Journal: Investigative Ophthalmology & Visual Science

Article Title: Myocilin Regulates Metalloprotease 2 Activity Through Interaction With TIMP3

doi: 10.1167/iovs.16-20336

Figure Lengend Snippet: TIMP3 is identified as a myocilin-binding protein. (A) Western blot analysis of immunoprecipitates with anti-myocilin antibodies or rabbit IgG from eye lysates of Myoc null (KO), WT, and Tg mice expressing WT (wt MYOC) or mutated mouse myocilin (m MYOC). The efficiency and specificity of immunoprecipitation were confirmed using anti-myocilin antibodies. The peptide number (N) indicates the number of peptides associated with myocilin (MYOC) or TIMP3 identified in the immunoprecipitates by shotgun proteomics. (B) Myocilin interacts with TIMP3 in HEK293 cells. HEK293 cells were cotransfected with indicated HA-tagged human TIMP constructs and a FLAG-tagged human myocilin construct. The lysates were immunoprecipitated with anti-HA antibodies and the precipitates were probed with anti-FLAG antibodies. (C) Myocilin interacts with TIMP3 in HepG2 cells. HepG2 cells were cotransfected with a myc-tagged TIMP3 construct and indicated FLAG-tagged myocilin constructs. The lysates were immunoprecipitated with anti-FLAG antibodies and the precipitates were probed with anti-myc antibodies. The arrowhead marks TIMP3 band and the arrow marks immunoglobulin band. (D) Model of TIMP3–OLF interaction based on computational docking, suggesting that OLF does not compete for the MMP binding site on TIMP3, which is near its N-terminus.

Article Snippet: Sections were fixed with 4% PFA for 10 minutes on ice, blocked with Blocker Casein PBS (Thermo Fisher) for 30 minutes at room temperature, and then incubated with primary antibodies against mouse myocilin or TIMP3 (Novus Biologicals) at 4°C overnight.

Techniques: Binding Assay, Western Blot, Expressing, Immunoprecipitation, Construct

Journal: Investigative Ophthalmology & Visual Science

Article Title: Myocilin Regulates Metalloprotease 2 Activity Through Interaction With TIMP3

doi: 10.1167/iovs.16-20336

Figure Lengend Snippet: The distribution of Myoc and Timp3 mRNAs in the eye drainage structures and in the posterior part of the eye. (A–D) Eye drainage structures: (A) Myoc mRNA (green puncta) is abundant in TM cells. Timp3 mRNA (magenta puncta) is abundant in the ciliary body and is also present in TM cells. (B) The image of the same area shown in (A) obtained using differential interference contrast (DIC) microscopy for better visualization of the tissue orientation. It also reveals that Timp3 mRNA is present in nonpigmented ciliary epithelium. (C) A magnified image of the TM area boxed in (A). (D) A further magnified image of the TM area. TM cells contain both Myoc and Timp3 mRNAs. CB, ciliary body; IR, iris; NR, neural retina; SC, sclera; TM, trabecular meshwork. (E–H) Posterior part of the eye: (E) Myoc mRNA was detected in choroid in the proximity of sclera and sclera. Timp3 mRNA is abundant in the inner nuclear layer of the retina and RPE, and is also detected in choroid in the proximity of sclera. (F) The image of the same area as in (E) obtained using DIC microscopy. (G) A magnified image of the area boxed in (E). (H) The image of the same area as in (G) obtained using DIC microscopy to show the tissue orientation. CH, choroid; GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; RPE, retinal pigmented epithelium; SC, sclera.

Article Snippet: Sections were fixed with 4% PFA for 10 minutes on ice, blocked with Blocker Casein PBS (Thermo Fisher) for 30 minutes at room temperature, and then incubated with primary antibodies against mouse myocilin or TIMP3 (Novus Biologicals) at 4°C overnight.

Techniques: Microscopy

Journal: Investigative Ophthalmology & Visual Science

Article Title: Myocilin Regulates Metalloprotease 2 Activity Through Interaction With TIMP3

doi: 10.1167/iovs.16-20336

Figure Lengend Snippet: Distribution of myocilin and TIMP3 in the ocular tissues. Cryosections of the FVRD mouse eyes were stained with anti-myocilin and TIMP3 antibodies. (A–C, G–I) Eye drainage structures: (A) Myocilin is detected mainly in the TM. (B) The image of the same area as in (A) obtained using DIC microscopy for better visualization of the tissue orientation. (C) Enlarged image of the TM area boxed in (A). (G) TIMP3 is detected in the TM and ciliary body. (H) The image of the same area as in (D) obtained using DIC microscopy. (H) Enlarged image of the TM area boxed in (G). (D–F, J–L) Posterior part of the eye: (D) Myocilin was detected in the choroid and sclera. (E) The image of the same area as in (D) obtained using DIC microscopy. (F) Enlarged image of the RPE–sclera region. (J) TIMP3 was detected in the RPE, Bruch's membrane, choroid, and sclera. (K) The image of the same area as in (J) obtained using DIC microscopy. (L) An enlarged image of RPE–sclera region. Abbreviations are as in . BrM, Bruch's membrane.

Article Snippet: Sections were fixed with 4% PFA for 10 minutes on ice, blocked with Blocker Casein PBS (Thermo Fisher) for 30 minutes at room temperature, and then incubated with primary antibodies against mouse myocilin or TIMP3 (Novus Biologicals) at 4°C overnight.

Techniques: Staining, Microscopy, Membrane

Journal: Investigative Ophthalmology & Visual Science

Article Title: Myocilin Regulates Metalloprotease 2 Activity Through Interaction With TIMP3

doi: 10.1167/iovs.16-20336

Figure Lengend Snippet: Myocilin enhances the inhibitory activity of TIMP3 toward MMP2. (A) The purity of TIMP3 and myocilin was estimated by SDS-PAGE. (B) MMP2 (50 nM) was coincubated with indicated different concentrations of TIMP3. The fluorescence signals representing the protease activity of MMP2 were monitored for 2 hours. Arrow indicates TIMP3 concentration selected for further studies. (C) Myocilin (200 nM) or control IgG (200 nM) was preincubated with TIMP3 (100 nM). TIMP3 alone or the protein mixtures were added to the MMP2 proteolytic reaction. Error bars represent ±SD of triplicate reactions. Statistically significant differences between two groups (+ MMP2, TIMP3 and + MMP2, TIMP3, MYOC) at different time points are indicated by asterisks (P < 0.05).

Article Snippet: Sections were fixed with 4% PFA for 10 minutes on ice, blocked with Blocker Casein PBS (Thermo Fisher) for 30 minutes at room temperature, and then incubated with primary antibodies against mouse myocilin or TIMP3 (Novus Biologicals) at 4°C overnight.

Techniques: Activity Assay, SDS Page, Fluorescence, Concentration Assay, Control

Journal: Frontiers in Pharmacology

Article Title: Direct Thrombin Inhibitor Dabigatran Compromises Pulmonary Endothelial Integrity in a Murine Model of Breast Cancer Metastasis to the Lungs; the Role of Platelets and Inflammation-Associated Haemostasis

doi: 10.3389/fphar.2022.834472

Figure Lengend Snippet: Effects of dabigatran on pulmonary metastasis of 4T1 breast cancer cells injected intravenously into BALB/c mice. Mice were treated with vehicle (black symbols) or dabigatran etexilate (grey symbols) as described in , injected with 4T1 breast cancer cells, and euthanized 24 h, 2 days, and 7 days after injection. In (A) quantitative analysis of pulmonary metastasis in mice, based on CellTracker Red fluorescence in murine lungs isolated from animals 24 h and 2 days after injection of 4T1 cancer cells, is shown. The representative pictures of lung parenchyma illustrating metastatic count in untreated mice 24 h and 2 days after i.v. injection (100 x) are shown in (B,D) , respectively; the representative images of dabigatran-treated mice 24 h and 2 days after injection are shown in (C,E) , respectively. In (F) , quantitative analysis of pulmonary metastasis based on haematoxylin and eosin (H&E) staining 7 days after i.v. injection is shown. The representative images of H&E-stained lung cross-sections (100×) of mice not treated and treated with dabigatran etexilate are shown in (G,I) , respectively, while the results of Ilastik segmentation of (G,I) are shown in (H,J) (blue: lung parenchyma, green: pulmonary metastases), respectively. Statistical analysis in (A) was performed using a two-way ANOVA test followed by Bonferroni post hoc tests, while the results in (F) were analysed with two-sided unpaired T test. The results are presented as the median ± IQR. The symbol *** denotes statistical significance at p < .001.

Article Snippet: Thirty mice were pre-treated with dabigatran etexilate (Biorbyt, cat. no orb180748) by oral gavage twice daily at a total dose of 100 mg per kg of body weight per day in .05% natrosol, and the same number of mice simultaneously received the vehicle (.05% natrosol) for 3 consecutive days.

Techniques: Injection, Fluorescence, Isolation, Staining

Journal: Frontiers in Pharmacology

Article Title: Direct Thrombin Inhibitor Dabigatran Compromises Pulmonary Endothelial Integrity in a Murine Model of Breast Cancer Metastasis to the Lungs; the Role of Platelets and Inflammation-Associated Haemostasis

doi: 10.3389/fphar.2022.834472

Figure Lengend Snippet: Pulmonary fibrin deposition, lag time of thrombin generation in plasma, and innate immunity activation markers. Mice were treated with vehicle (black symbols) or dabigatran etexilate (grey symbols) as described in , injected with 4T1 breast cancer cells, and euthanized at 24 h, 2 days, and 7 days after injection. (A) Lung cross-sections were stained with biotin-conjugated IgG fraction of polyclonal goat antiserum to mouse fibrin antibody as described in . The representative images of fibrin deposition in the lungs of 4T1 breast cancer cell-injected mice are given in AI (untreated, 24 h post i.v.), AII (dabigatran-treated, 24 h post i.v.), AIII (untreated, 2 days post i.v.), AIV (dabigatran-treated, 2 days post i.v.), AV (untreated, 7 days post i.v.), AVI (dabigatran-treated, 7 days post i.v.) (200x). Scanned images were segmented using Ilastik software and counted in ImageJ to determine the number of pixels corresponding to fibrin signal; (B) the thrombin activity was determined by thrombin generation assay in murine plasma, and expressed as a lag time to cleavage of fluorogenic substrate by free thrombin; (C) concentrations of interferon γ (IFNγ) and (D) complement iC3b subunit were determined in lung homogenates by ELISA kits. Statistical analysis was performed with Kruskal-Wallis (A) and two-way ANOVA (B–D) tests followed by appropriate post hoc tests, and the results were presented as median ± IQR. The symbols *, **, and *** denote statistical significance at p < .05, p < .01, and p < 0.001, respectively.

Article Snippet: Thirty mice were pre-treated with dabigatran etexilate (Biorbyt, cat. no orb180748) by oral gavage twice daily at a total dose of 100 mg per kg of body weight per day in .05% natrosol, and the same number of mice simultaneously received the vehicle (.05% natrosol) for 3 consecutive days.

Techniques: Clinical Proteomics, Activation Assay, Injection, Staining, Software, Activity Assay, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Pharmacology

Article Title: Direct Thrombin Inhibitor Dabigatran Compromises Pulmonary Endothelial Integrity in a Murine Model of Breast Cancer Metastasis to the Lungs; the Role of Platelets and Inflammation-Associated Haemostasis

doi: 10.3389/fphar.2022.834472

Figure Lengend Snippet: Effects of dabigatran on lung permeability and inflammation markers in the lungs of BALB/c mice injected with 4T1 breast cancer cells. Mice were treated with vehicle (black symbols) or dabigatran etexilate (grey symbols) as described in . (A) depicts retention of Evans blue (EB) in the lung parenchyma, reflecting lung permeability. Control mice did not receive cancer cells and were euthanized at the end of the dabigatran pre-treatment to exclude the possibility that dabigatran affected pulmonary permeability of healthy animals before their inoculation with 4T1 cancer cells. After dabigatran/vehicle pre-treatment, the other mice were injected intravenously with 4T1 breast cancer cells or the vehicle and euthanized 24 h, 2 days, and 7 days after injection. (B–D) show the levels of Ang-2, E-selectin, and MMP-9, respectively. (E) shows representative Western blot images of Ang-2, E-selectin, and MMP-9 with densitometric data after their normalization to the total protein used as loading control based on the stain-free technique as described in Materials and Methods . For (A–D) , statistical analysis was performed with two-way ANOVA and an appropriate post hoc test. The symbols *, **, and *** denote statistical significance at p < .05, p < .01, and p < .001, respectively, and the data are depicted as the median ± IQR.

Article Snippet: Thirty mice were pre-treated with dabigatran etexilate (Biorbyt, cat. no orb180748) by oral gavage twice daily at a total dose of 100 mg per kg of body weight per day in .05% natrosol, and the same number of mice simultaneously received the vehicle (.05% natrosol) for 3 consecutive days.

Techniques: Permeability, Injection, Control, Western Blot, Staining

Journal: Frontiers in Pharmacology

Article Title: Direct Thrombin Inhibitor Dabigatran Compromises Pulmonary Endothelial Integrity in a Murine Model of Breast Cancer Metastasis to the Lungs; the Role of Platelets and Inflammation-Associated Haemostasis

doi: 10.3389/fphar.2022.834472

Figure Lengend Snippet: Effects of dabigatran on endothelium function based on selected biomarkers of endothelial dysfunction in BALB/c mice injected with 4T1 breast cancer cells. Mice were treated with vehicle (black symbols) or dabigatran etexilate (grey symbols) as described in , injected with 4T1 breast cancer cells, and euthanized at 24 h, 2 days, and 7 days after 4T1 breast cancer cell injection. The panel of selected biomarkers of endothelial dysfunction (Ang-1 (A) , Ang-2 (B) , sTie-2 (C) , sFLT-1 (D) , SDC-1 (E) , sE-sel (F) , sICAM-1 (G) , vWF (H) , PAI-1 (I) , t-PA (J) , sP-sel (K) , and THBS-1 (L) ) was measured in the plasma using the microLC/MS-MRM method as described in . The data are presented as the median ± IQR. Statistical analysis was performed with two-way ANOVA (A,C–H,J–L) or Kruskal-Wallis (B,I) tests followed by appropriate post hoc tests. The symbols *, **, and *** denote statistical significance at p < .05, p < .01, and p < .001, respectively.

Article Snippet: Thirty mice were pre-treated with dabigatran etexilate (Biorbyt, cat. no orb180748) by oral gavage twice daily at a total dose of 100 mg per kg of body weight per day in .05% natrosol, and the same number of mice simultaneously received the vehicle (.05% natrosol) for 3 consecutive days.

Techniques: Injection, Clinical Proteomics

Journal: Frontiers in Pharmacology

Article Title: Direct Thrombin Inhibitor Dabigatran Compromises Pulmonary Endothelial Integrity in a Murine Model of Breast Cancer Metastasis to the Lungs; the Role of Platelets and Inflammation-Associated Haemostasis

doi: 10.3389/fphar.2022.834472

Figure Lengend Snippet: Effects of dabigatran on thrombin-induced platelet reactivity measured ex vivo . In (A,D–F) , mice were treated with vehicle (black symbols) or dabigatran etexilate (grey symbols) as described in , injected with 4T1 breast cancer cells, and euthanized at 24 h, 2 days, and 7 days after injection, whereas in (B,C) , washed blood samples of healthy mice were pre-incubated with dabigatran ex vivo . (A) shows the mean concentration of dabigatran in the animal plasma at the time of the euthanasia (approximately 6–8 h after the last oral dabigatran gavage). (B) shows the expression of an active form of GPIIb/IIIa on the surface of platelets in whole blood obtained from healthy animals. The blood samples isolated from these healthy animals were washed, pre-treated ex vivo with dabigatran at concentrations of 1, 10, 30, and 100 ng ml −1 , and activated with bovine thrombin at a dose of .025 or .1 U ml −1 . The data in (B) are presented as the mean and SEM of 2–3 independent experiments. Dabigatran alone at these concentrations did not affect the expression of the active form of GPIIb/IIIa (data not shown). (C) shows the representative flow cytometry results presented in (B) . In (D) and (E) , expression of an active form of GPIIb/IIIa on the platelet surface is shown as the percentage of the total (parent) platelet population and median fluorescence intensity (MFI) on the platelet surface, respectively, presented after activation of with .025 U ml −1 of bovine thrombin of washed blood samples obtained from untreated and dabigatran-treated 4T1 breast cancer cell-injected mice. In (F) , the representative flow cytometry data of (D,E) are shown. The data were analysed with one-way ANOVA (A) or two-way ANOVA (D,E) followed by appropriate post hoc test, and the results are presented as median ± IQR. The symbols *, **, and *** denote statistical significance at p < .05, p < .01, and p < .001, respectively.

Article Snippet: Thirty mice were pre-treated with dabigatran etexilate (Biorbyt, cat. no orb180748) by oral gavage twice daily at a total dose of 100 mg per kg of body weight per day in .05% natrosol, and the same number of mice simultaneously received the vehicle (.05% natrosol) for 3 consecutive days.

Techniques: Ex Vivo, Injection, Incubation, Concentration Assay, Clinical Proteomics, Expressing, Isolation, Flow Cytometry, Fluorescence, Activation Assay

Journal: Frontiers in Pharmacology

Article Title: Direct Thrombin Inhibitor Dabigatran Compromises Pulmonary Endothelial Integrity in a Murine Model of Breast Cancer Metastasis to the Lungs; the Role of Platelets and Inflammation-Associated Haemostasis

doi: 10.3389/fphar.2022.834472

Figure Lengend Snippet: Protection of human pulmonary endothelial barrier compromised by IL-1β stimulation afforded by releasates of washed human platelets and the reversal of this effect by dabigatran. Human lung microvascular endothelial cell (HLMVEC) monolayers were treated with releasates derived from non-stimulated PLT (R [PLT q ]) (A) or PLT stimulated with .1 U ml −1 thrombin (R [PLT thr ]) (B) in the presence of 10 ng ml −1 IL-1β or without IL-1β. The effect of releasates derived from thrombin-stimulated PLT on the barrier integrity formed by HLMVECs in the presence of 30 ng ml −1 of dabigatran (R [PLT thr + D]) was also tested without IL-1β (C) or in the presence of IL-1β (D) . The data are shown as the mean of 3–4 independent experiments and were analysed using analysis of covariance (ANCOVA) in the stable phase (II). Thrombin alone added in Tyrode buffer (.1 U ml −1 ; the final concentration of .01 U ml −1 ) did not affect endothelial barrier integrity.

Article Snippet: Thirty mice were pre-treated with dabigatran etexilate (Biorbyt, cat. no orb180748) by oral gavage twice daily at a total dose of 100 mg per kg of body weight per day in .05% natrosol, and the same number of mice simultaneously received the vehicle (.05% natrosol) for 3 consecutive days.

Techniques: Derivative Assay, Concentration Assay

Journal: The Journal of biological chemistry

Article Title: The C-terminal domains of ADAMTS1 contain exosites involved in its proteoglycanase activity.

doi: 10.1016/j.jbc.2023.103048

Figure Lengend Snippet: Figure 2. Inhibition of ADAMTS1 by TIMPs. A, inhibition of versicanase activity by TIMP family members. TIMP1, TIMP2, TIMP3, and TIMP4 (each at 500 nM) were incubated with ADAMTS1 (100 nM) for 1 h at 37 C before addition of V1-5GAG and digestion for 2 h. Following SDS-PAGE under reducing conditions (5% β-mercaptoethanol) and immunoblotting, FL V1-5GAG and versikine (VSK) were detected by the anti-Vc antibody. A representative immunoblot is shown (n = 2 independent experiments). B, inhibition of peptidolytic activity. TIMPs (each 25 nM) were incubated with a nominal con- centration of 25 nM ADAMTS1 for 1 h at 37 C before addition of the QF peptide substrate fluorescein-5(6)-carbonyl-Ala-Glu-Leu-Asn-Gly-Arg-Pro-Ile-Ser-Ile- Ala-Lys (5(6)-TAMRA) (3.5 μM) and digestion for 2 h. Following subtraction of the background (reactions not containing ADAMTS1), values were converted into percentage of ADAMTS1 activity in the absence of TIMPs and reported as average ± SD (n = 3, each point representing a technical replicate), p < 0.05 by Mann-Whitney test. C, titration of ADAMTS1 with TIMP3. TIMP3 (0–16 nM) was incubated with ADAMTS1 (20 nM nominal concentration) at 37 C for 1 h, and residual activity against the QF peptide FAM-AELNGRPISIAK-Tamra (3.5 μM) was determined. A representative titration curve is shown, each point representing a mean of two technical replicates. Final concentration of ADAMTS1 following titration was 10 nM. FL, full-length; No E, no enzyme; No I, no inhibitor; QF, Quenched-Fluorescent; TIMP, tissue inhibitor of metalloproteinase.

Article Snippet: Semiquantitative proteoglycan cleavage assays Purified V1-5GAG (100 nM) was digested with ADAMTS1, in TNC-B buffer at 37 C for 2 h. Where indicated, 500 μM recombinant human TIMP1, TIMP2, TIMP3, or TIMP4 (R&D Systems, Cat. n.: 970-TM, 971-TM, 973-TM, 974-TSF) were preincubated with 100 nM ADAMTS1 for 1 h at 37 C before digestion.

Techniques: Inhibition, Activity Assay, Incubation, SDS Page, Western Blot, MANN-WHITNEY, Titration, Concentration Assay

Journal: Pharmacology Research & Perspectives

Article Title: Utility of Glycosylated TIMP3 molecules: Inhibition of MMPs and TACE to improve cardiac function in rat myocardial infarct model

doi: 10.1002/prp2.442

Figure Lengend Snippet: Summary of MMP inhibition potency (IC 50 ) and serum half‐life of engineered TIMP3 molecules

Article Snippet: The TIMP3 and fusion constructs were next purified through a three‐column chromatography procedure, each utilizing a specific capture column; TIMP3 and glycosylation mutants with Capto MMC (GE Healthcare, Freiburg, Germany): HSA fusions with Cibacron Blue (Merck KGaA, Darmstadt, Germany): and Fc or Ab fusions with MabSelect Sure (GE Healthcare, Pittsburg, PA).

Techniques: Inhibition

Journal: Pharmacology Research & Perspectives

Article Title: Utility of Glycosylated TIMP3 molecules: Inhibition of MMPs and TACE to improve cardiac function in rat myocardial infarct model

doi: 10.1002/prp2.442

Figure Lengend Snippet: Rat PK profiles of TIMP3 molecules. N‐TIMP3 and TIMP3v2 (K22S/F34N) were continuously infused for 6‐8 h (3 and 10 mg/kg/hr) via jugular vein in Sprague Daley rats (n = 3 per group)

Article Snippet: The TIMP3 and fusion constructs were next purified through a three‐column chromatography procedure, each utilizing a specific capture column; TIMP3 and glycosylation mutants with Capto MMC (GE Healthcare, Freiburg, Germany): HSA fusions with Cibacron Blue (Merck KGaA, Darmstadt, Germany): and Fc or Ab fusions with MabSelect Sure (GE Healthcare, Pittsburg, PA).

Techniques: Concentration Assay

Journal: Pharmacology Research & Perspectives

Article Title: Utility of Glycosylated TIMP3 molecules: Inhibition of MMPs and TACE to improve cardiac function in rat myocardial infarct model

doi: 10.1002/prp2.442

Figure Lengend Snippet: Summary of rat cardiac function by intramyocardial delivery of TIMP3 molecules

Article Snippet: The TIMP3 and fusion constructs were next purified through a three‐column chromatography procedure, each utilizing a specific capture column; TIMP3 and glycosylation mutants with Capto MMC (GE Healthcare, Freiburg, Germany): HSA fusions with Cibacron Blue (Merck KGaA, Darmstadt, Germany): and Fc or Ab fusions with MabSelect Sure (GE Healthcare, Pittsburg, PA).

Techniques: