Fig 1: 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.
Fig 2: Recombinant TIMP3 increased phosphorylated ERK1/2 level and decreased estradiol production. (A) The PathDetect in vivo signal transduction pathway trans-reporting systems (Promega) was used to measure transcription activator and ERK1/2 signal transduction pathway after the addition of Recombinant TIMP3. First, the cultured granulosa cells were co-transfected with a pFR-luciferase reporter with a pFA2-Elk1 plasmid. 24h after the transfection, the recombinant TIMP3(rTIMP3) was added into the culture plate well. Granulosa cell sample was collected after 6h treatment then used for luciferase assay. Luciferase activity is indicative of transcription factor-dependent activation, is expressed as relative light units compared to control. (B) Estradiol production was measured by ELISA assay. The addition of TIMP3 suppresses estradiol production in the cultured granulosa cells. The data represents the mean ± SE of three or four independent experiments. Asterisks denote statistically significant differences between the blank control and the rTIMP3 group (p <0.05).
Fig 3: 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).
Fig 4: miR-574 regulates TIMP3 in the cultured granulosa cell. (A) Relative levels of TIMP3 mRNA in the granulosa cell after overexpression of miR-574. (B) Representative western blot of TIMP3 protein in the granulosa cell after lentivirus transduction. (C) Densitometric quantitation depicting decreased expression of TIMP3 protein after over-expression of miR-574. GAPDH was used for western blot and mRNA normalization. The data represents the mean ± SE of three or four independent experiments. Asterisks denote statistically significant differences between the negative control (RFP) and miR-574 group (p <0.05). (D) Relative levels of TIMP3 mRNA after transfection with specific siRNA for miR-574 (miR-574 siRNA) and negative control siRNA (NC) in the granulosa cells. (E) Representative western blot image of TIMP3 protein after siRNA of miR-574 transfection. (F) Densitometric quantitation depicting increased expression of TIMP3 protein. (G) Relative levels of Aromatase mRNA in the granulosa cell after overexpression or knock-down of miR-574. (H) Relative levels of Cyp450scc mRNA in the granulosa cell after overexpression and knock-down of miR-574. GAPDH mRNA and protein were used for western blot and real-time qPCR normalization, respectively. The data represents the mean ± SE of three independent experiments. Asterisks denote statistically significant differences between NC and miR-574-siRNA groups (p <0.05). NS, not significant.
Fig 5: Müller cells were left untreated or treated with high-glucose (HG, 25 mM) [panel (A)] cobalt chloride (CoCl2) (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, CoCl2, 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, CoCl2, or TNF-α.
Supplier Page from R&D Systems, a Bio-Techne Brand for Recombinant Human TIMP-3 Protein, CF