Journal: Journal of Cellular and Molecular Medicine

Article Title: E10A, an adenovirus carrying human endostatin gene, in combination with docetaxel treatment inhibits prostate cancer growth and metastases

doi: 10.1111/j.1582-4934.2008.00548.x

Figure Lengend Snippet: (A) LYVE-1-positive lymphatic vessels in orthotopic tumours received the different treatment. Representative images were shown (×200). (B) Serum endostatin levels in the mice with orthotopic tumours. After systemic delivery of E10A, serum endostatin levels were determined by ELISA. Serum endostatin levels in mice treated with Ad-LacZ were used as control. (C) Serum VEGF levels in different groups of mice. Mice were killed after 4 weeks of treatment and serum were collected for determination of VEGF levels by ELISA. Columns, mean ± S.D. * P < 0.05, compared with Ad-LacZ control.

Article Snippet: The supernatants of PC-3 cells after 72 hrs of infection with E10A or Ad-LacZ at MOI of 100 were harvested as conditioned medium (CM) and evaluated endostatin protein using a human endostatin ELISA kit (R&D systems, Minneapolis, MN, USA) according to the recommendation of the manufacturer.

Techniques: Enzyme-linked Immunosorbent Assay, Control

Journal: Experimental eye research

Article Title: Decreased endostatin in db/db retinas is associated with optic disc intravitreal vascularization.

doi: 10.1016/j.exer.2021.108801

Figure Lengend Snippet: Fig. 1. Endostatin staining and antibody specificity in mouse retina. (A) Immunofluorescence with a rabbit polyclonal anti-endostatin antibody in mice retina showed staining in the ganglion cell layer, outer plexiform layer, and in the outer segments of photoreceptors (left panel). Blocking peptide assay (Blocking PA) using recombinant mouse endostatin was performed to assess antibody specificity. Anti-endostatin antibody neutralization was complete and non-specific signal was observed in the retina (middle panel). Negative control was done by omission of the primary antibody (right panel). (B) FcR blocking assay confirmed the specificity of endostatin staining (left panel) when compared to retinas incubated with Mouse Seroblock FcR (middle panel). Negative control was done by omission of the primary antibody (right panel). (C) Isotype control assay using an anti-collagen I antibody from the same isotype and host as the anti-endostatin antibody was performed. Collagen I is absent from retina and very abundant in cornea. In comparison with the anti-endostatin antibody retinal signal (left panel), non-signal was observed in the retina when using anti-collagen I antibody (middle panel). Positive control for collagen I was performed in the cornea of the same eye in a consecutive section (right panel). Nuclei were counterstained with Hoechst. GCL: ganglion cell layer; INL: inner nuclear layer; ONL: outer nuclear layer; Co: cornea. Scale bars: 37.03 μm.

Article Snippet: For blocking peptide assay, the neutralization of the primary anti-endostatin antibody was done in a previous incubation step of 1 h before immunohistochemistry by adding 10-times excess of recombinant mouse endostatin (R&D Systems, MN, Minneapolis), in contrast to the control incubated with the anti-endostatin antibody alone.

Techniques: Staining, Immunofluorescence, Blocking Assay, Recombinant, Neutralization, Negative Control, Incubation, Control, Comparison, Positive Control

Journal: Experimental eye research

Article Title: Decreased endostatin in db/db retinas is associated with optic disc intravitreal vascularization.

doi: 10.1016/j.exer.2021.108801

Figure Lengend Snippet: Fig. 3. Endostatin in mouse neuroretina. (A) Immunostaining with anti-endostatin antibody revealed that endostatin is accumulated in the inner part of neuroretina, however, it was not observable at the optic disc (B) No endostatin signal was detected in blood vessel basement membranes or in the internal limiting membrane. Nuclei were counterstained with Hoechst. GCL: ganglion cell layer; INL: inner nuclear layer; ONL: outer nuclear layer; BBM: blood vessel basement membrane; ILM: internal limiting membrane. Scale bars: 43.48 μm (A); 33.33 μm (B).

Article Snippet: For blocking peptide assay, the neutralization of the primary anti-endostatin antibody was done in a previous incubation step of 1 h before immunohistochemistry by adding 10-times excess of recombinant mouse endostatin (R&D Systems, MN, Minneapolis), in contrast to the control incubated with the anti-endostatin antibody alone.

Techniques: Immunostaining, Membrane

Journal: Experimental eye research

Article Title: Decreased endostatin in db/db retinas is associated with optic disc intravitreal vascularization.

doi: 10.1016/j.exer.2021.108801

Figure Lengend Snippet: Fig. 2. Western blot analysis of mouse retinal endostatin. Retinal endostatin shows a main band between 15 and 20 kD (lane 1), very close to the 21 kDa band from the recombinant endostatin (lane 2), confirming the antibody specificity for cleaved endostatin. α-tubulin was used as a loading control.

Article Snippet: For blocking peptide assay, the neutralization of the primary anti-endostatin antibody was done in a previous incubation step of 1 h before immunohistochemistry by adding 10-times excess of recombinant mouse endostatin (R&D Systems, MN, Minneapolis), in contrast to the control incubated with the anti-endostatin antibody alone.

Techniques: Western Blot, Recombinant, Control

Journal: Experimental eye research

Article Title: Decreased endostatin in db/db retinas is associated with optic disc intravitreal vascularization.

doi: 10.1016/j.exer.2021.108801

Figure Lengend Snippet: Fig. 4. Endostatin is accumulated in bipolar cells of mouse retina. Double immunostaining with anti-PKCα antibody confirmed that endostatin was accumulated mainly in bipolar cells. Endostatin was found in their axon terminals (arrowheads), but not in their axons themselves (arrows) (A) and in the perikaryon and dendrites (arrowheads) (B). Nuclei were counterstained with Hoechst. GCL: ganglion cell layer; IPL: inner plexiform layer; INL: inner nuclear layer; OPL: outer plexiform layer; ONL: outer nuclear layer. Scale bar: 25.42 μm; 9.41 μm (A); 10.17 μm (B).

Article Snippet: For blocking peptide assay, the neutralization of the primary anti-endostatin antibody was done in a previous incubation step of 1 h before immunohistochemistry by adding 10-times excess of recombinant mouse endostatin (R&D Systems, MN, Minneapolis), in contrast to the control incubated with the anti-endostatin antibody alone.

Techniques: Double Immunostaining

Journal: Experimental eye research

Article Title: Decreased endostatin in db/db retinas is associated with optic disc intravitreal vascularization.

doi: 10.1016/j.exer.2021.108801

Figure Lengend Snippet: Fig. 5. Endostatin is present in cone photoreceptors of mice retina. Double immunostaining with anti-cone arrestin antibody showed endostatin in the inner seg ments of cone photoreceptors (arrows). Cone synaptic terminals did not show endostatin expression (arrowheads). Nuclei were counterstained with Hoechst. GCL: ganglion cell layer; INL: inner nuclear layer; OPL: outer plexiform layer; ONL: outer nuclear layer; IS: inner segments of photoreceptors. Scale bar: 25.88 μm; 17.25 μm (inset).

Article Snippet: For blocking peptide assay, the neutralization of the primary anti-endostatin antibody was done in a previous incubation step of 1 h before immunohistochemistry by adding 10-times excess of recombinant mouse endostatin (R&D Systems, MN, Minneapolis), in contrast to the control incubated with the anti-endostatin antibody alone.

Techniques: Double Immunostaining, Expressing

Journal: Experimental eye research

Article Title: Decreased endostatin in db/db retinas is associated with optic disc intravitreal vascularization.

doi: 10.1016/j.exer.2021.108801

Figure Lengend Snippet: Fig. 6. Human optic disc was practically devoid of endostatin. Double immunostainig with anti- endostatin and anti-collagen IV antibodies showed a markedly endostatin accumulation in the neuroretina, whereas as happens in mouse retina, endostatin was unobservable in the optic disc. The figure is a mosaic composite obtained from different captures at 63X in the laser scan ning confocal microscope. OD: Optic disc; ON: Optic nerve; CR: Central retinal artery and vein; R: Retina. Scale bar: 474.92 μm; 226.15 μm (inset).

Article Snippet: For blocking peptide assay, the neutralization of the primary anti-endostatin antibody was done in a previous incubation step of 1 h before immunohistochemistry by adding 10-times excess of recombinant mouse endostatin (R&D Systems, MN, Minneapolis), in contrast to the control incubated with the anti-endostatin antibody alone.

Techniques: Microscopy

Journal: Experimental eye research

Article Title: Decreased endostatin in db/db retinas is associated with optic disc intravitreal vascularization.

doi: 10.1016/j.exer.2021.108801

Figure Lengend Snippet: Fig. 7. Comparison between human and mouse endostatin distribution in neuro retina. Immunolabelling with anti- endostatin antibody in human and mouse retinas showed a similar, although with some differences, endostatin distribution in both species, mainly located at bipolar cells and photoreceptors. (A) Human bipolar ter minal axons (arrows) showed lower expres sion of endostatin compared to mouse retina. (B) Bipolar cell perikaryal were stained with endostatin in both human and mice retina with a similar intensity. (C) In contrast with mice, where only scarce cones (arrowheads) showed endostatin accumula tion in their inner segments, human retina showed a higher signal of endostatin at photoreceptors, suggesting that both cone and photoreceptors accumulated endostatin. The same anti-endostatin antibody was used on both human and mice retinal sections. GCL: ganglion cell layer; IPL: inner plexi form layer; INL, inner nuclear layer; OPL: outer plexiform layer; ONL: outer nuclear layer; IS: inner segments of photoreceptors. Scale bars: 38.3 μm; 10.64 μm (A, B, C).

Article Snippet: For blocking peptide assay, the neutralization of the primary anti-endostatin antibody was done in a previous incubation step of 1 h before immunohistochemistry by adding 10-times excess of recombinant mouse endostatin (R&D Systems, MN, Minneapolis), in contrast to the control incubated with the anti-endostatin antibody alone.

Techniques: Comparison, Staining

Journal: Experimental eye research

Article Title: Decreased endostatin in db/db retinas is associated with optic disc intravitreal vascularization.

doi: 10.1016/j.exer.2021.108801

Figure Lengend Snippet: Fig. 8. Endostatin is decreased in db/db mouse retinas. (A) Western blot analysis revealed that endostatin protein expression was 0.5-fold decreased in 12 weeks-old diabetic mouse retinas, especially in the ganglion cell layer. Statistical analysis showed significant differences in endostatin band intensity between db/+ and db/db mice (1.275 ± 0.126 arbitrary units in db/+ mice vs. 0.753 ± 0.055 arbitrary units in db/db mice; p = 0.019; n = 3). α-tubulin was used as a loading control (B) Immunofluorescence representative images showing decreased endostatin expression in db/db retinas. Nuclei were coun terstained with Hoechst. OD: Optic disc; CR: Central retinal vessel; R: Retina. Scale bar: 22.22 μm.

Article Snippet: For blocking peptide assay, the neutralization of the primary anti-endostatin antibody was done in a previous incubation step of 1 h before immunohistochemistry by adding 10-times excess of recombinant mouse endostatin (R&D Systems, MN, Minneapolis), in contrast to the control incubated with the anti-endostatin antibody alone.

Techniques: Western Blot, Expressing, Control, Immunofluorescence

Journal: Tissue Engineering. Part A

Article Title: Engineering Endostatin-Producing Cartilaginous Constructs for Cartilage Repair Using Nonviral Transfection of Chondrocyte-Seeded and Mesenchymal-Stem-Cell-Seeded Collagen Scaffolds

doi: 10.1089/ten.tea.2009.0771

Figure Lengend Snippet: Endostatin in the medium of cell-seeded, lipoplex-supplemented collagen constructs and control constructs. (a) Mesenchymal-stem-cell (MSC)-seeded type I/III constructs cultured in chondrogenic medium (CM) and nonchondrogenic medium (NCM) (n = 3 for first 6 days of lipoplex-supplemented scaffolds; n = 2 for all others). (b) Modification of the scaffold via crosslinking none, half, or all of the lipoplexes (n = 6), in MSC-seeded type I/III constructs cultured in CM. (c) MSCs cultured at standard and low oxygen in type I/III and type II collagen scaffolds in CM (n = 3). (d) Chondrocytes cultured at standard and low oxygen in type I/III and type II collagen scaffolds in CM (n = 3).

Article Snippet: Endostatin detection in the medium Endostatin protein in the culture medium was measured using a sandwich enzyme-linked immunosorbent assay (ELISA) kit for human endostatin protein (R&D Systems) following the manufacturer's instructions.

Techniques: Construct, Control, Cell Culture, Modification

Journal: Tissue Engineering. Part A

Article Title: Engineering Endostatin-Producing Cartilaginous Constructs for Cartilage Repair Using Nonviral Transfection of Chondrocyte-Seeded and Mesenchymal-Stem-Cell-Seeded Collagen Scaffolds

doi: 10.1089/ten.tea.2009.0771

Figure Lengend Snippet: Micrographs of histological sections of chondrocyte- and MSC-seeded constructs cultured in CM and NCM at standard oxygen. (a) Endostatin immunohistochemistry of MSC-seeded CI scaffold supplemented with 20 μg pEndo after 6 days of culture in NCM (red chromogen [arrows] indicates positive stain). Scale bar, 50 μm, 40 × magnification. (b) Hematoxylin and eosin stain of the interior of MSC-seeded CI scaffold supplemented with 4 μg pEndo after 22 days of culture in CM. Scale bar, 50 μm, 40 × magnification. Inset shows the section at lower magnification, scale bar, 100 μm, 10 × magnification. (c) Hematoxylin and eosin stain of the surface of MSC-seeded CI scaffold supplemented with 4 μg pEndo after 22 days of culture in CM. Scale bar, 100 μm, 10 × magnification. (d) Safranin-O staining of glycosaminoglycan (GAG) for MSC-seeded CI scaffold supplemented with 4 μg pEndo after 22 days of culture in CM (red chromogen indicates sulfated GAGs). Scale bar, 200 μm, 10× magnification. (e) Type II collagen immunohistochemistry of MSC-seeded CI scaffold supplemented with 4 μg pEndo after 22 days of culture in CM (red indicates positive stain). Scale bar, 200 μm, 10 × magnification. (f) Safranin-O staining of GAG for chondrocyte-seeded CI scaffold supplemented with 20 μg pEndo after 20 days of culture in CM (red chromogen indicates sulfated GAGs). Scale bar, 50 μm, 40 × magnification. Inset shows the section at lower magnification, scale bar, 500 μm, 4 × magnification. (g) Type II collagen immunohistochemistry of chondrocyte-seeded CI scaffold supplemented with 20 μg pEndo after 20 days of culture in CM (brownish-red chromogen indicates positive stain). Scale bar, 500 μm, 4 × magnification. (h) Masson's trichrome staining of chondrocyte-seeded CI scaffold supplemented with 20 μg pEndo after 20 days of culture in CM (blue chromogen indicates collagen, red chromogen indicates cytoplasm, and black chromogen indicates nuclei). Scale bar, 500 μm, 4 × magnification. Color images available online at www.liebertonline.com/ten.

Article Snippet: Endostatin detection in the medium Endostatin protein in the culture medium was measured using a sandwich enzyme-linked immunosorbent assay (ELISA) kit for human endostatin protein (R&D Systems) following the manufacturer's instructions.

Techniques: Construct, Cell Culture, Immunohistochemistry, Staining, H&E Stain

Journal: The Journal of Neuroscience

Article Title: Aberrant Neuronal and Paracellular Deposition of Endostatin in Brains of Patients with Alzheimer's Disease

doi: 10.1523/JNEUROSCI.22-24-10621.2002

Figure Lengend Snippet: Endostatin immunoreactive cells in AD brains and controls

Article Snippet: Recombinant endostatin was produced in BL21(DE3) Escherichia coli cells (Angewandte Gentechnologie Systeme) by induction with isopropylthio-β- d -galactoside for 4 hr.

Techniques: Biomarker Discovery

Journal: The Journal of Neuroscience

Article Title: Aberrant Neuronal and Paracellular Deposition of Endostatin in Brains of Patients with Alzheimer's Disease

doi: 10.1523/JNEUROSCI.22-24-10621.2002

Figure Lengend Snippet: Prominent endostatin deposition (brown) was observed in neurons of all cortical layers in AD brains (A). Counterstain was hemalaun. The bottom inset shows intraneuronal deposition of endostatin (brown). A double-labeling experiment with neurofilament (blue) confirmed the neuronal origin of endostatin+ cells (brown). Paracellular endostatin deposition (brown) was detected in the immediate vicinity of blood vessels in an Aβ(1–40)/endostatin double-labeled section (B). The inset shows a paracellular intraparenchymal endostatin deposit (brown). Extracellular endostatin deposits (brown) were detected in areas of focal glioses (blue) (C), colocalized with Aβ(1–40) fragments (blue) (inset) and tau protein (blue) (D). No endostatin immunoreactivity (blue) was observed in tau+ neurons (brown) with neurofibrillary tangles (bottom inset). Endostatin+ deposits (brown), however, colocalized in PAS-positive plaques (top inset).

Article Snippet: Recombinant endostatin was produced in BL21(DE3) Escherichia coli cells (Angewandte Gentechnologie Systeme) by induction with isopropylthio-β- d -galactoside for 4 hr.

Techniques: Labeling

Journal: The Journal of Neuroscience

Article Title: Aberrant Neuronal and Paracellular Deposition of Endostatin in Brains of Patients with Alzheimer's Disease

doi: 10.1523/JNEUROSCI.22-24-10621.2002

Figure Lengend Snippet: Flow cytometry (top) shows an increase in the number of endostatin-labeled cells after H2O2 and CoCl2 stimulation (A). Western blotting of cellular lysates (middle) and supernatants (bottom) reveals a more accentuated 10 kDa endostatin-immunoreactive band in CoCl2-stimulated than in unstimulated SKNSH cellular lysates and a more accentuated 20 kDa band in their supernatants (A). Western blotting demonstrates a more accentuated 20 kDa endostatin-immunoreactive band in an AD patient compared with a control (B). Surprisingly, Aβ(1–40) (80 μg/ml) supplementation to SKNSH neurons for 24 hr completely abolished the release of the 20 kDa band (C).

Article Snippet: Recombinant endostatin was produced in BL21(DE3) Escherichia coli cells (Angewandte Gentechnologie Systeme) by induction with isopropylthio-β- d -galactoside for 4 hr.

Techniques: Flow Cytometry, Labeling, Western Blot, Control

Journal: The Journal of Neuroscience

Article Title: Aberrant Neuronal and Paracellular Deposition of Endostatin in Brains of Patients with Alzheimer's Disease

doi: 10.1523/JNEUROSCI.22-24-10621.2002

Figure Lengend Snippet: Schematic diagram demonstrating the observed phenomena. Endostatin and Aβ(1–40) are released by neurons (solid line) and cooperate in the mediation of hypoperfusion that leads to accentuated hypoxia in yet unaffected neurons (dotted line). This in turn accelerates neuronal endostatin release, consequent hypoperfusion, and continuing hypoxia.

Article Snippet: Recombinant endostatin was produced in BL21(DE3) Escherichia coli cells (Angewandte Gentechnologie Systeme) by induction with isopropylthio-β- d -galactoside for 4 hr.

Techniques: