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timp 3 (R&D Systems)

Name: timp 3
Brand: R&D Systems
Rating: 94 (32 reviews)

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R&D Systems timp 3
Timp 3, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 32 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 94 stars, based on 32 article reviews

timp 3 - by Bioz Stars, 2026-02

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Fig. 3. Transcriptional and protein expression of Timp3. (A) Schematic illustration of TIMP3′s inhibitory activity on matrix metalloproteinases (MMPs) and on disintegrin and metalloprotease 17 (ADAM17), also called TACE (tumor necrosis factor-α-converting enzyme). (B, C) RNAscope localization of Timp3, Mmp14 and Adam17 mRNA expression in DRG of Plp1-Cre/tdTomato mice, wherein Cre recombinase is expressed in satellite glial cells (SGCs) (B), and in naïve CD1 mice (C). Arrowheads indicate mRNA colocalization of Mmp14 and Adam17 with Timp3 in SGCs, * indicates neurons. Scale bars = 25 μm in (B) and 5 μm in (C). (D) PCR in mouse and human DRG tissues. Samples with omitted RT (reverse transcriptase) show no bands, confirming the specificity of the amplification. (E) Immunofluo rescence of TIMP3 in human DRG tissue. Scale bars = 50 μm. DAPI was used as counterstain. # indicates the fluorescent signal due to the presence of lipofuscins in human DRG neurons.

Journal: Brain, behavior, and immunity

Article Title: Single-cell analysis of dorsal root ganglia reveals metalloproteinase signaling in satellite glial cells and pain.

doi: 10.1016/j.bbi.2023.08.005

Figure Lengend Snippet: Fig. 3. Transcriptional and protein expression of Timp3. (A) Schematic illustration of TIMP3′s inhibitory activity on matrix metalloproteinases (MMPs) and on disintegrin and metalloprotease 17 (ADAM17), also called TACE (tumor necrosis factor-α-converting enzyme). (B, C) RNAscope localization of Timp3, Mmp14 and Adam17 mRNA expression in DRG of Plp1-Cre/tdTomato mice, wherein Cre recombinase is expressed in satellite glial cells (SGCs) (B), and in naïve CD1 mice (C). Arrowheads indicate mRNA colocalization of Mmp14 and Adam17 with Timp3 in SGCs, * indicates neurons. Scale bars = 25 μm in (B) and 5 μm in (C). (D) PCR in mouse and human DRG tissues. Samples with omitted RT (reverse transcriptase) show no bands, confirming the specificity of the amplification. (E) Immunofluo rescence of TIMP3 in human DRG tissue. Scale bars = 50 μm. DAPI was used as counterstain. # indicates the fluorescent signal due to the presence of lipofuscins in human DRG neurons.

Article Snippet: Tissues and cultures were washed in PBS, blocked with 1% BSA, 0.2% Triton X-100 in PBS (BSA solution), and incubated in the BSA solution overnight at 4 ◦C with the following primary antibodies: TIMP3 Ab (1:500, Cat# MAB973, R&D Systems), FABP7 Ab (1:1000, Cat# PA524949, Thermo Fisher Scientific) or GFAP Ab (1:1000, Cat# MAB360, Millipore Sigma).

Techniques: Expressing, Activity Assay, RNAscope, Reverse Transcription, Amplification

Fig. 4. Timp3 controls mechanical and thermal sensitivities in naïve mice. (A) Schematic illustration of the experiment showing the timeline of siRNA injections, pharmacological and biochemical studies. (B) Western blot representative image and quantification show that Timp3 siRNA significantly decreases Timp3 protein levels in DRGs tissues (n = 4). (C) Timp3 siRNA injections do not cause locomotor dysfunction in the Rota-rod test (n = 5). (D) Mechanical and thermal (von Frey, Hargreaves, and dry ice) allodynia induced by Timp3 siRNA compared to a control (Ctrl) non-targeting siRNA (2 µg of siRNA per delivery in the transfection agent PEI, n = 7). (E) Anti-allodynic effect of exogenous recombinant TIMP3 (rTIMP3, 100 ng/site, i.t.), general endogenous tissue inhibitor of MMPs (TIMP-1, 4 pmol/ site), MMP2 and MMP14 inhibitors (10 µg/site, i.t.), TACE/ADAM17 inhibitor (TAPI-2, 1 µg/site, i.t.), and a neutralizing antibody for TNF-α (5 µg/site, i.t.) on mechanical allodynia induced by Timp3 siRNA on day 2. (F) Anti-TIMP3 antibody (TIMP3 Ab, 10 µg/site, i.t.) induces mechanical allodynia compared to IgG control in male and female mice. BL = baseline. Data are expressed as mean ± SEM and statistically analyzed by two-tailed t-test (B, C, E) and Two-way ANOVA followed by Sidak’s post hoc test (D, F). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Journal: Brain, behavior, and immunity

Article Title: Single-cell analysis of dorsal root ganglia reveals metalloproteinase signaling in satellite glial cells and pain.

doi: 10.1016/j.bbi.2023.08.005

Figure Lengend Snippet: Fig. 4. Timp3 controls mechanical and thermal sensitivities in naïve mice. (A) Schematic illustration of the experiment showing the timeline of siRNA injections, pharmacological and biochemical studies. (B) Western blot representative image and quantification show that Timp3 siRNA significantly decreases Timp3 protein levels in DRGs tissues (n = 4). (C) Timp3 siRNA injections do not cause locomotor dysfunction in the Rota-rod test (n = 5). (D) Mechanical and thermal (von Frey, Hargreaves, and dry ice) allodynia induced by Timp3 siRNA compared to a control (Ctrl) non-targeting siRNA (2 µg of siRNA per delivery in the transfection agent PEI, n = 7). (E) Anti-allodynic effect of exogenous recombinant TIMP3 (rTIMP3, 100 ng/site, i.t.), general endogenous tissue inhibitor of MMPs (TIMP-1, 4 pmol/ site), MMP2 and MMP14 inhibitors (10 µg/site, i.t.), TACE/ADAM17 inhibitor (TAPI-2, 1 µg/site, i.t.), and a neutralizing antibody for TNF-α (5 µg/site, i.t.) on mechanical allodynia induced by Timp3 siRNA on day 2. (F) Anti-TIMP3 antibody (TIMP3 Ab, 10 µg/site, i.t.) induces mechanical allodynia compared to IgG control in male and female mice. BL = baseline. Data are expressed as mean ± SEM and statistically analyzed by two-tailed t-test (B, C, E) and Two-way ANOVA followed by Sidak’s post hoc test (D, F). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Techniques: Western Blot, Control, Transfection, Recombinant, Two Tailed Test

Fig. 5. Recombinant TIMP3 protein reverses and prevents mechanical and cold allodynia in a mouse model of chemotherapy-induced neuropathic pain. (A) Schematic of the experiment showing the timeline of paclitaxel (PAX) or vehicle injections, pharmacological studies, and immunohistochemistry (IHC) analysis. (B) Representative image and (C) quantification of Timp3 protein in mouse DRG tissue 14 days after first injection of PAX or vehicle control (n = 5). (D, E) Paclitaxel- induced mechanical (von Frey) and (F) cold allodynia (dry ice) are significantly, dose-dependently reversed up to 6 h by single intrathecal administration of re combinant TIMP3 protein (rTIMP3, 3–100 ng/site) delivered at day 14 after chemotherapy injection (n = 4–5). (G) Schematic of experiment showing timeline of PAX injections concomitantly with rTIMP3 or PBS, behavioral tests, transcriptional, and histological analysis. (H, I) Repeated administrations of rTIMP3 (100 ng/site, i.t.) prevent paclitaxel-induced mechanical and cold allodynia (n = 5–6). BL = baseline. Data expressed as mean ± SEM, statistically analyzed by two-tailed t-test (C), two-way ANOVA followed by Sidak’s post hoc test (D, F, H, I), and one-way ANOVA followed by Tukey’s post hoc test (E). *P < 0.05, **P < 0.01, ***P < 0.001.

Journal: Brain, behavior, and immunity

Article Title: Single-cell analysis of dorsal root ganglia reveals metalloproteinase signaling in satellite glial cells and pain.

doi: 10.1016/j.bbi.2023.08.005

Figure Lengend Snippet: Fig. 5. Recombinant TIMP3 protein reverses and prevents mechanical and cold allodynia in a mouse model of chemotherapy-induced neuropathic pain. (A) Schematic of the experiment showing the timeline of paclitaxel (PAX) or vehicle injections, pharmacological studies, and immunohistochemistry (IHC) analysis. (B) Representative image and (C) quantification of Timp3 protein in mouse DRG tissue 14 days after first injection of PAX or vehicle control (n = 5). (D, E) Paclitaxel- induced mechanical (von Frey) and (F) cold allodynia (dry ice) are significantly, dose-dependently reversed up to 6 h by single intrathecal administration of re combinant TIMP3 protein (rTIMP3, 3–100 ng/site) delivered at day 14 after chemotherapy injection (n = 4–5). (G) Schematic of experiment showing timeline of PAX injections concomitantly with rTIMP3 or PBS, behavioral tests, transcriptional, and histological analysis. (H, I) Repeated administrations of rTIMP3 (100 ng/site, i.t.) prevent paclitaxel-induced mechanical and cold allodynia (n = 5–6). BL = baseline. Data expressed as mean ± SEM, statistically analyzed by two-tailed t-test (C), two-way ANOVA followed by Sidak’s post hoc test (D, F, H, I), and one-way ANOVA followed by Tukey’s post hoc test (E). *P < 0.05, **P < 0.01, ***P < 0.001.

Techniques: Recombinant, Immunohistochemistry, Injection, Control, Two Tailed Test

Fig. 6. Transcriptional analyses of TIMP3 signaling in cultured SGCs after paclitaxel treatment. (A) Schematic of the experi mental design used in cultured SGCs. (B) Representative image and quantification of immunofluorescence intensity of GFAP protein in SGC culture after 24 h of incu bation with paclitaxel (PAX, 300 nM) or vehicle control (Veh; n = 4). (C) Quantifi cation of SGC culture viability 24 h after PAX or Veh treatment (n = 6). (D) Quan tification of mRNA expression levels of Timp3, Mmp2, Mmp14 and Adam17 in SGC culture after PAX or Veh incubation (n = 6). (E) Heat map of mRNA expression of SGC and metalloprotease signaling markers in SGC culture after incubation with pro saptide Tx14 (1 µM) or pioglitazone (PGZ, 10 µM) and PAX compared to vehicle (n = 3). (F) Schematic illustrating the timeline of Tx14, PGZ, or PBS concomitantly treated with PAX, and the behavioral assay. Repeated injections of (G) Tx14 (10 µg/ site, i.t.) or (H) PGZ (100 µg/site, i.t.) prevent paclitaxel-induced mechanical allodynia (n = 6). BL = baseline. Data are expressed as mean ± SEM and statistically analyzed by two-tailed t-test (B, C, D), and Two-way ANOVA followed by Sidak’s post hoc test (G, H): *P < 0.05, **P < 0.01, ***P < 0.001.

Journal: Brain, behavior, and immunity

Article Title: Single-cell analysis of dorsal root ganglia reveals metalloproteinase signaling in satellite glial cells and pain.

doi: 10.1016/j.bbi.2023.08.005

Figure Lengend Snippet: Fig. 6. Transcriptional analyses of TIMP3 signaling in cultured SGCs after paclitaxel treatment. (A) Schematic of the experi mental design used in cultured SGCs. (B) Representative image and quantification of immunofluorescence intensity of GFAP protein in SGC culture after 24 h of incu bation with paclitaxel (PAX, 300 nM) or vehicle control (Veh; n = 4). (C) Quantifi cation of SGC culture viability 24 h after PAX or Veh treatment (n = 6). (D) Quan tification of mRNA expression levels of Timp3, Mmp2, Mmp14 and Adam17 in SGC culture after PAX or Veh incubation (n = 6). (E) Heat map of mRNA expression of SGC and metalloprotease signaling markers in SGC culture after incubation with pro saptide Tx14 (1 µM) or pioglitazone (PGZ, 10 µM) and PAX compared to vehicle (n = 3). (F) Schematic illustrating the timeline of Tx14, PGZ, or PBS concomitantly treated with PAX, and the behavioral assay. Repeated injections of (G) Tx14 (10 µg/ site, i.t.) or (H) PGZ (100 µg/site, i.t.) prevent paclitaxel-induced mechanical allodynia (n = 6). BL = baseline. Data are expressed as mean ± SEM and statistically analyzed by two-tailed t-test (B, C, D), and Two-way ANOVA followed by Sidak’s post hoc test (G, H): *P < 0.05, **P < 0.01, ***P < 0.001.

Techniques: Cell Culture, Immunofluorescence, Control, Expressing, Incubation, Behavioral Assay, Two Tailed Test

The immunohistochemical stains for matrix metalloproteinase (MMP)-3, -7, and -9 and tissue inhibitor of matrix metalloproteinase (TIMP)-1 in the Helicobacter pylori- ( H. pylori- ) infected group, in the nonsteroidal anti-inflammatory drug- (NSAID-) related group, and in the combined H. pylori -infection and NSAID-use group, in superficial epithelial cells and inflammatory cells of the lamina propria of gastric tissues (600x). H. pylori- infected gastric ulcers express higher MMP-7, MMP-9, and TIMP-1 than NSAID-related ulcers. Arrows indicate positive staining.

Journal: The Scientific World Journal

Article Title: Expressions of MMPs and TIMP-1 in Gastric Ulcers May Differentiate H. pylori -Infected from NSAID-Related Ulcers

doi: 10.1100/2012/539316

Figure Lengend Snippet: The immunohistochemical stains for matrix metalloproteinase (MMP)-3, -7, and -9 and tissue inhibitor of matrix metalloproteinase (TIMP)-1 in the Helicobacter pylori- ( H. pylori- ) infected group, in the nonsteroidal anti-inflammatory drug- (NSAID-) related group, and in the combined H. pylori -infection and NSAID-use group, in superficial epithelial cells and inflammatory cells of the lamina propria of gastric tissues (600x). H. pylori- infected gastric ulcers express higher MMP-7, MMP-9, and TIMP-1 than NSAID-related ulcers. Arrows indicate positive staining.

Article Snippet: Tissue immunohistochemical staining was performed using mouse monoclonal antibodies of anti-human-MMP-3, -7, -9, or TIMP-1 (Chemicon International, Inc., Temecula, CA, USA).

Techniques: Immunohistochemical staining, Infection, Staining

Box plots of the distributions and median scores of MMP-7, and -9 and TIMP-1 in the superficial epithelium of gastric ulcer tissues and nonulcer tissues in different groups, respectively. The P values compared scores according to patients in the H. pylori -infected, NSAID-related, and combined H. pylori -infection and NSAID-use groups by Mann-Whitney U test, and also compared scores according to ulcer tissues and nonulcer tissues by Wilcoxon signed-rank test. In the box plots, the 75th and 25th percentiles are represented by the top and bottom of the box, respectively. The horizontal lines refer to the medians. Abbreviations are as in .

Journal: The Scientific World Journal

Article Title: Expressions of MMPs and TIMP-1 in Gastric Ulcers May Differentiate H. pylori -Infected from NSAID-Related Ulcers

doi: 10.1100/2012/539316

Figure Lengend Snippet: Box plots of the distributions and median scores of MMP-7, and -9 and TIMP-1 in the superficial epithelium of gastric ulcer tissues and nonulcer tissues in different groups, respectively. The P values compared scores according to patients in the H. pylori -infected, NSAID-related, and combined H. pylori -infection and NSAID-use groups by Mann-Whitney U test, and also compared scores according to ulcer tissues and nonulcer tissues by Wilcoxon signed-rank test. In the box plots, the 75th and 25th percentiles are represented by the top and bottom of the box, respectively. The horizontal lines refer to the medians. Abbreviations are as in .

Article Snippet: Tissue immunohistochemical staining was performed using mouse monoclonal antibodies of anti-human-MMP-3, -7, -9, or TIMP-1 (Chemicon International, Inc., Temecula, CA, USA).

Techniques: Infection, MANN-WHITNEY

Box plots of the distributions and median scores of MMP-3, MMP-9, and TIMP-1 in inflammatory cells of the lamina propria of gastric ulcer tissues and nonulcer tissues in different groups, respectively. The P values compared scores according to patients in the H. pylori -infected, NSAID-related, and combined H. pylori -infection and NSAID-use groups by Mann-Whitney U test and also compared scores according to ulcer tissues and nonulcer tissues by Wilcoxon signed-rank test. In the box plots, the 75th and 25th percentiles are represented by the top and bottom of the box, respectively. The horizontal lines refer to the medians. Abbreviations are as in .

Journal: The Scientific World Journal

Article Title: Expressions of MMPs and TIMP-1 in Gastric Ulcers May Differentiate H. pylori -Infected from NSAID-Related Ulcers

doi: 10.1100/2012/539316

Figure Lengend Snippet: Box plots of the distributions and median scores of MMP-3, MMP-9, and TIMP-1 in inflammatory cells of the lamina propria of gastric ulcer tissues and nonulcer tissues in different groups, respectively. The P values compared scores according to patients in the H. pylori -infected, NSAID-related, and combined H. pylori -infection and NSAID-use groups by Mann-Whitney U test and also compared scores according to ulcer tissues and nonulcer tissues by Wilcoxon signed-rank test. In the box plots, the 75th and 25th percentiles are represented by the top and bottom of the box, respectively. The horizontal lines refer to the medians. Abbreviations are as in .

Article Snippet: Tissue immunohistochemical staining was performed using mouse monoclonal antibodies of anti-human-MMP-3, -7, -9, or TIMP-1 (Chemicon International, Inc., Temecula, CA, USA).

Techniques: Infection, MANN-WHITNEY

Expression of TIMP-3 in vitreous fluid samples. Equal volumes (15 μl) of vitreous fluid samples from patients with proliferative diabetic retinopathy (PDR) ( n = 12) and non-diabetic patients with rhegmatogenous retinal detachment (RD) ( n = 12) were subjected to gel electrophoresis and the presence of TIMP-3 was detected by Western blot analysis. A representative set of samples is shown.

Journal: Frontiers in Physiology

Article Title: Tissue Inhibitor of Metalloproteinase-3 Ameliorates Diabetes-Induced Retinal Inflammation

doi: 10.3389/fphys.2021.807747

Figure Lengend Snippet: Expression of TIMP-3 in vitreous fluid samples. Equal volumes (15 μl) of vitreous fluid samples from patients with proliferative diabetic retinopathy (PDR) ( n = 12) and non-diabetic patients with rhegmatogenous retinal detachment (RD) ( n = 12) were subjected to gel electrophoresis and the presence of TIMP-3 was detected by Western blot analysis. A representative set of samples is shown.

Article Snippet: The antibodies used for Western blot analysis in the present study were as follows: rabbit monoclonal anti-phospho-extracellular signal-regulated kinase (ERK)1/2 antibody (1:1,500, MAB1018, R&D Systems, Minneapolis, MA, United States), mouse monoclonal anti-phospho-p65 subunit of nuclear factor-kappa B (NF-κB) antibody (1:500, sc-136548, Santa Cruz Biotechnology Inc., Santa Cruz, CA, United States), rabbit polyclonal anti-phospho vascular endothelial growth factor receptor (VEGFR-2) antibody (1:1,000, ab194806, Abcam, Cambridge, United Kingdom), mouse monoclonal anti-intercellular adhesion molecule-1 (ICAM-1) antibody (1:1,000, sc-8439, Santa Cruz Biotechnology Inc.), mouse monoclonal anti-vascular cell adhesion protein-1 (VCAM-1) antibody (1:1,000, sc-13160, Santa Cruz Biotechnology Inc.), mouse monoclonal anti-VEGF antibody (1:1,500, MAB293, R&D Systems), rabbit polyclonal caspase-3 antibody (1:1,000, sc-7148, Santa Cruz Biotechnology Inc.), rabbit polyclonal ADAM-17 antibody (1:1,000, ab39163, Abcam), and rabbit monoclonal anti-TIMP-3 antibody (1:1,000, ab277794, Abcam).

Techniques: Expressing, Nucleic Acid Electrophoresis, Western Blot

Tissue inhibitor of matrix metalloproteinase-3 (TIMP-3) attenuates retinal inflammation and prevents diabetes-induced blood-retinal barrier (BRB) breakdown. The BRB breakdown [panel (A) ] was quantified with the fluorescein isothiocyanate-conjugated dextran technique after treatment with intravitreal injection of 350 μM TIMP-3 in 5 μl in one eye and the same volume of phosphate-buffered saline (PBS) in the contralateral eye of rats 10 weeks after induction of diabetes. Results are expressed as mean ± standard deviation of five rats in each group. Evaluation of inflammatory mediators was evaluated shortly after diabetes induction (see section “Materials and Methods”). Western blot analysis of rat retinas was performed to evaluate protein expression levels of phospho-ERK1/2 [panel (B) ], the p65 subunit of NF-κB [panel (C) ], intercellular adhesion molecule-1 (ICAM-1) [panel (D) ], and vascular endothelial growth factor (VEGF) [panel (E) ]. Results are expressed as mean ± standard deviation of 12 rats in each group. One-way ANOVA and independent t -test were used for comparisons between the three and two groups, respectively, panels (A–E) . * p < 0.05 compared with non-diabetic controls. # p < 0.05 compared with PBS-treated diabetic rats.

Journal: Frontiers in Physiology

Article Title: Tissue Inhibitor of Metalloproteinase-3 Ameliorates Diabetes-Induced Retinal Inflammation

doi: 10.3389/fphys.2021.807747

Figure Lengend Snippet: Tissue inhibitor of matrix metalloproteinase-3 (TIMP-3) attenuates retinal inflammation and prevents diabetes-induced blood-retinal barrier (BRB) breakdown. The BRB breakdown [panel (A) ] was quantified with the fluorescein isothiocyanate-conjugated dextran technique after treatment with intravitreal injection of 350 μM TIMP-3 in 5 μl in one eye and the same volume of phosphate-buffered saline (PBS) in the contralateral eye of rats 10 weeks after induction of diabetes. Results are expressed as mean ± standard deviation of five rats in each group. Evaluation of inflammatory mediators was evaluated shortly after diabetes induction (see section “Materials and Methods”). Western blot analysis of rat retinas was performed to evaluate protein expression levels of phospho-ERK1/2 [panel (B) ], the p65 subunit of NF-κB [panel (C) ], intercellular adhesion molecule-1 (ICAM-1) [panel (D) ], and vascular endothelial growth factor (VEGF) [panel (E) ]. Results are expressed as mean ± standard deviation of 12 rats in each group. One-way ANOVA and independent t -test were used for comparisons between the three and two groups, respectively, panels (A–E) . * p < 0.05 compared with non-diabetic controls. # p < 0.05 compared with PBS-treated diabetic rats.

Techniques: Injection, Standard Deviation, Western Blot, Expressing

Müller cells were left untreated or treated with high-glucose (HG, 25 mM) [panel (A) ] cobalt chloride (CoCl 2 ) (300 μM) [panel (B) ] or tumor necrosis factor -α (TNF-α) (50 ng/ml) [panel (C) ] for 24 h or TIMP-3 (100 ng/ml) for 1 h followed by HG, CoCl 2 , or TNF-α. For HG treatment, cultures containing 25 mM mannitol were used as a control. Levels of vascular endothelial growth factor (VEGF) and monocyte chemotactic protein-1 (MCP-1) were quantified in the culture media by ELISA. Results are expressed as median (interquartile range) from three different experiments performed in triplicate. Kruskal-Wallis test and Mann-Whitney test were used for comparison between three groups and two groups, respectively. * p < 0.05 compared with values obtained from control cells. # p < 0.05 compared with values obtained from cells treated with HG, CoCl 2 , or TNF-α.

Journal: Frontiers in Physiology

Article Title: Tissue Inhibitor of Metalloproteinase-3 Ameliorates Diabetes-Induced Retinal Inflammation

doi: 10.3389/fphys.2021.807747

Figure Lengend Snippet: Müller cells were left untreated or treated with high-glucose (HG, 25 mM) [panel (A) ] cobalt chloride (CoCl 2 ) (300 μM) [panel (B) ] or tumor necrosis factor -α (TNF-α) (50 ng/ml) [panel (C) ] for 24 h or TIMP-3 (100 ng/ml) for 1 h followed by HG, CoCl 2 , or TNF-α. For HG treatment, cultures containing 25 mM mannitol were used as a control. Levels of vascular endothelial growth factor (VEGF) and monocyte chemotactic protein-1 (MCP-1) were quantified in the culture media by ELISA. Results are expressed as median (interquartile range) from three different experiments performed in triplicate. Kruskal-Wallis test and Mann-Whitney test were used for comparison between three groups and two groups, respectively. * p < 0.05 compared with values obtained from control cells. # p < 0.05 compared with values obtained from cells treated with HG, CoCl 2 , or TNF-α.

Techniques: Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Müller cells were pre-incubated with TIMP-3 (100 ng/ml) or dilution medium for 1 h before increasing the sugar content of the cultures [25 mM of mannitol as control or 25 mM of glucose (HG)]. After 24 h levels of the p65 subunit of NF-κB [panel (A) ], phospho-ERK1/2 [panel (B) ], caspase-3 [panel (C) ], and ADAM17 [panel (D) ] in cell lysates was determined by Western blot analysis. Results are expressed as median (interquartile range) from three different experiments performed in triplicate. Kruskal-Wallis test and Mann-Whitney test were used for comparisons between three groups and two groups, respectively. * p < 0.05 compared with values obtained from cells treated with mannitol. # p < 0.05 compared with values obtained from cells treated with HG.

Journal: Frontiers in Physiology

Article Title: Tissue Inhibitor of Metalloproteinase-3 Ameliorates Diabetes-Induced Retinal Inflammation

doi: 10.3389/fphys.2021.807747

Figure Lengend Snippet: Müller cells were pre-incubated with TIMP-3 (100 ng/ml) or dilution medium for 1 h before increasing the sugar content of the cultures [25 mM of mannitol as control or 25 mM of glucose (HG)]. After 24 h levels of the p65 subunit of NF-κB [panel (A) ], phospho-ERK1/2 [panel (B) ], caspase-3 [panel (C) ], and ADAM17 [panel (D) ] in cell lysates was determined by Western blot analysis. Results are expressed as median (interquartile range) from three different experiments performed in triplicate. Kruskal-Wallis test and Mann-Whitney test were used for comparisons between three groups and two groups, respectively. * p < 0.05 compared with values obtained from cells treated with mannitol. # p < 0.05 compared with values obtained from cells treated with HG.

Techniques: Incubation, Western Blot, MANN-WHITNEY

THP-1 monocytes were left untreated or treated with TIMP-3 (100 ng/ml) for 24 h. Subsequently, the THP-1 cells were fluorescently labeled and adhesion to a human retinal microvascular endothelial cell (HRMEC) monolayer was assessed [panel (A) ]. Results are expressed as median (interquartile range) from two independent experiments (each treatment condition: 6 wells) (* p < 0.05; Mann-Whitney test). Alternatively, HRMECs were pre-incubated with TIMP-3 (100 ng/ml) or dilution medium for 1 h before stimulation with dilution medium, tumor necrosis factor-α (TNF-α) (25 ng/ml) [panel (B) ] or vascular endothelial growth factor (VEGF) (50 ng/ml) [panel (C) ] for 24 h. Adhesion of fluorescently labeled monocytic cells to the HRMEC monolayer was assessed. Results are expressed as median (interquartile range) from three independent experiments (each treatment condition: 6 wells). Kruskal-Wallis test and Mann-Whitney test were used for comparisons between three groups and two groups, respectively (RFU = relative fluorescence unit). HRMECs were left untreated or were stimulated with TNF-α (50 ng/ml) for 24 h with/without a 1-h pre-incubation with TIMP-3 (100 ng/ml). Protein expression of vascular cell adhesion molecule-1 (VCAM-1) [panel (D) ] and intercellular adhesion molecule-1 (ICAM-1) [panel (E) ] was determined by Western blot analysis. Results are expressed as mean ± standard deviation from three independent experiments (each treatment condition: 8 wells). One-way ANOVA and independent t -test were used for comparisons between three groups and 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 or VEGF.

Journal: Frontiers in Physiology

Article Title: Tissue Inhibitor of Metalloproteinase-3 Ameliorates Diabetes-Induced Retinal Inflammation

doi: 10.3389/fphys.2021.807747

Figure Lengend Snippet: THP-1 monocytes were left untreated or treated with TIMP-3 (100 ng/ml) for 24 h. Subsequently, the THP-1 cells were fluorescently labeled and adhesion to a human retinal microvascular endothelial cell (HRMEC) monolayer was assessed [panel (A) ]. Results are expressed as median (interquartile range) from two independent experiments (each treatment condition: 6 wells) (* p < 0.05; Mann-Whitney test). Alternatively, HRMECs were pre-incubated with TIMP-3 (100 ng/ml) or dilution medium for 1 h before stimulation with dilution medium, tumor necrosis factor-α (TNF-α) (25 ng/ml) [panel (B) ] or vascular endothelial growth factor (VEGF) (50 ng/ml) [panel (C) ] for 24 h. Adhesion of fluorescently labeled monocytic cells to the HRMEC monolayer was assessed. Results are expressed as median (interquartile range) from three independent experiments (each treatment condition: 6 wells). Kruskal-Wallis test and Mann-Whitney test were used for comparisons between three groups and two groups, respectively (RFU = relative fluorescence unit). HRMECs were left untreated or were stimulated with TNF-α (50 ng/ml) for 24 h with/without a 1-h pre-incubation with TIMP-3 (100 ng/ml). Protein expression of vascular cell adhesion molecule-1 (VCAM-1) [panel (D) ] and intercellular adhesion molecule-1 (ICAM-1) [panel (E) ] was determined by Western blot analysis. Results are expressed as mean ± standard deviation from three independent experiments (each treatment condition: 8 wells). One-way ANOVA and independent t -test were used for comparisons between three groups and 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 or VEGF.

Techniques: Labeling, MANN-WHITNEY, Incubation, Fluorescence, Expressing, Western Blot, Standard Deviation

Tissue inhibitor of matrix metalloproteinase-3 (TIMP-3) inhibits vascular endothelial growth factor (VEGF)-mediated human retinal microvascular endothelial cell (HRMEC) migration, chemotaxis and proliferation. A scratch was made in confluent monolayers of overnight starved HRMECs with a micropipette tip. Subsequently, the cultures were pre-incubated with TIMP-3 (100 ng/ml) or dilution medium for 1 h before stimulation with dilution medium or vascular endothelial growth factor (VEGF) (50 ng/ml) for 24 h. Cells were visualized using an inverted microscope. Three independent experiments were performed. Each experiment was done in triplicate and 6–8 independent field images were taken for the migration analysis which was done by using image J software. In the figure one representative image is shown [panel (A) ]. Results are expressed as median (interquartile range). Kruskal-Wallis test and Mann-Whitney test were used for comparisons between three groups and two groups, respectively. * p < 0.05 compared with untreated cells. # p < 0.05 compared with VEGF-treated cells. Chemotaxis and proliferation of HRMECs stimulated with 10 ng/ml VEGF was modulated by a 30-min pre-incubation with TIMP-3 (10 or 100 ng/ml). Cell migration was monitored using the xCELLigence RTCA-DP system. The median (interquartile range) percentage of inhibition of VEGF-induced chemotaxis [panel (B) ] or proliferation [panel (C) ] is shown. In total five chemotaxis experiments were performed and conditions were tested in duplicate or triplicate within 1 experiment. For proliferation, 6 experiments were performed and conditions were tested at least in triplicate within 1 experiment. * p < 0.05; Mann-Whitney test (compared with VEGF).

Journal: Frontiers in Physiology

Article Title: Tissue Inhibitor of Metalloproteinase-3 Ameliorates Diabetes-Induced Retinal Inflammation

doi: 10.3389/fphys.2021.807747

Figure Lengend Snippet: Tissue inhibitor of matrix metalloproteinase-3 (TIMP-3) inhibits vascular endothelial growth factor (VEGF)-mediated human retinal microvascular endothelial cell (HRMEC) migration, chemotaxis and proliferation. A scratch was made in confluent monolayers of overnight starved HRMECs with a micropipette tip. Subsequently, the cultures were pre-incubated with TIMP-3 (100 ng/ml) or dilution medium for 1 h before stimulation with dilution medium or vascular endothelial growth factor (VEGF) (50 ng/ml) for 24 h. Cells were visualized using an inverted microscope. Three independent experiments were performed. Each experiment was done in triplicate and 6–8 independent field images were taken for the migration analysis which was done by using image J software. In the figure one representative image is shown [panel (A) ]. Results are expressed as median (interquartile range). Kruskal-Wallis test and Mann-Whitney test were used for comparisons between three groups and two groups, respectively. * p < 0.05 compared with untreated cells. # p < 0.05 compared with VEGF-treated cells. Chemotaxis and proliferation of HRMECs stimulated with 10 ng/ml VEGF was modulated by a 30-min pre-incubation with TIMP-3 (10 or 100 ng/ml). Cell migration was monitored using the xCELLigence RTCA-DP system. The median (interquartile range) percentage of inhibition of VEGF-induced chemotaxis [panel (B) ] or proliferation [panel (C) ] is shown. In total five chemotaxis experiments were performed and conditions were tested in duplicate or triplicate within 1 experiment. For proliferation, 6 experiments were performed and conditions were tested at least in triplicate within 1 experiment. * p < 0.05; Mann-Whitney test (compared with VEGF).

Techniques: Migration, Chemotaxis Assay, Incubation, Inverted Microscopy, Software, MANN-WHITNEY, Inhibition

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