5 mRNA fold change in brain EC of room air (RA) vs. hyperoxia exposure (HOX) relative to Tubb5, female (F) and male (M). N = 5 mice/sex/group; female, ∗P < 0.05; male, ∗∗P < 0.01. B, representative immunofluorescence double staining images of CLDN5 (green) in the CTX of female mice exposed to RA (top) and HOX (bottom) in females. Images were taken using a Keyence microscope at 20× magnification. Scale bar = 100 μm. C, quantitative analysis of CLDN5 densities in cortex. N = 4 mice/sex/group. ∗P < 0.05 for both sexes. D, representative immunofluorescence triple staining images of DLL4 (green), CLDN5 (red) and CD31 (white) in the CTX of male mice exposed to RA (top) and HOX (bottom). Red arrows pointed to DLL4 positive vessels. Images were taken using a Keyence microscope at 60× magnification. Image exposure times for both the RA and HOX groups were configured as: 5 s for green, 1.2 s for red, 8 s for white and 1/3 s for blue, respectively. Image contrast was adjusted less than 10% for all images. E, DLL4 mRNA time course change in HBMEC exposed to normoxia vs. hyperoxia. Data were collected from three different experiments. ∗P < 0.05.

Article Snippet: Antibodies against mouse DLL4 (#MAB1389-SP; R&D Systems Minneapolis, MN, USA), NICD (#4147; Cell Signaling, Danvers, MA, USA), HEY1 (#19929-1-AP; Thermo Fisher, Rockford, IL, USA), VEGFA (#ab46154; Abcam, Waltham, MA, USA), VEGFR2 (KDR) (#9698S; Cell Signaling), JAG1(#70109; Cell Signaling), HES1 (#11988; Cell Signaling), albumin (#16475-1-AP; Thermo Fisher), CLDN5 (#PA5-99415; Thermo Fisher) and ZO1 (TJP1, #ab276131; Abcam) were used to determine the protein abundances.

Techniques: Quantitative Proteomics, Immunofluorescence, Double Staining, Microscopy, Staining

Journal: mAbs

Article Title: Simultaneous blockade of VEGF and Dll4 by HD105, a bispecific antibody, inhibits tumor progression and angiogenesis

doi: 10.1080/19420862.2016.1171432

Figure Lengend Snippet: Simultaneous binding to VEGF and Dll4 by HD105 bispecific antibody leads to effective blockade of VEGF/VEGFR2 and Dll4/Notch1 interactions. The HD105 bispecific antibody was constructed of the C-terminal of the anti-VEGF (bevacizumab-similar) IgG backbone linked with a single-chain Fv targeting Dll4 (A). The binding affinity of the HD105 bispecific antibody against human VEGF or human Dll4 was determined by Biacore assays (B) and ELISAs (C, D). The KD values of each antibody against VEGF or Dll4 are summarized in Table (B). The HD105 bispecific antibody (closed circle) dose-dependently bound to human VEGF (C) or Dll4 (D). In addition, the HD105 bispecific antibody simultaneously bound to each antigen, human VEGF and human Dll4, in dual-antigen capture ELISAs (E). The anti-Dll4 antibody (open circle in C) or the anti-VEGF (bevacizumab-similar) antibody (open circle in D, E) was used as negative control. Competitive ELISAs demonstrated that the HD105 bispecific antibody inhibited the interaction between VEGF/VEGFR2 (F) or Dll4/Notch1 (G) in a dose-dependent manner. The EC50 (half maximal effective concentration) values of the anti-VEGF (bevacizumab-similar) antibody (open circle) and HD105 bispecific antibody (closed circle) for VEGF/VEGFR2 inhibition were 2.98 ± 0.5 nM and 2.84 ± 0.41 nM, respectively (F). The EC50 values of the anti-Dll4 antibody (open circle) and HD105 bispecific antibody (closed circle) were 0.65 ± 0.06 nM and 1.14 ± 0.06 nM, respectively (G).

Article Snippet: Antibodies and cell culture An anti-VEGF (bevacizumab-similar) antibody, an anti-Dll4 monoclonal antibody, the HD105 bispecific antibody, an anti-mouse Dll4 monoclonal antibody, and a mouse version of the HD105 bispecific antibody were produced by Hanwha Chemical, Biologics R&D Center (Daejeon, South Korea).

Techniques: Binding Assay, Construct, Negative Control, Concentration Assay, Inhibition

Journal: mAbs

Article Title: Simultaneous blockade of VEGF and Dll4 by HD105, a bispecific antibody, inhibits tumor progression and angiogenesis

doi: 10.1080/19420862.2016.1171432

Figure Lengend Snippet: Blockade of both VEGF/VEGFR2 and Dll4/Notch1 signaling pathways by HD105 bispecific antibody leads to inhibition of each signaling-induced cellular response. The HD105 bispecific antibody inhibited both the VEGF/VEGFR2 and the Dll4/Notch1 signaling pathways in HUVECs (A). The VEGF/VEGFR2 signaling pathway was monitored by the activation of VEGFR2 and ERK (phosphorylation). The Dll4/Notch1 signaling pathway was monitored by the generation of NICD (Notch-induced intracellular domain). HUVEC sprouting assays were performed in a fibrin gel in the presence of PBS (B), anti-VEGF (bevacizumab-similar) antibody (C), anti-Dll4 antibody (D), or HD105 bispecific antibody (E). Representative images show sprouting tip cells of HUVECs from the beads under basal media (B, arrowheads) and more sprouting under anti-Dll4 antibody treatment (D, arrows) but much less sprouting under anti-VEGF antibody (C) or HD105 bispecific antibody treatment (E). Scale bar (B-E), 150 μm. The bar graph (F) shows the measurement of sprouting HUVECs at 225 μm from beads (n = 20 beads/group, mean ± SE). *, P < 0.05 versus PBS. †, P < 0.05vs. anti-Dll4 antibody. The HD105 bispecific antibody inhibited VEGF-dependent HUVEC proliferation (G) and Dll4-induced Notch-1-dependent activation of luciferase in SKOV-3-RBP-J Κ luciferase cells (H) in a dose-dependent manner. The IC50 values of the anti-VEGF (bevacizumab-similar) antibody (open circle) and HD105 bispecific antibody (closed circle) on HUVEC proliferation were 1.49 ± 0.04 nM and 1.58 ± 0.08 nM, respectively (G). The IC50 values of the HD105 bispecific antibody (closed circle) and the anti-Dll4 antibody (open circle) on luciferase activation were determined to be 0.62 ± 0.23 nM and 0.58 ± 0.03 nM, respectively (H).

Article Snippet: Antibodies and cell culture An anti-VEGF (bevacizumab-similar) antibody, an anti-Dll4 monoclonal antibody, the HD105 bispecific antibody, an anti-mouse Dll4 monoclonal antibody, and a mouse version of the HD105 bispecific antibody were produced by Hanwha Chemical, Biologics R&D Center (Daejeon, South Korea).

Techniques: Inhibition, Activation Assay, Luciferase

Journal: mAbs

Article Title: Simultaneous blockade of VEGF and Dll4 by HD105, a bispecific antibody, inhibits tumor progression and angiogenesis

doi: 10.1080/19420862.2016.1171432

Figure Lengend Snippet: Suppression of tumor progression in several cancer xenograft models by HD105 bispecific antibody. Human A549 lung cancer (A) or human SCH gastric cancer (B, C) was subcutaneously implanted into nude mice. After tumors were grown to an average volume of 150–200 mm3, PBS (open triangle), anti-VEGF (bevacizumab-similar) antibody (2.5 mg/kg, open circle), anti-mouse Dll4 antibody (2.5 mg/kg, closed triangle), or mouse HD105 bispecific antibody (3.25 mg/kg, closed circle) was intraperitoneally injected twice (A549) or once (SCH) per week (A, B). Tumor volume was calculated by the formula width2 × length × 0.52. The dose dependency of the mouse HD105 bispecific antibody was evaluated in human SCH gastric cancer xenograft model (C). PBS (open triangle) or mouse HD105 bispecific antibody (0.361 mg/kg, closed triangle; 1.083 mg/kg, open circle; 3.25 mg/kg, closed circle) was intraperitoneally injected once per week. The response to mouse HD105 bispecific antibody (6.5 mg/kg, once per week, closed circle) was also determined using other human gastric cancer xenograft models, including MKN-74 (D), SNU-5 (E), and SNU-16 (F). Tumor progression was not inhibited by the mouse HD105 bispecific antibody in MKN-74 and SNU-5 but was inhibited in SNU-16 similarly to SCH.

Article Snippet: Antibodies and cell culture An anti-VEGF (bevacizumab-similar) antibody, an anti-Dll4 monoclonal antibody, the HD105 bispecific antibody, an anti-mouse Dll4 monoclonal antibody, and a mouse version of the HD105 bispecific antibody were produced by Hanwha Chemical, Biologics R&D Center (Daejeon, South Korea).

Techniques: Injection

Journal: mAbs

Article Title: Simultaneous blockade of VEGF and Dll4 by HD105, a bispecific antibody, inhibits tumor progression and angiogenesis

doi: 10.1080/19420862.2016.1171432

Figure Lengend Snippet: Suppression of tumor angiogenesis in cancer xenograft models by HD105 bispecific antibody. Fluorescence micrographs compare the vasculature of A549 human lung cancer tissues in xenograft mice after treatment with PBS (A), anti-VEGF (bevacizumab-similar) antibody (B), anti-mouse Dll4 antibody (C), or mouse HD105 bispecific antibody (D). Scale bar (A-D), 50 μm. The tumor vasculature was stained for CD31 immunoreactivity (green), and the vascular basement was stained for type IV collagen (red). Tumor vessels were decreased after treatment with anti-VEGF (bevacizumab-similar) antibody or mouse HD105 bispecific antibody, whereas tumor vessels were markedly increased after treatment with anti-mouse Dll4 antibody compared to PBS. Higher-resolution images compare the phenotype changes of tumor vessels in detail after PBS (E), anti-VEGF (bevacizumab-similar) antibody (F), anti-mouse Dll4 antibody (G), or mouse HD105 bispecific antibody treatment (H). Scale bar (E-H), 20 μm. The tumor vasculature was stained for CD31 immunoreactivity (red), and the perivascular pericyte was stained for NG2 (green). The nuclei of the tumor tissues were stained by DAPI (4′,6-diamidino-2-phenylindole). Tumor vessels after treatment with anti-mouse Dll4 antibody were conspicuously thinner and more branched than the tumor vessels of other groups. Bar graph (I) measuring tumor vessel density of A549 tumor tissues in xenograft mice confirms the conspicuous increase of tumor vessels after anti-mouse Dll4 antibody treatment but decreases after anti-VEGF (bevacizumab-similar) antibody, mouse HD105 bispecific antibody, or combination treatment with anti-mouse Dll4 antibody and anti-VEGF (bevacizumab-similar) antibody. †, P < 0.05 versus PBS. *, P < 0.05vs. anti-Dll4 antibody. However, the functional tumor vessels in SCH gastric cancer tissues assessed by intravenous FITC-labeled Lycopersicon esculentum (Tomato) lectin staining were significantly decreased after treatment with anti-VEGF (bevacizumab-similar) antibody as well as anti-mouse Dll4 antibody (J). †, P < 0.05 versus PBS. ‡, < 0.05vs. anti-VEGF (bevacizumab-similar) antibody. *, P < 0.05 versus anti-Dll4 antibody. Functional tumor vessels were more decreased after treatment with mouse HD105 bispecific antibody compared to the other groups.

Article Snippet: Antibodies and cell culture An anti-VEGF (bevacizumab-similar) antibody, an anti-Dll4 monoclonal antibody, the HD105 bispecific antibody, an anti-mouse Dll4 monoclonal antibody, and a mouse version of the HD105 bispecific antibody were produced by Hanwha Chemical, Biologics R&D Center (Daejeon, South Korea).

Techniques: Fluorescence, Staining, Functional Assay, Labeling

Journal: mAbs

Article Title: Simultaneous blockade of VEGF and Dll4 by HD105, a bispecific antibody, inhibits tumor progression and angiogenesis

doi: 10.1080/19420862.2016.1171432

Figure Lengend Snippet: Increase in apoptotic tumor cells in cancer xenograft models treated with HD105 bispecific antibody. Fluorescence micrographs show apoptotic cells stained for activated caspase-3 antibody (red) in SCH human gastric cancer tissues in xenograft mice after treatment with PBS (A), anti-VEGF (bevacizumab-similar) antibody (B), anti-mouse Dll4 antibody (C), and mouse HD105 bispecific antibody (D and E). Scale bar (A-D), 50 μm; (E), 20 μm. Nuclei of the tumor tissues were stained by DAPI (4′,6-diamidino-2-phenylindole, blue). The higher-resolution image confirms that activated caspase-3 antibody was stained in the cytoplasm of the apoptotic cells after mouse HD105 bispecific antibody treatment (E). The bar graph (F) measuring the cell density of apoptotic cells in SCH cancer tissues confirms the significant increase in apoptotic cells after mouse HD105 bispecific antibody treatment. *, P < 0.05vs. PBS. ‡, < 0.05 versus anti-VEGF (bevacizumab-similar)) antibody. *, P < 0.05vs. anti-Dll4 antibody.

Article Snippet: Antibodies and cell culture An anti-VEGF (bevacizumab-similar) antibody, an anti-Dll4 monoclonal antibody, the HD105 bispecific antibody, an anti-mouse Dll4 monoclonal antibody, and a mouse version of the HD105 bispecific antibody were produced by Hanwha Chemical, Biologics R&D Center (Daejeon, South Korea).

Techniques: Fluorescence, Staining

Journal: mAbs

Article Title: Simultaneous blockade of VEGF and Dll4 by HD105, a bispecific antibody, inhibits tumor progression and angiogenesis

doi: 10.1080/19420862.2016.1171432

Figure Lengend Snippet: Purification and stability of HD105 bispecific antibody. The HD105 bispecific antibody was produced by CHO cells and then purified by several chromatographic steps. The purity of the antibody in each purification step was analyzed by SEC-HPLC (A). The stability of the purified HD105 bispecific antibody (20 mg/ml) was monitored by SEC-HPLC (B) and SDS-PAGE (C) after 4 weeks' incubation at 4°C, 25°C, or 40°C. The binding affinity against each target of the HD105 bispecific antibody was also monitored by DACE analysis (D) after 4 weeks' incubation at 4°C, 25°C, or 40°C.

Article Snippet: Antibodies and cell culture An anti-VEGF (bevacizumab-similar) antibody, an anti-Dll4 monoclonal antibody, the HD105 bispecific antibody, an anti-mouse Dll4 monoclonal antibody, and a mouse version of the HD105 bispecific antibody were produced by Hanwha Chemical, Biologics R&D Center (Daejeon, South Korea).

Techniques: Purification, Produced, SDS Page, Incubation, Binding Assay