Journal: Cells

Article Title: ADAMTS13 Improves Endothelial Function and Reduces Inflammation in Diabetic Retinopathy.

doi: 10.3390/cells14020085

Figure Lengend Snippet: Figure 1. Epiretinal membranes from PDR patients contain endothelial and stromal cells express- ing ADAMTS13. (A) Negative control slide showing no staining. (B) Staining for the endothelial cell marker CD31 showing new blood vessels (arrows). (C) Staining for CD68 identifying mono- cytes/macrophages in the stroma (arrows). (D) Staining for ADAMTS13 showing immunoreactivity in vascular endothelial cells (arrows) and in stromal cells (arrowheads). (E) Double immunohisto- chemical staining for ADAMTS13 (red) and CD68 (brown) showing co-expression in stromal cells (arrows). No counterstain to visualize the cell nuclei was applied (black scale bar, 10 µM).

Article Snippet: The immunodetection of specific molecules was conducted with the following reagents and conditions: VWF with a mouse monoclonal anti-VWF antibody (1:1000, sc-365712, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), CD41 with a mouse monoclonal antiCD41 antibody (1:1000, sc-365938, Santa Cruz Biotechnology Inc.), β-catenin with a goat polyclonal anti-ß-catenin antibody (1:1000, AF1329, R&D system, Minneapolis, MN, USA), ADAMTS13 with a rabbit monoclonal anti-ADAMTS13 antibody (1:1000, NBP3-16038, Novus Biologicals, Littleton, CO, USA) and with the three mouse monoclonal antibodies 3H9, 5C11, and 12H6, as described above, HMGB1 with a rabbit polyclonal anti-high mobility group box-1 (HMGB1) (1:1000, Cat. no. ab18256, Abcam, Cambridge, UK), ERK1/2 with a rabbit monoclonal anti-phospho-extracellular signal-regulated kinase (ERK)1/2 antibody (1:1000, MAB1018, R&D Systems), ICAM-1 with a mouse monoclonal anti-intercellular adhesion molecule-1 (ICAM-1) antibody (1:100, sc-8439, Santa Cruz Biotechnology Inc.), and VCAM-1 with a mouse monoclonal anti-vascular cell adhesion-1 (VCAM-1) antibody (1:100, sc-13160, Santa Cruz Biotechnology Inc.).

Techniques: Negative Control, Staining, Marker, Expressing

Journal: Cells

Article Title: ADAMTS13 Improves Endothelial Function and Reduces Inflammation in Diabetic Retinopathy.

doi: 10.3390/cells14020085

Figure Lengend Snippet: Figure 4. ADAMTS13 expression levels in diabetic rat retinas and effects of exogenously administered ADAMTS13. ADAMTS13 expression levels in the retinal lysates of diabetic rats (D) (n = 12) and nondiabetic control animals (n = 12) were determined by western blot analysis. After the measurement of the intensities of ADAMTS13 proteoform bands, the immunoblots were stripped and reprobed to evaluate ß-tubulin intensities in all sample panels (A). Results are expressed as means ± standard deviation of the ratios between ADAMTS13 and ß-tubulin (* p < 0.05; independent t-test). The effects of intravitreal ADAMTS13 injection on vascular permeability and markers of hemostasis and inflammation in rat retinas after streptozotocin-induced diabetes were evaluated by quantifications of the BRB breakdown by detection of FITC dextran seeped into the retina after the systemic injection (B). Retinal protein expression levels of the von Willebrand factor (VWF) (C), the platelet marker CD41 (D), vascular endothelial (VE)-cadherin (E), and ß-catenin (F) were determined by immunoblot analysis. Statistical comparisons (mean ± standard deviation of 8–10 rats) were performed as described in Section 2.12. * p < 0.05 compared with values obtained from nondiabetic controls. # p < 0.05 compared with values obtained from diabetic rats.

Article Snippet: The immunodetection of specific molecules was conducted with the following reagents and conditions: VWF with a mouse monoclonal anti-VWF antibody (1:1000, sc-365712, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), CD41 with a mouse monoclonal antiCD41 antibody (1:1000, sc-365938, Santa Cruz Biotechnology Inc.), β-catenin with a goat polyclonal anti-ß-catenin antibody (1:1000, AF1329, R&D system, Minneapolis, MN, USA), ADAMTS13 with a rabbit monoclonal anti-ADAMTS13 antibody (1:1000, NBP3-16038, Novus Biologicals, Littleton, CO, USA) and with the three mouse monoclonal antibodies 3H9, 5C11, and 12H6, as described above, HMGB1 with a rabbit polyclonal anti-high mobility group box-1 (HMGB1) (1:1000, Cat. no. ab18256, Abcam, Cambridge, UK), ERK1/2 with a rabbit monoclonal anti-phospho-extracellular signal-regulated kinase (ERK)1/2 antibody (1:1000, MAB1018, R&D Systems), ICAM-1 with a mouse monoclonal anti-intercellular adhesion molecule-1 (ICAM-1) antibody (1:100, sc-8439, Santa Cruz Biotechnology Inc.), and VCAM-1 with a mouse monoclonal anti-vascular cell adhesion-1 (VCAM-1) antibody (1:100, sc-13160, Santa Cruz Biotechnology Inc.).

Techniques: Expressing, Control, Western Blot, Standard Deviation, Injection, Permeability, Marker

Journal: Cells

Article Title: ADAMTS13 Improves Endothelial Function and Reduces Inflammation in Diabetic Retinopathy.

doi: 10.3390/cells14020085

Figure Lengend Snippet: Figure 5. Intravitreal administration of ADAMTS13 reduces retinal inflammation in diabetic rats. The relative protein amounts of phospho-ERK1/2 (A), high-mobility group box-1 (HMGB1) (B), vascular cell adhesion molecule-1 (VCAM-1) (C), and intercellular adhesion molecule-1 (ICAM-1) (D) were determined in rat retinas with the use of western blots. The animals were made diabetic with the use of a single streptozotocin bolus, ADAMTS13 was injected in the vitreous, and its effects on inflammation markers were evaluated by comparison of ADAMTS13-injected with the contralateral PBS-injected eyes in single animals. Statistical comparisons (mean standard deviation of 8–10 rats in each group) were performed as described in Section 2.12. * p < 0.05 compared with nondiabetic controls. # p < 0.05 compared with diabetic rats.

Article Snippet: The immunodetection of specific molecules was conducted with the following reagents and conditions: VWF with a mouse monoclonal anti-VWF antibody (1:1000, sc-365712, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), CD41 with a mouse monoclonal antiCD41 antibody (1:1000, sc-365938, Santa Cruz Biotechnology Inc.), β-catenin with a goat polyclonal anti-ß-catenin antibody (1:1000, AF1329, R&D system, Minneapolis, MN, USA), ADAMTS13 with a rabbit monoclonal anti-ADAMTS13 antibody (1:1000, NBP3-16038, Novus Biologicals, Littleton, CO, USA) and with the three mouse monoclonal antibodies 3H9, 5C11, and 12H6, as described above, HMGB1 with a rabbit polyclonal anti-high mobility group box-1 (HMGB1) (1:1000, Cat. no. ab18256, Abcam, Cambridge, UK), ERK1/2 with a rabbit monoclonal anti-phospho-extracellular signal-regulated kinase (ERK)1/2 antibody (1:1000, MAB1018, R&D Systems), ICAM-1 with a mouse monoclonal anti-intercellular adhesion molecule-1 (ICAM-1) antibody (1:100, sc-8439, Santa Cruz Biotechnology Inc.), and VCAM-1 with a mouse monoclonal anti-vascular cell adhesion-1 (VCAM-1) antibody (1:100, sc-13160, Santa Cruz Biotechnology Inc.).

Techniques: Western Blot, Injection, Comparison, Standard Deviation

Journal: Cells

Article Title: ADAMTS13 Improves Endothelial Function and Reduces Inflammation in Diabetic Retinopathy.

doi: 10.3390/cells14020085

Figure Lengend Snippet: Figure 6. Regulation of proangiogenic and inflammatory molecule expression by ADAMTS13 in human retinal Müller glial cells. Human glial cells were left untreated or treated with high glucose (HG) (25 mM), tumor necrosis factor-α (TNF-α) (1 ng/mL), or cobalt chloride (CoCl2) (300 µM) for 24 h or ADAMTS13 (100 ng/mL) for 1h followed by HG, CoCl2, or TNF-α. A total of 25 mM of mannitol was used as an inert control for osmotic effects by HG treatment. Levels of monocyte chemotactic protein-1 (MCP-1), matrix metalloproteinase-9 (MMP-9), and vascular endothelial growth factor (VEGF) were quantified in the culture media by ELISA. The present data were generated from three different experiments, each performed in triplicates, and the results are provided as means ± standard deviation; statistical comparisons were performed as described in Section 2.12. * p < 0.05 indicates the comparisons with values obtained from control cells. # p < 0.05 documents the differences with values obtained from cells treated with HG, TNF-α, or CoCl2.

Article Snippet: The immunodetection of specific molecules was conducted with the following reagents and conditions: VWF with a mouse monoclonal anti-VWF antibody (1:1000, sc-365712, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), CD41 with a mouse monoclonal antiCD41 antibody (1:1000, sc-365938, Santa Cruz Biotechnology Inc.), β-catenin with a goat polyclonal anti-ß-catenin antibody (1:1000, AF1329, R&D system, Minneapolis, MN, USA), ADAMTS13 with a rabbit monoclonal anti-ADAMTS13 antibody (1:1000, NBP3-16038, Novus Biologicals, Littleton, CO, USA) and with the three mouse monoclonal antibodies 3H9, 5C11, and 12H6, as described above, HMGB1 with a rabbit polyclonal anti-high mobility group box-1 (HMGB1) (1:1000, Cat. no. ab18256, Abcam, Cambridge, UK), ERK1/2 with a rabbit monoclonal anti-phospho-extracellular signal-regulated kinase (ERK)1/2 antibody (1:1000, MAB1018, R&D Systems), ICAM-1 with a mouse monoclonal anti-intercellular adhesion molecule-1 (ICAM-1) antibody (1:100, sc-8439, Santa Cruz Biotechnology Inc.), and VCAM-1 with a mouse monoclonal anti-vascular cell adhesion-1 (VCAM-1) antibody (1:100, sc-13160, Santa Cruz Biotechnology Inc.).

Techniques: Expressing, Control, Enzyme-linked Immunosorbent Assay, Generated, Standard Deviation

Journal: Cells

Article Title: ADAMTS13 Improves Endothelial Function and Reduces Inflammation in Diabetic Retinopathy.

doi: 10.3390/cells14020085

Figure Lengend Snippet: Figure 7. ADAMTS13 promotes the integrity of adherens junctions in human retinal microvascular endothelial cells. Retinal endothelial cells were left untreated (control) or were cultured in the presence of 25 mM of high glucose (HG) concentrations (A), 300 µM of cobalt chloride (CoCl2) (B), or 1 ng/mL of TNF-α (C) for 24 h. A third type of treatment consisted of the pretreatment with 100 ng/mL of ADAMTS13 for 1 h followed by HG, CoCl2, or TNF-α. A total of 25 mM of mannitol was used as a control for the treatment with high glucose. Levels of soluble VE-cadherin (left histograms) and soluble syndecan-1 (right histograms) in cell culture media were quantified with the use of specific ELISAs. The data represent means ± standard deviations from different (n = 3) experiments performed in triplicates, and statistical comparisons were performed as described in Section 2.12. * p < 0.05 indicates comparisons with values obtained from control cells. # p < 0.05 provides the comparisons with values obtained from cells treated with HG, CoCl2, or TNF-α.

Article Snippet: The immunodetection of specific molecules was conducted with the following reagents and conditions: VWF with a mouse monoclonal anti-VWF antibody (1:1000, sc-365712, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), CD41 with a mouse monoclonal antiCD41 antibody (1:1000, sc-365938, Santa Cruz Biotechnology Inc.), β-catenin with a goat polyclonal anti-ß-catenin antibody (1:1000, AF1329, R&D system, Minneapolis, MN, USA), ADAMTS13 with a rabbit monoclonal anti-ADAMTS13 antibody (1:1000, NBP3-16038, Novus Biologicals, Littleton, CO, USA) and with the three mouse monoclonal antibodies 3H9, 5C11, and 12H6, as described above, HMGB1 with a rabbit polyclonal anti-high mobility group box-1 (HMGB1) (1:1000, Cat. no. ab18256, Abcam, Cambridge, UK), ERK1/2 with a rabbit monoclonal anti-phospho-extracellular signal-regulated kinase (ERK)1/2 antibody (1:1000, MAB1018, R&D Systems), ICAM-1 with a mouse monoclonal anti-intercellular adhesion molecule-1 (ICAM-1) antibody (1:100, sc-8439, Santa Cruz Biotechnology Inc.), and VCAM-1 with a mouse monoclonal anti-vascular cell adhesion-1 (VCAM-1) antibody (1:100, sc-13160, Santa Cruz Biotechnology Inc.).

Techniques: Control, Cell Culture

Journal: Cells

Article Title: ADAMTS13 Improves Endothelial Function and Reduces Inflammation in Diabetic Retinopathy.

doi: 10.3390/cells14020085

Figure Lengend Snippet: Figure 8. Regulation of inflammatory cytokine expression by ADAMTS13 in human retinal microvas- cular endothelial cells. Endothelial cultures were left untreated (control) or were treated with 25 mM of high glucose (HG), 300 µM of cobalt chloride (CoCl2), or 1 ng/mL of tumor necrosis factor-α (TNF-α) for 24 h with or without a 1 h preincubation with 100 ng/mL of ADAMTS13. A total of 25 mM of mannitol was used as control for cell damage induced by high glucose levels. In panel (A), signaling events were probed by measurement of phospho-ERK1/2 levels in cell lysates. In panel (B), cell culture medium levels of monocyte chemotactic protein-1 (MCP-1) and, in panel (C), cell culture medium levels of fractalkine were quantified with the use of specific ELISAs. Data are expressed as means ± standard deviation from independent (n = 3) experiments with triplicates per experiment. Statistical comparisons were performed as described in Section 2.12. * p < 0.05 indicates comparisons with values obtained from control cells. # p < 0.05 indicates comparisons with values obtained from stimulated cells.

Article Snippet: The immunodetection of specific molecules was conducted with the following reagents and conditions: VWF with a mouse monoclonal anti-VWF antibody (1:1000, sc-365712, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), CD41 with a mouse monoclonal antiCD41 antibody (1:1000, sc-365938, Santa Cruz Biotechnology Inc.), β-catenin with a goat polyclonal anti-ß-catenin antibody (1:1000, AF1329, R&D system, Minneapolis, MN, USA), ADAMTS13 with a rabbit monoclonal anti-ADAMTS13 antibody (1:1000, NBP3-16038, Novus Biologicals, Littleton, CO, USA) and with the three mouse monoclonal antibodies 3H9, 5C11, and 12H6, as described above, HMGB1 with a rabbit polyclonal anti-high mobility group box-1 (HMGB1) (1:1000, Cat. no. ab18256, Abcam, Cambridge, UK), ERK1/2 with a rabbit monoclonal anti-phospho-extracellular signal-regulated kinase (ERK)1/2 antibody (1:1000, MAB1018, R&D Systems), ICAM-1 with a mouse monoclonal anti-intercellular adhesion molecule-1 (ICAM-1) antibody (1:100, sc-8439, Santa Cruz Biotechnology Inc.), and VCAM-1 with a mouse monoclonal anti-vascular cell adhesion-1 (VCAM-1) antibody (1:100, sc-13160, Santa Cruz Biotechnology Inc.).

Techniques: Expressing, Control, Cell Culture, Standard Deviation

Journal: Cells

Article Title: ADAMTS13 Improves Endothelial Function and Reduces Inflammation in Diabetic Retinopathy.

doi: 10.3390/cells14020085

Figure Lengend Snippet: Figure 9. ADAMTS13 affects leukocyte adhesion to human retinal microvascular endothelial cells (HRMECs). HRMECs were left untreated or were stimulated with 1 ng/mL of tumor necrosis factor-α (TNF-α) for 24 h with or without a 1 h preincubation with ADAMTS13 (100 ng/mL). Monocyte adhesion to HRMEC monolayers was assessed with the use of fluorescently labeled THP-1 monocytic cells (A). The effects of exogenous ADAMTS13 on protein expression levels of intercellular adhesion molecule-1 (ICAM-1) (B) and vascular cell adhesion molecule-1 (VCAM-1) (C) were determined with the use of western blots. Results are expressed as means ± standard deviation from three different experiments performed in triplicates. One-way ANOVA and independent t-tests were used for comparisons among three groups and between two groups, respectively. * p < 0.05 compared with values obtained from untreated cells. # p < 0.05 compared with values obtained from cells treated with TNF-α (RFU = relative fluorescence units).

Article Snippet: The immunodetection of specific molecules was conducted with the following reagents and conditions: VWF with a mouse monoclonal anti-VWF antibody (1:1000, sc-365712, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), CD41 with a mouse monoclonal antiCD41 antibody (1:1000, sc-365938, Santa Cruz Biotechnology Inc.), β-catenin with a goat polyclonal anti-ß-catenin antibody (1:1000, AF1329, R&D system, Minneapolis, MN, USA), ADAMTS13 with a rabbit monoclonal anti-ADAMTS13 antibody (1:1000, NBP3-16038, Novus Biologicals, Littleton, CO, USA) and with the three mouse monoclonal antibodies 3H9, 5C11, and 12H6, as described above, HMGB1 with a rabbit polyclonal anti-high mobility group box-1 (HMGB1) (1:1000, Cat. no. ab18256, Abcam, Cambridge, UK), ERK1/2 with a rabbit monoclonal anti-phospho-extracellular signal-regulated kinase (ERK)1/2 antibody (1:1000, MAB1018, R&D Systems), ICAM-1 with a mouse monoclonal anti-intercellular adhesion molecule-1 (ICAM-1) antibody (1:100, sc-8439, Santa Cruz Biotechnology Inc.), and VCAM-1 with a mouse monoclonal anti-vascular cell adhesion-1 (VCAM-1) antibody (1:100, sc-13160, Santa Cruz Biotechnology Inc.).

Techniques: Labeling, Expressing, Western Blot, Standard Deviation, Fluorescence

Journal: Cells

Article Title: ADAMTS13 Improves Endothelial Function and Reduces Inflammation in Diabetic Retinopathy.

doi: 10.3390/cells14020085

Figure Lengend Snippet: Figure 10. ADAMTS13 reduces cellular oxidative stress. Oxidative stress was induced in HRMECs and human retinal Müller glial cells and monitored with the use of 2′-7′-dichlorofluorescein (DCF) flu- orescence intensity analysis. The in vitro effects of cell pretreatment with 100 ng/mL of ADAMTS13 for 1 h were quantified. HRMECs were left untreated (control) or were treated with 25 mM of high glucose (HG) for 24 h. A total of 25 mM of mannitol was used as a control condition for the HG treatment (A). Human retinal Müller glial cells (B) and HRMECs (C) were left untreated or were treated with 10 mM of hydrogen peroxide (H2O2) for 1 h. Data are provided as medians (interquartile range) from independent (n = 3) experiments, each performed in triplicates. Statistical comparisons were performed as described in Section 2.12. * p < 0.5 indicates comparisons with values obtained from control cells. # p < 0.05 indicates comparisons with values obtained from cells treated with HG or H2O2.

Article Snippet: The immunodetection of specific molecules was conducted with the following reagents and conditions: VWF with a mouse monoclonal anti-VWF antibody (1:1000, sc-365712, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), CD41 with a mouse monoclonal antiCD41 antibody (1:1000, sc-365938, Santa Cruz Biotechnology Inc.), β-catenin with a goat polyclonal anti-ß-catenin antibody (1:1000, AF1329, R&D system, Minneapolis, MN, USA), ADAMTS13 with a rabbit monoclonal anti-ADAMTS13 antibody (1:1000, NBP3-16038, Novus Biologicals, Littleton, CO, USA) and with the three mouse monoclonal antibodies 3H9, 5C11, and 12H6, as described above, HMGB1 with a rabbit polyclonal anti-high mobility group box-1 (HMGB1) (1:1000, Cat. no. ab18256, Abcam, Cambridge, UK), ERK1/2 with a rabbit monoclonal anti-phospho-extracellular signal-regulated kinase (ERK)1/2 antibody (1:1000, MAB1018, R&D Systems), ICAM-1 with a mouse monoclonal anti-intercellular adhesion molecule-1 (ICAM-1) antibody (1:100, sc-8439, Santa Cruz Biotechnology Inc.), and VCAM-1 with a mouse monoclonal anti-vascular cell adhesion-1 (VCAM-1) antibody (1:100, sc-13160, Santa Cruz Biotechnology Inc.).

Techniques: In Vitro, Control

Journal: Cells

Article Title: ADAMTS13 Improves Endothelial Function and Reduces Inflammation in Diabetic Retinopathy.

doi: 10.3390/cells14020085

Figure Lengend Snippet: Figure 11. Inhibition of endothelial cell migration by ADAMTS13. (A,B) Confluent monolayers of overnight starved HRMECs were scratched with sterile micropipette tips and monolayer regeneration was microscopically monitored, subject to various treatments. In one set of experiments, the cultures were pretreated with a dilution medium or ADAMTS13 (60, 200, or 600 ng/mL) for 1 h, followed by stimulation with vascular endothelial growth factor (VEGF) (10 ng/mL) for 16 h. Two independent experiments were performed in duplicates and two-to-three independent field images were taken for the migration analysis with the Image J software (summarized in (B)). In panel (A), representative images illustrate the effect of ADAMTS13, at a dose of 600 ng/mL, on VEGF-induced cell migration. (C) In a second set of experiments, endothelial cell migration through 8 µm pores of polyethylene terephthalate (PET) membranes in response to VEGF (10 ng/mL) with or without pretreatment with ADAMTS13 (6 to 600 ng/mL) was analyzed with the xCELLigence instrument (three or four independent experiments in duplicates). Results are expressed as means ± standard deviation. One- way ANOVA and independent t-tests were used for comparisons among five groups and between two groups, respectively. * p < 0.05 compared with values obtained from untreated cells. # p < 0.05 compared with values obtained from cells treated with VEGF only.

Article Snippet: The immunodetection of specific molecules was conducted with the following reagents and conditions: VWF with a mouse monoclonal anti-VWF antibody (1:1000, sc-365712, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), CD41 with a mouse monoclonal antiCD41 antibody (1:1000, sc-365938, Santa Cruz Biotechnology Inc.), β-catenin with a goat polyclonal anti-ß-catenin antibody (1:1000, AF1329, R&D system, Minneapolis, MN, USA), ADAMTS13 with a rabbit monoclonal anti-ADAMTS13 antibody (1:1000, NBP3-16038, Novus Biologicals, Littleton, CO, USA) and with the three mouse monoclonal antibodies 3H9, 5C11, and 12H6, as described above, HMGB1 with a rabbit polyclonal anti-high mobility group box-1 (HMGB1) (1:1000, Cat. no. ab18256, Abcam, Cambridge, UK), ERK1/2 with a rabbit monoclonal anti-phospho-extracellular signal-regulated kinase (ERK)1/2 antibody (1:1000, MAB1018, R&D Systems), ICAM-1 with a mouse monoclonal anti-intercellular adhesion molecule-1 (ICAM-1) antibody (1:100, sc-8439, Santa Cruz Biotechnology Inc.), and VCAM-1 with a mouse monoclonal anti-vascular cell adhesion-1 (VCAM-1) antibody (1:100, sc-13160, Santa Cruz Biotechnology Inc.).

Techniques: Inhibition, Migration, Sterility, Software, Standard Deviation

Journal: Cells

Article Title: ADAMTS13 Improves Endothelial Function and Reduces Inflammation in Diabetic Retinopathy.

doi: 10.3390/cells14020085

Figure Lengend Snippet: Figure 1. Epiretinal membranes from PDR patients contain endothelial and stromal cells express- ing ADAMTS13. (A) Negative control slide showing no staining. (B) Staining for the endothelial cell marker CD31 showing new blood vessels (arrows). (C) Staining for CD68 identifying mono- cytes/macrophages in the stroma (arrows). (D) Staining for ADAMTS13 showing immunoreactivity in vascular endothelial cells (arrows) and in stromal cells (arrowheads). (E) Double immunohisto- chemical staining for ADAMTS13 (red) and CD68 (brown) showing co-expression in stromal cells (arrows). No counterstain to visualize the cell nuclei was applied (black scale bar, 10 µM).

Article Snippet: However, we were not able to detect ADAMTS13 expression in these experiments with the commercial antibodies against ADAMTS13 from Abcam and Novus Biologicals, nor with the monoclonal antibodies 3H9, 12H6, and 5C11 against human ADAMTS13.

Techniques: Negative Control, Staining, Marker, Expressing

Journal: Cells

Article Title: ADAMTS13 Improves Endothelial Function and Reduces Inflammation in Diabetic Retinopathy.

doi: 10.3390/cells14020085

Figure Lengend Snippet: Figure 4. ADAMTS13 expression levels in diabetic rat retinas and effects of exogenously administered ADAMTS13. ADAMTS13 expression levels in the retinal lysates of diabetic rats (D) (n = 12) and nondiabetic control animals (n = 12) were determined by western blot analysis. After the measurement of the intensities of ADAMTS13 proteoform bands, the immunoblots were stripped and reprobed to evaluate ß-tubulin intensities in all sample panels (A). Results are expressed as means ± standard deviation of the ratios between ADAMTS13 and ß-tubulin (* p < 0.05; independent t-test). The effects of intravitreal ADAMTS13 injection on vascular permeability and markers of hemostasis and inflammation in rat retinas after streptozotocin-induced diabetes were evaluated by quantifications of the BRB breakdown by detection of FITC dextran seeped into the retina after the systemic injection (B). Retinal protein expression levels of the von Willebrand factor (VWF) (C), the platelet marker CD41 (D), vascular endothelial (VE)-cadherin (E), and ß-catenin (F) were determined by immunoblot analysis. Statistical comparisons (mean ± standard deviation of 8–10 rats) were performed as described in Section 2.12. * p < 0.05 compared with values obtained from nondiabetic controls. # p < 0.05 compared with values obtained from diabetic rats.

Article Snippet: However, we were not able to detect ADAMTS13 expression in these experiments with the commercial antibodies against ADAMTS13 from Abcam and Novus Biologicals, nor with the monoclonal antibodies 3H9, 12H6, and 5C11 against human ADAMTS13.

Techniques: Expressing, Control, Western Blot, Standard Deviation, Injection, Permeability, Marker

Journal: Cells

Article Title: ADAMTS13 Improves Endothelial Function and Reduces Inflammation in Diabetic Retinopathy.

doi: 10.3390/cells14020085

Figure Lengend Snippet: Figure 5. Intravitreal administration of ADAMTS13 reduces retinal inflammation in diabetic rats. The relative protein amounts of phospho-ERK1/2 (A), high-mobility group box-1 (HMGB1) (B), vascular cell adhesion molecule-1 (VCAM-1) (C), and intercellular adhesion molecule-1 (ICAM-1) (D) were determined in rat retinas with the use of western blots. The animals were made diabetic with the use of a single streptozotocin bolus, ADAMTS13 was injected in the vitreous, and its effects on inflammation markers were evaluated by comparison of ADAMTS13-injected with the contralateral PBS-injected eyes in single animals. Statistical comparisons (mean standard deviation of 8–10 rats in each group) were performed as described in Section 2.12. * p < 0.05 compared with nondiabetic controls. # p < 0.05 compared with diabetic rats.

Article Snippet: However, we were not able to detect ADAMTS13 expression in these experiments with the commercial antibodies against ADAMTS13 from Abcam and Novus Biologicals, nor with the monoclonal antibodies 3H9, 12H6, and 5C11 against human ADAMTS13.

Techniques: Western Blot, Injection, Comparison, Standard Deviation

Journal: Cells

Article Title: ADAMTS13 Improves Endothelial Function and Reduces Inflammation in Diabetic Retinopathy.

doi: 10.3390/cells14020085

Figure Lengend Snippet: Figure 6. Regulation of proangiogenic and inflammatory molecule expression by ADAMTS13 in human retinal Müller glial cells. Human glial cells were left untreated or treated with high glucose (HG) (25 mM), tumor necrosis factor-α (TNF-α) (1 ng/mL), or cobalt chloride (CoCl2) (300 µM) for 24 h or ADAMTS13 (100 ng/mL) for 1h followed by HG, CoCl2, or TNF-α. A total of 25 mM of mannitol was used as an inert control for osmotic effects by HG treatment. Levels of monocyte chemotactic protein-1 (MCP-1), matrix metalloproteinase-9 (MMP-9), and vascular endothelial growth factor (VEGF) were quantified in the culture media by ELISA. The present data were generated from three different experiments, each performed in triplicates, and the results are provided as means ± standard deviation; statistical comparisons were performed as described in Section 2.12. * p < 0.05 indicates the comparisons with values obtained from control cells. # p < 0.05 documents the differences with values obtained from cells treated with HG, TNF-α, or CoCl2.

Article Snippet: However, we were not able to detect ADAMTS13 expression in these experiments with the commercial antibodies against ADAMTS13 from Abcam and Novus Biologicals, nor with the monoclonal antibodies 3H9, 12H6, and 5C11 against human ADAMTS13.

Techniques: Expressing, Control, Enzyme-linked Immunosorbent Assay, Generated, Standard Deviation

Journal: Cells

Article Title: ADAMTS13 Improves Endothelial Function and Reduces Inflammation in Diabetic Retinopathy.

doi: 10.3390/cells14020085

Figure Lengend Snippet: Figure 7. ADAMTS13 promotes the integrity of adherens junctions in human retinal microvascular endothelial cells. Retinal endothelial cells were left untreated (control) or were cultured in the presence of 25 mM of high glucose (HG) concentrations (A), 300 µM of cobalt chloride (CoCl2) (B), or 1 ng/mL of TNF-α (C) for 24 h. A third type of treatment consisted of the pretreatment with 100 ng/mL of ADAMTS13 for 1 h followed by HG, CoCl2, or TNF-α. A total of 25 mM of mannitol was used as a control for the treatment with high glucose. Levels of soluble VE-cadherin (left histograms) and soluble syndecan-1 (right histograms) in cell culture media were quantified with the use of specific ELISAs. The data represent means ± standard deviations from different (n = 3) experiments performed in triplicates, and statistical comparisons were performed as described in Section 2.12. * p < 0.05 indicates comparisons with values obtained from control cells. # p < 0.05 provides the comparisons with values obtained from cells treated with HG, CoCl2, or TNF-α.

Article Snippet: However, we were not able to detect ADAMTS13 expression in these experiments with the commercial antibodies against ADAMTS13 from Abcam and Novus Biologicals, nor with the monoclonal antibodies 3H9, 12H6, and 5C11 against human ADAMTS13.

Techniques: Control, Cell Culture

Journal: Cells

Article Title: ADAMTS13 Improves Endothelial Function and Reduces Inflammation in Diabetic Retinopathy.

doi: 10.3390/cells14020085

Figure Lengend Snippet: Figure 8. Regulation of inflammatory cytokine expression by ADAMTS13 in human retinal microvas- cular endothelial cells. Endothelial cultures were left untreated (control) or were treated with 25 mM of high glucose (HG), 300 µM of cobalt chloride (CoCl2), or 1 ng/mL of tumor necrosis factor-α (TNF-α) for 24 h with or without a 1 h preincubation with 100 ng/mL of ADAMTS13. A total of 25 mM of mannitol was used as control for cell damage induced by high glucose levels. In panel (A), signaling events were probed by measurement of phospho-ERK1/2 levels in cell lysates. In panel (B), cell culture medium levels of monocyte chemotactic protein-1 (MCP-1) and, in panel (C), cell culture medium levels of fractalkine were quantified with the use of specific ELISAs. Data are expressed as means ± standard deviation from independent (n = 3) experiments with triplicates per experiment. Statistical comparisons were performed as described in Section 2.12. * p < 0.05 indicates comparisons with values obtained from control cells. # p < 0.05 indicates comparisons with values obtained from stimulated cells.

Article Snippet: However, we were not able to detect ADAMTS13 expression in these experiments with the commercial antibodies against ADAMTS13 from Abcam and Novus Biologicals, nor with the monoclonal antibodies 3H9, 12H6, and 5C11 against human ADAMTS13.

Techniques: Expressing, Control, Cell Culture, Standard Deviation

Journal: Cells

Article Title: ADAMTS13 Improves Endothelial Function and Reduces Inflammation in Diabetic Retinopathy.

doi: 10.3390/cells14020085

Figure Lengend Snippet: Figure 9. ADAMTS13 affects leukocyte adhesion to human retinal microvascular endothelial cells (HRMECs). HRMECs were left untreated or were stimulated with 1 ng/mL of tumor necrosis factor-α (TNF-α) for 24 h with or without a 1 h preincubation with ADAMTS13 (100 ng/mL). Monocyte adhesion to HRMEC monolayers was assessed with the use of fluorescently labeled THP-1 monocytic cells (A). The effects of exogenous ADAMTS13 on protein expression levels of intercellular adhesion molecule-1 (ICAM-1) (B) and vascular cell adhesion molecule-1 (VCAM-1) (C) were determined with the use of western blots. Results are expressed as means ± standard deviation from three different experiments performed in triplicates. One-way ANOVA and independent t-tests were used for comparisons among three groups and between two groups, respectively. * p < 0.05 compared with values obtained from untreated cells. # p < 0.05 compared with values obtained from cells treated with TNF-α (RFU = relative fluorescence units).

Article Snippet: However, we were not able to detect ADAMTS13 expression in these experiments with the commercial antibodies against ADAMTS13 from Abcam and Novus Biologicals, nor with the monoclonal antibodies 3H9, 12H6, and 5C11 against human ADAMTS13.

Techniques: Labeling, Expressing, Western Blot, Standard Deviation, Fluorescence

Journal: Cells

Article Title: ADAMTS13 Improves Endothelial Function and Reduces Inflammation in Diabetic Retinopathy.

doi: 10.3390/cells14020085

Figure Lengend Snippet: Figure 10. ADAMTS13 reduces cellular oxidative stress. Oxidative stress was induced in HRMECs and human retinal Müller glial cells and monitored with the use of 2′-7′-dichlorofluorescein (DCF) flu- orescence intensity analysis. The in vitro effects of cell pretreatment with 100 ng/mL of ADAMTS13 for 1 h were quantified. HRMECs were left untreated (control) or were treated with 25 mM of high glucose (HG) for 24 h. A total of 25 mM of mannitol was used as a control condition for the HG treatment (A). Human retinal Müller glial cells (B) and HRMECs (C) were left untreated or were treated with 10 mM of hydrogen peroxide (H2O2) for 1 h. Data are provided as medians (interquartile range) from independent (n = 3) experiments, each performed in triplicates. Statistical comparisons were performed as described in Section 2.12. * p < 0.5 indicates comparisons with values obtained from control cells. # p < 0.05 indicates comparisons with values obtained from cells treated with HG or H2O2.

Article Snippet: However, we were not able to detect ADAMTS13 expression in these experiments with the commercial antibodies against ADAMTS13 from Abcam and Novus Biologicals, nor with the monoclonal antibodies 3H9, 12H6, and 5C11 against human ADAMTS13.

Techniques: In Vitro, Control

Journal: Cells

Article Title: ADAMTS13 Improves Endothelial Function and Reduces Inflammation in Diabetic Retinopathy.

doi: 10.3390/cells14020085

Figure Lengend Snippet: Figure 11. Inhibition of endothelial cell migration by ADAMTS13. (A,B) Confluent monolayers of overnight starved HRMECs were scratched with sterile micropipette tips and monolayer regeneration was microscopically monitored, subject to various treatments. In one set of experiments, the cultures were pretreated with a dilution medium or ADAMTS13 (60, 200, or 600 ng/mL) for 1 h, followed by stimulation with vascular endothelial growth factor (VEGF) (10 ng/mL) for 16 h. Two independent experiments were performed in duplicates and two-to-three independent field images were taken for the migration analysis with the Image J software (summarized in (B)). In panel (A), representative images illustrate the effect of ADAMTS13, at a dose of 600 ng/mL, on VEGF-induced cell migration. (C) In a second set of experiments, endothelial cell migration through 8 µm pores of polyethylene terephthalate (PET) membranes in response to VEGF (10 ng/mL) with or without pretreatment with ADAMTS13 (6 to 600 ng/mL) was analyzed with the xCELLigence instrument (three or four independent experiments in duplicates). Results are expressed as means ± standard deviation. One- way ANOVA and independent t-tests were used for comparisons among five groups and between two groups, respectively. * p < 0.05 compared with values obtained from untreated cells. # p < 0.05 compared with values obtained from cells treated with VEGF only.

Article Snippet: However, we were not able to detect ADAMTS13 expression in these experiments with the commercial antibodies against ADAMTS13 from Abcam and Novus Biologicals, nor with the monoclonal antibodies 3H9, 12H6, and 5C11 against human ADAMTS13.

Techniques: Inhibition, Migration, Sterility, Software, Standard Deviation