Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: A Role for MMP-10 (Matrix Metalloproteinase-10) in Calcific Aortic Valve Stenosis.

doi: 10.1161/ATVBAHA.120.314143

Figure Lengend Snippet: Figure 5. Effects of aMMP-10 (active matrix metalloproteinase-10) blockade in aortic valve interstitial cells (AVICs). IL (interleukin)-1β (A), Sox9 (SRY [sex-determining region Y]-box 9; B), BMP (bone morphogenetic protein)-4 (C), BMP-9 (D), and Smad 1/5/8 (E) protein expression was measured by Western Blot or ELISA in AVICs treated with aMMP-10 (10−9 M) alone or in combination with TIMP-1 (tissue inhibitor of metalloproteinases type 1; 10−9 M) for 24 h. Alizarin red staining of AVICs treated with aMMP-10 and TIMP-1 (100 µm; F). Secreted protein levels of Runx2 (runt-related transcription factor 2; (G), Sox9 (sex-determining region Y-box 9; H), OPN (osteopontin; I), BMP (bone morphogenetic protein)-4; J), BMP-9 (K), and Smad 1/5/8 (L) were measured by Western Blot or ELISA in AVICs cultured with osteogenic differentiation media (ODM) in presence or absence of MMP-10ab or OPG (osteoprotegerin) during 7 d. Alizarin red staining of AVICs cultivated with ODM, ODM+MMP-10ab, and ODM+OPG (M). All conditions were performed at least in triplicate. Whisker plots represent median and minimum to maximum range of 6 assays in arbitrary units (AU) normalized to total protein. *P<0.05 vs control; $P<0.05 vs ODM or aMMP-10, respectively.

Article Snippet: 24 Cells were also cultured with osteogenic differentiation media (DMEM 1× [Thermo Fisher], 20% FBS, 1% penicillin/streptomycin, 5 μg/mL insulin [Sigma-Aldrich]; 10 ng/mL FGF-2 [Novus biological], 0.2 M Na2HPO4 [SigmaAldrich], and 0.2 M NaH2PO4 [Sigma-Aldrich]), in the absence or presence of a neutralizing antibody against MMP-10 (10−9 M, MMP-10ab [R&D Systems]) or OPG (osteoprotegerin; 10−9 M [Preprotech]) during 7 days.

Techniques: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Staining, Cell Culture, Whisker Assay, Control

Journal: The Journal of Biological Chemistry

Article Title: Selective inhibition of matrix metalloproteinase 10 (MMP10) with a single-domain antibody

doi: 10.1074/jbc.RA119.011712

Figure Lengend Snippet: Active recombinant MMP10. A, schematic representing MMP10 immunogen as a bivalent Fc fusion protein; B, evidence for activation of proMMP10 by APMA. Top, cleavage of quenched fluorescent substrate by MMP10; bottom, SDS-PAGE showing generation of active MMP10 evidenced by loss of the propeptide. RFU, relative fluorescence units.

Article Snippet: Characterization of H3-mediated MMP10 inhibition mAb H3, mAb C10, and TIMP1 were titrated 1:2 starting at 400 n m across 23 points in the presence of 2 n m human MMP10 (R&D Systems; see above) and left to complex for 15 min at room temperature.

Techniques: Recombinant, Activation Assay, SDS Page, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Selective inhibition of matrix metalloproteinase 10 (MMP10) with a single-domain antibody

doi: 10.1074/jbc.RA119.011712

Figure Lengend Snippet: Screening of MMP10-Fc binders and inhibitors. A, antibody clones were tested for affinity to MMP10 using biolayer interferometry. B, four of the binders inhibited MMP10 activity as measured by a decrease in cleavage of a quenched fluorescence substrate of MMP10. Antibodies were also measured for inhibition of MMP3 to screen for selectivity. RFU, relative fluorescence units.

Article Snippet: Characterization of H3-mediated MMP10 inhibition mAb H3, mAb C10, and TIMP1 were titrated 1:2 starting at 400 n m across 23 points in the presence of 2 n m human MMP10 (R&D Systems; see above) and left to complex for 15 min at room temperature.

Techniques: Clone Assay, Activity Assay, Fluorescence, Inhibition

Journal: The Journal of Biological Chemistry

Article Title: Selective inhibition of matrix metalloproteinase 10 (MMP10) with a single-domain antibody

doi: 10.1074/jbc.RA119.011712

Figure Lengend Snippet: H3 selectivity toward MMP10. H3 (blue) and TIMP1 (red) were titrated against the indicated MMPs. Activity was monitored using a fluorescence plate reader. Error bars, S.D.; n = 3. RFU, relative fluorescence units.

Article Snippet: Characterization of H3-mediated MMP10 inhibition mAb H3, mAb C10, and TIMP1 were titrated 1:2 starting at 400 n m across 23 points in the presence of 2 n m human MMP10 (R&D Systems; see above) and left to complex for 15 min at room temperature.

Techniques: Activity Assay, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Selective inhibition of matrix metalloproteinase 10 (MMP10) with a single-domain antibody

doi: 10.1074/jbc.RA119.011712

Figure Lengend Snippet: In-depth kinetics of H3. H3, TIMP1, and C10 were titrated with MMP10 as described under “Experimental procedures” to determine kinetics of inhibition. Error bars, S.D.; n = 3. RFU, relative fluorescence units.

Article Snippet: Characterization of H3-mediated MMP10 inhibition mAb H3, mAb C10, and TIMP1 were titrated 1:2 starting at 400 n m across 23 points in the presence of 2 n m human MMP10 (R&D Systems; see above) and left to complex for 15 min at room temperature.

Techniques: Inhibition, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Selective inhibition of matrix metalloproteinase 10 (MMP10) with a single-domain antibody

doi: 10.1074/jbc.RA119.011712

Figure Lengend Snippet: Inhibition of MMP10 cleavage of endogenous substrates. A, H3 was serially diluted into wells containing a constant concentration of MMP10. Samples were then incubated with AAT to observe whether H3 prevents MMP10-mediated cleavage of AAT. Samples were analyzed by reducing SDS-PAGE. Data are representative of three separate experiments. B, NE activity was measured after the addition of H3/MMP10/AAT samples. Error bars. S.D.; n = 3. RFU, relative fluorescence units.

Article Snippet: Characterization of H3-mediated MMP10 inhibition mAb H3, mAb C10, and TIMP1 were titrated 1:2 starting at 400 n m across 23 points in the presence of 2 n m human MMP10 (R&D Systems; see above) and left to complex for 15 min at room temperature.

Techniques: Inhibition, Concentration Assay, Incubation, SDS Page, Activity Assay, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Selective inhibition of matrix metalloproteinase 10 (MMP10) with a single-domain antibody

doi: 10.1074/jbc.RA119.011712

Figure Lengend Snippet: Interaction of MMP10 derivatives with H3 as measured by BLI. A biotinylated protein (biotinylated MMP10-Fc in A and B, biotinylated H3 in C and D) was loaded onto a streptavidin sensor. Once the baseline response (BL) was established, the sensor was introduced into solutions containing the proteins indicated above the sensorgrams to measure the association phase. A, H3 binding to activated MMP10-Fc or proMMP10-Fc. B, H3 binding to active MMP10 or marimastat-inhibited MMP10-Fc. C, binding of TIMP1 to the H3/MMP10 complex. D, binding of TIMP2 to the H3/MMP10 complex.

Article Snippet: Characterization of H3-mediated MMP10 inhibition mAb H3, mAb C10, and TIMP1 were titrated 1:2 starting at 400 n m across 23 points in the presence of 2 n m human MMP10 (R&D Systems; see above) and left to complex for 15 min at room temperature.

Techniques: Binding Assay

Journal: JCI Insight

Article Title: Targetable mechanisms driving immunoevasion of persistent senescent cells link chemotherapy-resistant cancer to aging

doi: 10.1172/jci.insight.124716

Figure Lengend Snippet: (A) MMP mRNA levels measured by quantitative real-time PCR. (B) Secreted MMP levels measured by antibody arrays in conditioned media from fibroblast monocultures. (C) Immunofluorescence of MMP3 staining in naive (top) and persistent (bottom) XRA SEN WI-38 cells. Of note, the vast majority of DAPI-stained nuclei evident in the bottom panel correspond to the nuclei of PBMCs, i.e. not the persistent SnCs (exemplified by arrows indicating PBMC nuclei). Original magnification, ×20. (D) MMP3 expression levels in naive (left) versus persistent (right) cells senesced owing to p16 overexpression versus REP versus XRA versus p53-deficient XRA. MMP3 was detected by immunofluorescence and binned into one of 3 categories: undetectable or very low [MMP3 (–), gray], low to high [MMP3 (+) or (++), pink], and very high [MMP3 (+++), red]. WI-38 and IMR-90 cells were tested. (E) MMP mRNA levels measured by quantitative real-time PCR in SnCs that persisted after 1 or 2 (2x) rounds of PBMCs separated by 10 days. Values were normalized to the fibroblast marker CD90 and then compared between persistent and naive SnCs (fold change relative to naive SnCs). MMPs were not detected in PBMCs (Supplemental Table 2D).

Article Snippet: We used the following primary antibodies: p53 (Oncogene Research Products, DO-1), 53BP1 (Bethyl, BL182), RAS (BD Biosciences, 610001), p16 INK4a (Neomarkers, MS-889-P1 and MS-218-P1), ATM (Abcam Y-170), phospho-ATM (Upstate, 05-740), IL-6 (R&D Systems, MAB2061 and AF-206-NA), IL-8 (R&D Systems, MAB208), MICA/B (R&D Systems, MAB13001; Santa Cruz Biotechnology Inc., sc-5460), MICA (R&D Systems, AF1300 and MAB1300), MICB (R&D Systems, AF1599 and MAB1599), NKG2D (R&D Systems, MAB139; Santa Cruz Biotechnology Inc., sc-9621), NKG2D blocking peptide (Santa Cruz Biotechnology Inc., sc-9621p), CD155 (R&D Systems, MAB2530), CD112 (Abcam, 502-57), DNAM-1 (R&D Systems, MAB666), IL-7 (Abcam, ab175380), MMP1 (R&D Systems, AF901; Oncogene Research Products, IM35L), MMP3 (R&D Systems, AF513; Oncogene Research Products, IM70T; BioVision, 3523-100), MMP10 (R&D Systems, AF910; Oncogene Research Products, IM75T), MMP12 (R&D Systems, MAB917), IgG control (R&D Systems, G-101-C-ABS; MAB002).

Techniques: Real-time Polymerase Chain Reaction, Immunofluorescence, Staining, Expressing, Over Expression, Marker

Journal: JCI Insight

Article Title: Targetable mechanisms driving immunoevasion of persistent senescent cells link chemotherapy-resistant cancer to aging

doi: 10.1172/jci.insight.124716

Figure Lengend Snippet: (A) Survival of RAS-induced senescent WI-38, IMR-90, and HCA-2 cells with active or inactive p53 (p53–) assessed by cell number after 10 days of direct coculture with PBMCs. (B) mRNA levels of NKG2D ligands and MMPs in RAS SnCs were measured by quantitative real-time PCR, and normalized to the average expression in control and p53-deficient presenescent cells. The range of the color scale is 64-fold greater than in Figure 2, A, C, E, and G, and Figure 5A. The ratio of the average expression levels of MMPs relative to NKG2D ligands is ~130 in RAS SnCs compared with ~7 in XRA or REP SnCs. (C) MMP-1, -3, -10, -12 immunofluorescence comparing PRE, XRA, and RAS-induced senescent WI-38 cells. Original magnification, ×20. (D) Detection of soluble MICA by ELISA in conditioned media of RAS-induced senescent WI-38, IMR-90, and HCA2 cells (p53-deficient or WT). Left: MICA levels in monocultures (naive), with SEN (XRA) and PRE cells for comparison. Right, MICA levels in supernatants collected after 10 days of direct coculture with PBMCs (persistent SnCs). In A and D, box plot length: 25% and 75% of data; centerline: median; whiskers: 25% – (or 75% +) 1.5 × IQR; dots: outliers; color bars: average in A, median in D. (E) Proportion of cells with different MMP3 levels quantified from immunofluorescence of naive and persistent RAS SnCs (top: p53 WT; bottom: p53-deficient; quantified as in Figure 5D).

Article Snippet: We used the following primary antibodies: p53 (Oncogene Research Products, DO-1), 53BP1 (Bethyl, BL182), RAS (BD Biosciences, 610001), p16 INK4a (Neomarkers, MS-889-P1 and MS-218-P1), ATM (Abcam Y-170), phospho-ATM (Upstate, 05-740), IL-6 (R&D Systems, MAB2061 and AF-206-NA), IL-8 (R&D Systems, MAB208), MICA/B (R&D Systems, MAB13001; Santa Cruz Biotechnology Inc., sc-5460), MICA (R&D Systems, AF1300 and MAB1300), MICB (R&D Systems, AF1599 and MAB1599), NKG2D (R&D Systems, MAB139; Santa Cruz Biotechnology Inc., sc-9621), NKG2D blocking peptide (Santa Cruz Biotechnology Inc., sc-9621p), CD155 (R&D Systems, MAB2530), CD112 (Abcam, 502-57), DNAM-1 (R&D Systems, MAB666), IL-7 (Abcam, ab175380), MMP1 (R&D Systems, AF901; Oncogene Research Products, IM35L), MMP3 (R&D Systems, AF513; Oncogene Research Products, IM70T; BioVision, 3523-100), MMP10 (R&D Systems, AF910; Oncogene Research Products, IM75T), MMP12 (R&D Systems, MAB917), IgG control (R&D Systems, G-101-C-ABS; MAB002).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Control, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Comparison

Journal: JCI Insight

Article Title: Targetable mechanisms driving immunoevasion of persistent senescent cells link chemotherapy-resistant cancer to aging

doi: 10.1172/jci.insight.124716

Figure Lengend Snippet: (A) Pairwise comparison of MMP expression before and after genotoxic chemotherapy in patients with prostate cancer. Analyses and displays are explained in the Figure 1A legend. An additional set of 20 laser-captured tumor areas isolated from 10 patients before versus after neoadjuvant mitoxantrone therapy is included for validation (purple markers/lines). (B) MMP3 detection by IHC in prostate cancer tissues from patients treated or not with mitoxantrone/docetaxel (MIT/DTX; original magnification, ×4). Tumors from 26 patients not subjected to therapy were compared with tumors from 50 patients receiving MIT/DTX. Histopathology and staining assessment are color coded as 0 (no staining), +1 (detectable), +2 (intermediate), +3 (high). Distribution of each staining intensity level is shown as pie charts below a representative image of intense staining in either condition. (C) Pairwise comparison of NKG2D expression before and after chemotherapy, as described in A. (D) Comparison of MMP, NKG2D ligand, and NKG2D expression profiles in tumor versus normal prostate tissue (top vs. bottom panels) before and after chemotherapy. The 2 sets of 20 paired tissues collected from 10 patients were pooled and plotted by gene type: MMPs (MMP-1, -3, -10, -12; left), NKG2D ligands (MICA, -B, ULBP-1, -2, -3; middle), NKG2D receptor (right). Results are displayed as values before (x axis) compared with after (y axis) chemotherapy. Overall fold change (FC; averaged across patients) and significance (P value, Student’s t test, paired, 2 tails) across MMPs, NKG2D ligands, and NKG2D are shown.

Article Snippet: We used the following primary antibodies: p53 (Oncogene Research Products, DO-1), 53BP1 (Bethyl, BL182), RAS (BD Biosciences, 610001), p16 INK4a (Neomarkers, MS-889-P1 and MS-218-P1), ATM (Abcam Y-170), phospho-ATM (Upstate, 05-740), IL-6 (R&D Systems, MAB2061 and AF-206-NA), IL-8 (R&D Systems, MAB208), MICA/B (R&D Systems, MAB13001; Santa Cruz Biotechnology Inc., sc-5460), MICA (R&D Systems, AF1300 and MAB1300), MICB (R&D Systems, AF1599 and MAB1599), NKG2D (R&D Systems, MAB139; Santa Cruz Biotechnology Inc., sc-9621), NKG2D blocking peptide (Santa Cruz Biotechnology Inc., sc-9621p), CD155 (R&D Systems, MAB2530), CD112 (Abcam, 502-57), DNAM-1 (R&D Systems, MAB666), IL-7 (Abcam, ab175380), MMP1 (R&D Systems, AF901; Oncogene Research Products, IM35L), MMP3 (R&D Systems, AF513; Oncogene Research Products, IM70T; BioVision, 3523-100), MMP10 (R&D Systems, AF910; Oncogene Research Products, IM75T), MMP12 (R&D Systems, MAB917), IgG control (R&D Systems, G-101-C-ABS; MAB002).

Techniques: Comparison, Expressing, Isolation, Biomarker Discovery, Histopathology, Staining