Journal: Cancer gene therapy

Article Title: Mc-hES, a novel plasmid carrying human endostatin gene, inhibits nasopharyngeal carcinoma growth.

doi: 10.1038/cgt.2011.72

Figure Lengend Snippet: Figure 2 Expression profiles and biological function of endostatin in eukaryotic cells. (a) RT–PCR analysis of endostatin transcripts in CNE-2 cells transfected with plasmid. Lane 1, DL2000, bands of 2, 1, 0.75, 0.5 and 0.25 kb; lane 2, mc-hES; lane 3, pcDNA-hES; lane 4, pj-hES; lane 5, p2jc31; lane 6, empty control; lane 7, positive control. (b) Western blot assay. Lane 1, p2jc31; lane 2, mc-hES; lane 3, pcDNA-hES; lane 4, pj-hES. (c) Expression profiles of endostatin in the supernatant of cells transfected with plasmids. Columns represent the mean of three independent experiments, each conducted in triplicate; bars, s.e. Po 0.05, compared with pj-hES and pcDNA-hES treated group. (d) MTT assay. HUVECs were treated with CM from transfected CNE-2 cells and cell proliferation was evaluated after 72 h. Columns, cell proliferation normalized against p2jc31 control (mean±s.d.). Mc-hES versus pcDNA-hES, P ¼ 0.019; mc-hES versus pj-hES, P ¼ 0.005; pcDNA-hES versus p2jc31, P ¼ 0.237. (e) Tube formation assay. HUVECs were treated with CM. After 16 h of incubation images of tube formation were captured and tube formation was scored in one 50 microscopic field. The number of tubes formed was quantitated and the data are presented as the mean±s.d. per field ( 50). Data were normalized to p2jc31-treated control (n ¼ 5). Mc-hES versus pcDNA-hES, P ¼ 0.015; mc-hES versus pj-hES, P ¼ 0.008; pcDNA-hES versus p2jc31, P ¼ 0.009. (f) Migration assay. HUVECs were treated with CM and pipetted into inserts of matrigel-coated transwells. Addition of chemoattractant, HUVECs in CM containing 50 ng ml1 vascular endothelial growth factor, to the lower well of boyden chamber stimulated endothelial migration to the underside of the transwell membrane. After 6 h of incubation, migrated cells were stained by DAPI. The number of cells that migrated was counted by microscopy and the data are presented as the mean±s.d. per field ( 100). Data were normalized to p2jc31-treated control (n ¼ 5). Mc-hES versus pcDNA-hES, P ¼ 0.01; mc-hES versus pj-hES, P ¼ 0.015; pcDNA-hES versus p2jc31, P ¼ 0.019.

Article Snippet: CNE-2 cell supernatants were harvested as conditioned medium (CM) after 72 h of transfection and endostatin expression was evaluated using a human endostatin ELISA kit (R&D systems, Minneapolis, MN) according to the recommendation of the manufacturer.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Transfection, Plasmid Preparation, Control, Positive Control, Western Blot, MTT Assay, Tube Formation Assay, Incubation, Migration, Membrane, Staining, Microscopy

Journal: Cancer gene therapy

Article Title: Mc-hES, a novel plasmid carrying human endostatin gene, inhibits nasopharyngeal carcinoma growth.

doi: 10.1038/cgt.2011.72

Figure Lengend Snippet: Figure 4 (a) Endostatin expression profile in the tumor tissues. For expression profiles of endostatin, results are given in nanograms per mg of tumor tissue after single administration. Columns, mean of three mice; bars, s.d. mc-hES versus pcDNA-hES. Po0.001 at days 3, 7, 14 and 20. (b) Results from representative experiments are shown for RT–PCR analysis of endostatin transcripts. b-actin was used as loading control. b, blank; M, DL2000; , negative control; þ , positive control; s, saline.

Article Snippet: CNE-2 cell supernatants were harvested as conditioned medium (CM) after 72 h of transfection and endostatin expression was evaluated using a human endostatin ELISA kit (R&D systems, Minneapolis, MN) according to the recommendation of the manufacturer.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Negative Control, Positive Control, Saline

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: British Journal of Cancer

Article Title: Autoantibodies to endostatin in patients with breast cancer: correlation to endostatin levels and clinical outcome

doi: 10.1038/sj.bjc.6603037

Figure Lengend Snippet: Reactivity of serum samples from patients with metastatic breast cancer, control serum samples and anti-endostatin antibodies (positive control), with recombinant human endostatin. Western immunoblot shows that serum samples from patients 1, 4, 5, 7, 8, 9, 10 and positive control (anti-endostatin antibodies) were strongly reactive to recombinant endostatin, whereas serum samples from other patients and from normal controls were negative.

Article Snippet: The positive control was human endostatin exposed to antibodies to murine endostatin (R&D System, Abingdon, UK).

Techniques: Control, Positive Control, Recombinant, Western Blot

Journal: British Journal of Cancer

Article Title: Autoantibodies to endostatin in patients with breast cancer: correlation to endostatin levels and clinical outcome

doi: 10.1038/sj.bjc.6603037

Figure Lengend Snippet: Detection of endostatin by Western blot analysis. The sera of 12 patients evaluated for endostatin concentration by ELISA were separated on polyacrylamide gel and incubated with a specific endostatin antibody as described in Materials and Methods. Results of four patients are shown. Marker bands (10, 15, 25, 30 and 35 kDa) are depicted. No specific signal could be detected in the sera with 5.7 and 11.7 ng ml −1 of the inhibitor.

Article Snippet: The positive control was human endostatin exposed to antibodies to murine endostatin (R&D System, Abingdon, UK).

Techniques: Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay, Incubation, Marker

Journal: British Journal of Cancer

Article Title: Autoantibodies to endostatin in patients with breast cancer: correlation to endostatin levels and clinical outcome

doi: 10.1038/sj.bjc.6603037

Figure Lengend Snippet: ( A ) Survival of patients as a function of the detection of autoantibodies to endostatin. Kaplan–Meier analysis showed that patients with detectable endostatin autoantibodies had significantly longer overall survival ( P =0.03). ( B ) Survival of patients as a function of serum endostatin levels. Kaplan–Meier analysis showed that patients with lower serum endostatin levels had significantly longer overall survival ( P =0.007).

Article Snippet: The positive control was human endostatin exposed to antibodies to murine endostatin (R&D System, Abingdon, UK).

Techniques:

Journal: European Heart Journal Open

Article Title: Orthostatic hypotension is associated with higher levels of circulating endostatin

doi: 10.1093/ehjopen/oeae030

Figure Lengend Snippet: Comparison of the distribution of plasma endostatin levels in patients with orthostatic hypotension vs. controls ( n = 296). The box and whisker plot illustrates the distribution of endostatin levels across orthostatic hypotension-positive (left) and orthostatic hypotension-negative (right) groups. The plot reveals median, quartiles, and outliers within each group. Our findings indicate that endostatin levels are significantly higher in patients with orthostatic hypotension than in the control group ( P < 0.001).

Article Snippet: For endostatin analysis, the samples were thawed and examined by sandwich immunoassay using a commercially available ELISA kit for endostatin (DY1098, R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions.

Techniques: Comparison, Clinical Proteomics, Whisker Assay, Control

Journal: European Heart Journal Open

Article Title: Orthostatic hypotension is associated with higher levels of circulating endostatin

doi: 10.1093/ehjopen/oeae030

Figure Lengend Snippet: ( A ) Correlation between plasma endostatin levels and tertiles of orthostatic systolic blood pressure drop. The correlation between plasma endostatin levels stratified according to tertiles of orthostatic systolic blood pressure drop during head-up tilt. Boxplots represent pooled population, i.e. both orthostatic hypotension and controls. A negative systolic blood pressure value indicates an increase in systolic blood pressure upon standing, and a positive value systolic blood pressure reduction. Some subjects in the first tertile demonstrated increased systolic blood pressure upon standing, i.e. orthostatic hypertension, as such their orthostatic systolic blood pressure increased up to 25 mmHg, while in others it dropped by 12 mmHg. As demonstrated, levels of circulating endostatin were significantly higher across increasing tertiles of orthostatic systolic blood pressure drop, i.e. the higher the drop in systolic blood pressure, the higher the endostatin level (Kruskal–Wallis, P < 0.001). ( B ) Relationship between plasma endostatin levels and magnitude of orthostatic systolic blood pressure change. The scatter plot illustrates the relationship between plasma endostatin levels (measured in pg/mL) and the magnitude of orthostatic drop in systolic blood pressure (measured in mmHg), adjusted for baseline supine systolic blood pressure. Each dot represents individual patient data, indicating their endostatin level and the corresponding orthostatic systolic blood pressure drop. The regression line indicates a positive but weak correlation (Pearson’s r = 0.15, P < 0.001), suggesting that higher endostatin levels are associated with a greater drop in orthostatic systolic blood pressure.

Article Snippet: For endostatin analysis, the samples were thawed and examined by sandwich immunoassay using a commercially available ELISA kit for endostatin (DY1098, R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions.

Techniques: Clinical Proteomics

Journal: European Heart Journal Open

Article Title: Orthostatic hypotension is associated with higher levels of circulating endostatin

doi: 10.1093/ehjopen/oeae030

Figure Lengend Snippet: Multivariate logistic regression analysis of factors associated with orthostatic hypotension

Article Snippet: For endostatin analysis, the samples were thawed and examined by sandwich immunoassay using a commercially available ELISA kit for endostatin (DY1098, R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions.

Techniques:

Journal: European Heart Journal Open

Article Title: Orthostatic hypotension is associated with higher levels of circulating endostatin

doi: 10.1093/ehjopen/oeae030

Figure Lengend Snippet: Receiver operating characteristic analysis of plasma endostatin levels for predicting orthostatic hypotension. The receiver operating characteristic curve demonstrates the sensitivity and specificity of endostatin levels at various thresholds. The optimal cut-off value for endostatin was 47 652.8 pg/mL (marked by the dot) yielding a sensitivity of 61% and specificity of 71%. The area under the curve was 0.68, indicating a good discriminative power of endostatin in predicting orthostatic hypotension.

Article Snippet: For endostatin analysis, the samples were thawed and examined by sandwich immunoassay using a commercially available ELISA kit for endostatin (DY1098, R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions.

Techniques: Clinical Proteomics

Journal: Frontiers in Immunology

Article Title: Soluble CD147 regulates endostatin via its effects on the activities of MMP-9 and secreted proteasome 20S

doi: 10.3389/fimmu.2024.1319939

Figure Lengend Snippet: The angiogenic potential of the co-culture. The human HT1080 fibroblast cell line (3 × 10 5 cells) or the human U937 monocytic-like cell line (3 × 10 5 cells) were plated in 24-well plates alone or in co-culture (U937 cells were plated in inserts with 0.4-μm pores size) and incubated for 48 h in the presence of TNFα (1 ng/ml). Then, supernatants were collected, and the concentrations of (A) CD147, (B) VEGF, (C) MMP-9, (D) endostatin, (E) Tsp-1, and (F) TIMP-1 were assessed by ELISA (n = 8). Data are presented as means ± SE and were analyzed using one-way ANOVA followed by Bonferroni’s post-hoc test. (G, H) EaHy926 cells (2 × 10 4 cells) were grown to confluency, and then a scratch was applied using the edge of a tip. Detached cells were washed away, and supernatants derived from the experimental groups were diluted 1:2 with serum-starvation medium and applied onto the endothelial layer, with or without the presence of the rabbit anti-human CD147 antibody (h161-pAb, 2 ng/ml). Images were taken at the beginning of the experiment (0 h) and after 20 h. (G) Representative images and (H) the measured distance between the two sides of the scratch at 20 h subtracted from the distance at 0 h (n = 8–9 per group) are shown. Data are presented as means ± SE and were analyzed using one-way ANOVA followed by Bonferroni’s post-hoc test.

Article Snippet: The membranes were incubated for 1 h at room temperature with the primary anti-endostatin antibody (R&D Systems) diluted 1:3,000 in blocking solution, and after three washes with the wash buffer [1× Tris-buffered saline (TBS) with 0.05% Tween 20], they were incubated with a horseradish peroxidase (HRP)–conjugated secondary antibody [donkey anti-goat immunoglobulin G (IgG), Jackson ImmunoResearch Labs, West Grove, PA, USA] diluted 1:5,000 in blocking buffer.

Techniques: Co-Culture Assay, Incubation, Enzyme-linked Immunosorbent Assay, Derivative Assay

Journal: Frontiers in Immunology

Article Title: Soluble CD147 regulates endostatin via its effects on the activities of MMP-9 and secreted proteasome 20S

doi: 10.3389/fimmu.2024.1319939

Figure Lengend Snippet: CD147 regulates the accumulation of endostatin. HT1080 cells (3 × 10 4 cells) were incubated alone or in co-culture with U937 cells (3 × 10 4 cells) for 48 h in the presence of TNFα (1 ng/ml). (A) The cells were incubated alone, with the anti-CD147 antibody (h161-pAb, 2 ng/ml) or with an irrelevant antibody (rabbit IgG, 2 ng/ml). After 48-h incubation, supernatants were collected, and the concentrations of endostatin (n = 6) were determined by ELISA. Data are presented as means ± SE and were analyzed using two-way ANOVA followed by Bonferroni’s post-hoc test. (B) The human HT1080 fibroblast cell line (10 5 cells) was transfected with two CD147 siRNA molecules (10 nM each) and left for 24 h in full medium. Then, the medium was replaced with serum-starvation medium (antibiotics-free) with TNFα (1 ng/ml), and U937 cells (10 5 cells) were added in inserts (0.4-μm pore size). After 48-h incubation in co-cultures, supernatants were collected, and the concentrations of endostatin (n = 8) were assessed using ELISA. Data are presented as means ± SE and were analyzed using one-way ANOVA followed by Bonferroni’s post-hoc test. (C) HT1080 cells (3 × 10 4 cells), U937 cells (3 × 10 4 cells), and their co-culture were incubated in serum-starvation medium with TNFα (1 ng/ml), in the presence of the human recombinant CD147 (300 ng/ml) or the Fc fragment (300 ng/ml) for 48 h. After incubation, supernatants were collected, and the concentrations of endostatin (n = 6) were assessed by ELISA. Data are presented as means ± SE and were analyzed using two-way ANOVA followed by Bonferroni’s post-hoc test.

Article Snippet: The membranes were incubated for 1 h at room temperature with the primary anti-endostatin antibody (R&D Systems) diluted 1:3,000 in blocking solution, and after three washes with the wash buffer [1× Tris-buffered saline (TBS) with 0.05% Tween 20], they were incubated with a horseradish peroxidase (HRP)–conjugated secondary antibody [donkey anti-goat immunoglobulin G (IgG), Jackson ImmunoResearch Labs, West Grove, PA, USA] diluted 1:5,000 in blocking buffer.

Techniques: Incubation, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Transfection, Pore Size, Recombinant

Journal: Frontiers in Immunology

Article Title: Soluble CD147 regulates endostatin via its effects on the activities of MMP-9 and secreted proteasome 20S

doi: 10.3389/fimmu.2024.1319939

Figure Lengend Snippet: CD147 hormetically regulates endostatin. The HT1080 fibroblast cell line (3 × 10 5 cells) was cultured alone or co-cultured with U937 cells (3 × 10 5 cells) in serum-starvation medium in the presence of TNFα (1 ng/ml). (A, B) Supernatants were collected at different time points, and the concentrations of (A) endostatin (n = 6) and (B) VEGF (n = 6) were assessed by ELISA. Data are presented as means ± SE and were analyzed using two-way ANOVA followed by Bonferroni’s post-hoc test. (C–E) Increasing concentrations of recombinant CD147 were added, and the cultures were incubated for 48 h. Supernatants were collected, and concentrations of (C) endostatin, (D) TIMP-1 (n = 6), and (E) MMP-9 (n = 6) were assessed using ELISA. **, p < 0.01; ***, p < 0.001 relative to unstimulated HT1080 cells; $, p < 0.05, $$$, p < 0.001 relative to unstimulated co-culture; &, p < 0.05, &&, p < 0.01, &&&, p < 0.001 relative to HT1080 cells in each concentration. Data are presented as means ± SE and were analyzed using two-way ANOVA followed by Bonferroni’s post-hoc test.

Article Snippet: The membranes were incubated for 1 h at room temperature with the primary anti-endostatin antibody (R&D Systems) diluted 1:3,000 in blocking solution, and after three washes with the wash buffer [1× Tris-buffered saline (TBS) with 0.05% Tween 20], they were incubated with a horseradish peroxidase (HRP)–conjugated secondary antibody [donkey anti-goat immunoglobulin G (IgG), Jackson ImmunoResearch Labs, West Grove, PA, USA] diluted 1:5,000 in blocking buffer.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Recombinant, Incubation, Co-Culture Assay, Concentration Assay

Journal: Frontiers in Immunology

Article Title: Soluble CD147 regulates endostatin via its effects on the activities of MMP-9 and secreted proteasome 20S

doi: 10.3389/fimmu.2024.1319939

Figure Lengend Snippet: Endostatin is regulated in co-culture posttranslationally. HT1080 cells (3 × 10 6 cells) were incubated alone or in co-culture with U937 cells (3 × 10 6 cells) in serum-starvation medium for 48 h and in the presence of TNFα (1 ng/ml), separated by inserts (0.4-μm pore size) to allow separate extraction of RNA from each cell type. Supernatants were collected, and total RNA was extracted, transcribed to cDNA, and amplified by qPCR using primers specific for Col18A (n = 8). (A) Col18A mRNA in HT1080 cells in single cultures and co-cultures. (B) Col18A mRNA in U937 cells in single cultures and co-cultures (n = 8). Data are presented as means ± SE and analyzed using the two-tailed unpaired Student’s t -test. (C) A representative image of a Western blot of the ECM extracted from the plates, probed with an anti-endostatin antibody that also detects the precursor Col18A, and (D) its quantitation (n = 3). ns, not significant.

Article Snippet: The membranes were incubated for 1 h at room temperature with the primary anti-endostatin antibody (R&D Systems) diluted 1:3,000 in blocking solution, and after three washes with the wash buffer [1× Tris-buffered saline (TBS) with 0.05% Tween 20], they were incubated with a horseradish peroxidase (HRP)–conjugated secondary antibody [donkey anti-goat immunoglobulin G (IgG), Jackson ImmunoResearch Labs, West Grove, PA, USA] diluted 1:5,000 in blocking buffer.

Techniques: Co-Culture Assay, Incubation, Pore Size, Extraction, Amplification, Two Tailed Test, Western Blot, Quantitation Assay

Journal: Frontiers in Immunology

Article Title: Soluble CD147 regulates endostatin via its effects on the activities of MMP-9 and secreted proteasome 20S

doi: 10.3389/fimmu.2024.1319939

Figure Lengend Snippet: Proteasome 20S and MMP-9 are involved in endostatin generation. The HT1080 cells (3 × 10 4 cells) in single cultures or in co-culture with U937 cells (3 × 10 4 cells) were incubated in serum-starvation medium in the presence of TNFα (1 ng/ml), together with different protease inhibitors that were added at the following concentrations: leupeptin 1 μM, pepstatin A 5 μM, MG132 1 μM, AM114 1 μM, phenantroline 40 μM, NSC405020 100 μM, MMP-9 inhibitor I 5 nM, and disulfiram 20 μM. Endostatin levels were evaluated by ELISA in supernatants derived from (A) HT1080 single cultures and (B) co-cultures (n = 12). Data are presented as means ± SE and were analyzed using one-way ANOVA followed by Dunnett’s post-hoc test.

Article Snippet: The membranes were incubated for 1 h at room temperature with the primary anti-endostatin antibody (R&D Systems) diluted 1:3,000 in blocking solution, and after three washes with the wash buffer [1× Tris-buffered saline (TBS) with 0.05% Tween 20], they were incubated with a horseradish peroxidase (HRP)–conjugated secondary antibody [donkey anti-goat immunoglobulin G (IgG), Jackson ImmunoResearch Labs, West Grove, PA, USA] diluted 1:5,000 in blocking buffer.

Techniques: Co-Culture Assay, Incubation, Enzyme-linked Immunosorbent Assay, Derivative Assay

Journal: Frontiers in Immunology

Article Title: Soluble CD147 regulates endostatin via its effects on the activities of MMP-9 and secreted proteasome 20S

doi: 10.3389/fimmu.2024.1319939

Figure Lengend Snippet: MMP-9 and proteasome 20S synergistically enhance the cleavage of endostatin from Col18A, and CD147 regulates endostatin and the activity of proteasome 20S in a hormetic manner. HT1080 (3 × 10 4 cells) were incubated in serum-starvation medium with TNFα (1 ng/ml) for 72 h and allowed to deposit their ECM proteins. Cells were destroyed with DDW for 20 min, and cellular debris was washed away with PBS three times. The ECM was then incubated with the indicated concentrations of (A) HT1080-derived supernatants together with AM114 and MMP-9 inhibitor I (n = 6) or with (B) proteasome 20S that was activated with 0.035% SDS and recombinant MMP-9 that was activated by incubation with APMA (1 mM) (n = 12). The activity of (C) MMP-9 (n = 6) or (D) proteasome 20S (n = 6) was measured as well. The generation of endostatin and the activities of MMP-9 and proteasome 20S were synergistically enhanced in the presence of both enzymes. Additionally, HT1080-derived ECM was incubated with recombinant MMP-9 (0.2 ng/ml) and proteasome 20S (0.15 nM) and with increasing concentrations of recombinant CD147 for 48 h. Supernatants were collected, and (E) the levels of endostatin, (F) the activity of proteasome 20S, and (G) the activity of MMP-9 were evaluated (n = 6). Data are presented as means ± SE and were analyzed using one-way ANOVA followed by Bonferroni’s post-hoc test.

Article Snippet: The membranes were incubated for 1 h at room temperature with the primary anti-endostatin antibody (R&D Systems) diluted 1:3,000 in blocking solution, and after three washes with the wash buffer [1× Tris-buffered saline (TBS) with 0.05% Tween 20], they were incubated with a horseradish peroxidase (HRP)–conjugated secondary antibody [donkey anti-goat immunoglobulin G (IgG), Jackson ImmunoResearch Labs, West Grove, PA, USA] diluted 1:5,000 in blocking buffer.

Techniques: Activity Assay, Incubation, Derivative Assay, Recombinant

Journal: Frontiers in Immunology

Article Title: Soluble CD147 regulates endostatin via its effects on the activities of MMP-9 and secreted proteasome 20S

doi: 10.3389/fimmu.2024.1319939

Figure Lengend Snippet: The effect of disease on the serum levels of RA patients. Serum samples from patients with active RA were collected (RA, n = 23) and the concentrations of (A–C, E–G) the pro- and anti-angiogenic factors were compared to those in the serum of healthy volunteers (HC, n = 25). (D, H) Additionally, the activities of proteasome 20S and MMP-9 were evaluated in the serum samples. Data are presented as median values (red) and were analyzed using the non-parametric Mann–Whitney test. (I, J) The functional wound assay was carried out using cells of the confluent human endothelial cell line EaHy926 that were scratched. Serum samples from RA patients (n = 12) or controls (n = 12) were diluted 1:2 in full medium and applied onto the endothelial layer, with or without the presence of the rabbit anti-human CD147 antibody (h161-pAb, 2 ng/ml). Images were taken at the beginning of the experiment (0 h) and after 20 h. The distance between the two sides of the scratch at 20 h was subtracted from the distance at 0 h, to calculate the net distance that endothelial cells migrated to. Data are presented as means ± SE and were analyzed using the one-way ANOVA test followed by Bonferroni’s post-hoc test. (K–P) The human HT1080 fibroblast cell line (3 × 10 4 cells) was seeded in 96-well plates and incubated in serum-starvation medium for 48 h in the presence of TNFα (1 ng/ml) and the serum samples (diluted 1:2) obtained from controls or RA patients and with or without h161-pAb (2 ng/ml). Then, supernatants were collected, and the concentrations of (K) endostatin (n = 13), (L) MMP-9 (n = 13), (N) Tsp-1 (n = 13), and (O) TIMP-1 (n = 13) were determined by ELISA. The activities of (M) MMP-9 (n = 13) and (P) proteasome 20S (n = 13) were measured. Data are presented as means ± SE and were analyzed using the one-way ANOVA test followed by Bonferroni’s post-hoc test.

Article Snippet: The membranes were incubated for 1 h at room temperature with the primary anti-endostatin antibody (R&D Systems) diluted 1:3,000 in blocking solution, and after three washes with the wash buffer [1× Tris-buffered saline (TBS) with 0.05% Tween 20], they were incubated with a horseradish peroxidase (HRP)–conjugated secondary antibody [donkey anti-goat immunoglobulin G (IgG), Jackson ImmunoResearch Labs, West Grove, PA, USA] diluted 1:5,000 in blocking buffer.

Techniques: MANN-WHITNEY, Functional Assay, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Soluble CD147 regulates endostatin via its effects on the activities of MMP-9 and secreted proteasome 20S

doi: 10.3389/fimmu.2024.1319939

Figure Lengend Snippet: Schematic model of the regulatory role of soluble CD147 in the generation of endostatin. When CD147 is secreted in low amounts, it enhances the activities of both MMP-9 and secreted proteasome 20S. These two enzymes collaborate and synergize to cleave Col18A and generate endostatin. When CD147 is secreted in high concentrations, it inhibits TIMP-1, which in turn inhibits MMP-9 activity, and it inhibits proteasome 20S activity. Because proteasome 20S and MMP-9 must collaborate to generate endostatin, the strong inhibition of only one of them, proteasome 20S in this case, is sufficient to inhibit endostatin generation.

Article Snippet: The membranes were incubated for 1 h at room temperature with the primary anti-endostatin antibody (R&D Systems) diluted 1:3,000 in blocking solution, and after three washes with the wash buffer [1× Tris-buffered saline (TBS) with 0.05% Tween 20], they were incubated with a horseradish peroxidase (HRP)–conjugated secondary antibody [donkey anti-goat immunoglobulin G (IgG), Jackson ImmunoResearch Labs, West Grove, PA, USA] diluted 1:5,000 in blocking buffer.

Techniques: Activity Assay, Inhibition

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Immunological disruption of antiangiogenic signals by recruited allospecific T cells leads to corneal allograft rejection.

doi: 10.4049/jimmunol.1103216

Figure Lengend Snippet: FIGURE 1. Corneal graft survival curves of BALB/c mice correlated with the endostatin kinetics of the grafts. (A) Corneal graft survival curves (n = 12). All syngeneic corneal grafts survived (BALB/c [H-2d] to BALB/c [H-2d]), whereas 75% of allografts (C57BL/6 [H-2b] to BALB/c [H-2d]) were rejected between POD20 and POD60. (B) Kinetics of endostatin production. After corneal transplantation, upregulation of endostatin production occurred in both allogeneic and syngeneic transplants. Although syngeneic transplants retained high endostatin levels, endostatin production in allogeneic transplants started to decline after POD10 and was significantly decreased by POD20. *p , 0.05. (C) Kinetics of VEGF production. VEGF expression increased in both groups after transplantation, began decreasing after POD3, and increased again after POD10. Allografts retained higher VEGF production compared with syngeneic grafts (p , 0.05). (D) DiI staining identified a significant number of blood vessels in allogeneic grafts, whereas no vessels were detected in syngeneic grafts at POD40. Original magnification 35.

Article Snippet: Recombinant mouse endostatin (R&D Systems) was used as the standard protein.

Techniques: Transplantation Assay, Expressing, Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Immunological disruption of antiangiogenic signals by recruited allospecific T cells leads to corneal allograft rejection.

doi: 10.4049/jimmunol.1103216

Figure Lengend Snippet: FIGURE 2. Immunohistochemical analysis of endostatin expression and T cell infiltration. Upper panels, Endostatin staining showed that endostatin production in syngeneic grafts re- mained elevated, and no infiltrating T cells were detected. Lower panels, In allogeneic grafts, endostatin production peaked at POD10; con- currently, T cell recruitment into the allograft started around POD10 and increased thereafter, with concomitant graft rejection. Infiltrating T cells in the allografts surrounded the endo- statin-expressing cells. As the number of T cells increased, the number of endostatin-producing cells decreased. The tissue orientation shows the epithelium at the top and the endothelium at the bottom. Images are representative of three mice/group. Scale bar, 50 mm.

Article Snippet: Recombinant mouse endostatin (R&D Systems) was used as the standard protein.

Techniques: Immunohistochemical staining, Expressing, Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Immunological disruption of antiangiogenic signals by recruited allospecific T cells leads to corneal allograft rejection.

doi: 10.4049/jimmunol.1103216

Figure Lengend Snippet: FIGURE 5. Endostatin suppressed the neovascularization of the corneal allografts. (A) Real-time PCR showed that there was more relative mRNA expression of VEGFR2 in the allografts treated with PBS in comparison with allografts treated with endostatin. (B) Immunohistochemical staining showed that fewer CD31+ cells concentrated in the corneal stroma in the endostatin- treated group compared with the PBS-treated group (n = 3 mice/group). (C) DiI staining of blood vessels demonstrates that PBS-treated allografts had significant staining of vessels in the host and graft (left panel), whereas fewer vessels originating from the limbus were seen in the endostatin-treated group, in which the vessels extended to the graft margin but did not extend beyond it into the grafts (right panel). Original magnification 35.

Article Snippet: Recombinant mouse endostatin (R&D Systems) was used as the standard protein.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Comparison, Immunohistochemical staining, Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Immunological disruption of antiangiogenic signals by recruited allospecific T cells leads to corneal allograft rejection.

doi: 10.4049/jimmunol.1103216

Figure Lengend Snippet: FIGURE 6. T cell infiltration decreased in endostatin-treated corneal allografts at POD40. (A and B) Real-time PCR analysis showed increased CD4 and CD8 expression in the PBS-treated group compared with the endostatin-treated group. (C and D) Immunostaining of T cells showed that there were more CD4+ and CD8+ T cells infiltrated into the corneal stroma of the PBS-treated group compared with the endostatin-treated group (n = 3 mice/group). (E and F) DAPI nuclear staining of corneal grafts. The stroma of endostatin-treated allografts maintained its structural integrity, whereas PBS-treated allografts had thicker and structurally disorganized stroma. Original magnification 320.

Article Snippet: Recombinant mouse endostatin (R&D Systems) was used as the standard protein.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Immunostaining, Staining

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