Journal: Journal of Inflammation Research

Article Title: The β3/5 Integrin-MMP9 Axis Regulates Pulmonary Inflammatory Response and Endothelial Leakage in Acute Lung Injury

doi: 10.2147/JIR.S331939

Figure Lengend Snippet: MMP-9 regulates inflammation and permeability in LPS-treated cells. ( A – D ) PMVECs were treated with LPS (1μg/mL) with or without rMMP-9 (10 ng/mL) for 4 h. The mRNA levels of IL-6, CXCL-1, ICAM-1 , and VCAM-1 were detected. ( E ) The protein levels of VCAM-1, ICAM-1 and TRAF-6 were detected by Western blot analysis in PMVECs exposed to LPS with or without rMMP-9 treatment. ( F ) MMP-9 protein levels were detected by immunoblotting in PMVECs exposed to LPS with or without PDTC treatment. ( G ) 293T cells were transfected with Cignal NF-kB dual luciferase reporter plasmids for 48 h, and then cells were treated with LPS (1μg/mL) with or without rMMP-9 (10 ng/mL) for an additional 6 h or 18 h. ( H ) 293T cells were co-transfected with NF-kB dual luciferase reporter plasmids along with control siRNA, or MMP-9 siRNA for 48 h, and then cells were treated with LPS (1μg/mL) for an additional 6 h or 18 h. Cells were then collected and assayed for luciferase activity to evaluate NF-kB promoter activity. ( I ) PMVECs were transfected with control siRNA, or MMP-9 siRNA for 48 h, and then cells were treated with LPS (1μg/mL) in a time course. Supernatant IL-6 secretion was measured by ELISA. ( J ) PMVECs were treated by APMA (APMA was used to active rMMP9 in vitro, and served as a control of active rMMP9) or active rMMP9 (10 ng/mL) 1 h before LPS challenged at indicated times, and then supernatant IL-6 was detected. ( K and L ) The permeability of treated PMVEC monolayers grown on 0.4 mm filters was determined by the appearance of rhodamine-dextran, which was added to the top well at the beginning of the experiment and detected the absorbance at 590 nm in the bottom well during a 90 min time course. * P <0.05, ** P <0.01. All the results are from at least three independent experiments; Data represent means ±SEM.

Article Snippet: Murine recombinant MMP-9 (R&D, 909-MM), MMP-9 ELISA kits (MMPT90), IL-6 ELISA kits (M6000B), TNF-α ELISA kits (DY410), CXCL-1 ELISA kits (MKC00B), and MPO ELISA kits (DY3667) were from R&D Systems.

Techniques: Permeability, Western Blot, Transfection, Luciferase, Activity Assay, Enzyme-linked Immunosorbent Assay, In Vitro

Journal: Journal of Inflammation Research

Article Title: The β3/5 Integrin-MMP9 Axis Regulates Pulmonary Inflammatory Response and Endothelial Leakage in Acute Lung Injury

doi: 10.2147/JIR.S331939

Figure Lengend Snippet: MMP-9 worsens LPS-induced lung injury in vivo. Mice were treated with LPS (2mg/kg, i.t.), recombinant MMP-9 (rMMP-9, 50μg/kg, i.v.), or their combination. 18 h later, bronchoalveolar lavage fluid (BALF) and lung tissues were collected. The protein concentration ( A ), IL-6 ( B ), TNF-α ( C ), cell counts ( D ), neutrophils ( E ) in Bronchoalveolar lavage fluid (BALF), and MPO activity ( F ) from the lung of mice were measured. ( G ) EBA uptake and ( H ) Hematoxylin and eosin (H&E)–stained lung sections (×200 magnification) with the injury score ( I ) were detected. Mice were treated by a lethal dose of LPS (20mg/kg, i.t.) with or without rMMP-9 (50μg/kg, i.v.), and then survival rates ( J ) were investigated. Data were analyzed by the Mantel-Cox test (survival). * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001. Data represent means ±SEM.

Article Snippet: Murine recombinant MMP-9 (R&D, 909-MM), MMP-9 ELISA kits (MMPT90), IL-6 ELISA kits (M6000B), TNF-α ELISA kits (DY410), CXCL-1 ELISA kits (MKC00B), and MPO ELISA kits (DY3667) were from R&D Systems.

Techniques: In Vivo, Recombinant, Protein Concentration, Activity Assay, Staining

Journal: Journal of Inflammation Research

Article Title: The β3/5 Integrin-MMP9 Axis Regulates Pulmonary Inflammatory Response and Endothelial Leakage in Acute Lung Injury

doi: 10.2147/JIR.S331939

Figure Lengend Snippet: Integrin β3/5 regulates MMP-9 expression in LPS-treated PMVECs. ( A and B ) Heat map of downregulated (green) and upregulated (red) mRNAs from PMVECs transfected with Itgb3 siRNA ( siItgb3 ) or Itgb5 siRNA ( siItgb5 ) for 48 h, and then subjected to 18 h of LPS (1μg/mL) treatment. Numbers 1, 2, and 3 refer to three independent experimental analyses of log 2 [(LPS+siCon)/(LPS+siItgb3/5)] . ( C and D ) MMP-9 gene expressions were measured in PMVECs transfected with siItgb3/5 for 48 h prior to 18 h of LPS (1μg/mL) treatment. ( E and F ) Western blot analysis of MMP-9 protein levels from PMVECs transfected with control siRNA ( siCon ) or siItgb3/5 for 48 h prior to 18 h of PBS or LPS (1μg/mL) treatment. ( G and H ) Western blot analysis of MMP-9 protein levels from PMVECs transfected with Itgb3/5 plasmid in a dose course and then exposed to LPS (1μg/mL). ELISA analysis of MMP-9 secretion from PMVECs transfected with Itgb3/5 plasmid ( I ) or siItgb3/5 ( J ) and then exposed to PBS or LPS (1μg/mL). * P <0.05, ** P <0.01. All the results are from at least three independent experiments; Data represent means ±SEM.

Article Snippet: Murine recombinant MMP-9 (R&D, 909-MM), MMP-9 ELISA kits (MMPT90), IL-6 ELISA kits (M6000B), TNF-α ELISA kits (DY410), CXCL-1 ELISA kits (MKC00B), and MPO ELISA kits (DY3667) were from R&D Systems.

Techniques: Expressing, Transfection, Western Blot, Plasmid Preparation, Enzyme-linked Immunosorbent Assay

Journal: Journal of Inflammation Research

Article Title: The β3/5 Integrin-MMP9 Axis Regulates Pulmonary Inflammatory Response and Endothelial Leakage in Acute Lung Injury

doi: 10.2147/JIR.S331939

Figure Lengend Snippet: β5 Integrin/MMP-9 axis modulates inflammatory response and permeability in vitro. PMVECs were transfected with empty or Itgb5 plasmid, or control siRNA or Itgb5 siRNA for 48 h, and then cells were exposed to rMMP-9 (10 ng/mL) for 1h, followed by treatment with LPS (1 μg/mL) in a time course. ( A and B ) Immunoblot analysis of NF-κB signaling protein abundances (p-IKK, IKKβ, p-P65, P65, IκBα, and p-IκBα) was measured. ( D and F ) Supernatant IL-6 secretion was measured by ELISA. ( I and J ) The permeability of treated PMVECs was determined by the appearance of rhodamine-dextran. ( C and E ) 293T cells were co-transfected with NF-kB dual luciferase reporter plasmids along with empty or Itgb5 plasmid, or control siRNA or Itgb5 siRNA for 48 h, and then cells were treated with LPS (1 μg/mL) for an additional 6 h or 18 h. Cells were then collected and assayed for luciferase activity to evaluate NF-kB promoter activity. ( G ) PMVECs were transfected with control siRNA or Itgb5 siRNA for 48 h, and then cells were exposed to LPS for 18 h. The intercellular ROS levels were detected by flow cytometry. ( H ) PMVECs were treated with or without ROS scavenger, Nac (10 mM) for 1 h, prior to LPS (1 μg/mL) administration for 18 h, and immunoblot analysis of PMVECs and immunoprecipitation (IP) of Integrin β5. * P <0.05, ** P <0.01. All the results are from at least three independent experiments; Data represent means ±SEM.

Article Snippet: Murine recombinant MMP-9 (R&D, 909-MM), MMP-9 ELISA kits (MMPT90), IL-6 ELISA kits (M6000B), TNF-α ELISA kits (DY410), CXCL-1 ELISA kits (MKC00B), and MPO ELISA kits (DY3667) were from R&D Systems.

Techniques: Permeability, In Vitro, Transfection, Plasmid Preparation, Western Blot, Enzyme-linked Immunosorbent Assay, Luciferase, Activity Assay, Flow Cytometry, Immunoprecipitation

Journal: Journal of Inflammation Research

Article Title: The β3/5 Integrin-MMP9 Axis Regulates Pulmonary Inflammatory Response and Endothelial Leakage in Acute Lung Injury

doi: 10.2147/JIR.S331939

Figure Lengend Snippet: β5 Integrin/MMP-9 axis regulates acute lung injury in vivo. ( A ) Immunoblot analysis of β5 Integrin and MMP-9 from mice intratracheally treated with control or LPS for 18 h. Data and means ± SEM of 4 mice per group. ( B ) Linear regression of Itgb5 gene expression normalized to control mice vs BALF MMP-9 in ALI subjects (n=12). ( C ) EBA uptake, ( D ) protein concentrations, ( E ) total cell counts, and ( F ) neutrophils from BAL fluid of mice intratracheally treated with Lenti-Empty or Lenti-Itgb5 and then treated with LPS and PBS or recombinant MMP-9, as indicated. Data and means ± SEM pooled of 3 mice per group are from 2 independent experiments. ( G ) EBA uptake, ( H ) protein concentrations, ( I ) total cell counts, and ( J ) neutrophils from BAL fluid of mice intratracheally treated with Lenti-control shRNA or Lenti-Itgb5 shRNA and then treated with LPS and PBS or recombinant MMP-9, as indicated. Data and means ± SEM pooled of 3 mice per group are from 2 independent experiments. ( K ) Histological analysis of lung samples from mice treated as indicated. Images are representative of all independent experiments. ( L ) Survival rates of mice treated as indicated. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001.

Article Snippet: Murine recombinant MMP-9 (R&D, 909-MM), MMP-9 ELISA kits (MMPT90), IL-6 ELISA kits (M6000B), TNF-α ELISA kits (DY410), CXCL-1 ELISA kits (MKC00B), and MPO ELISA kits (DY3667) were from R&D Systems.

Techniques: In Vivo, Western Blot, Expressing, Recombinant, shRNA

Journal: Journal of Inflammation Research

Article Title: The β3/5 Integrin-MMP9 Axis Regulates Pulmonary Inflammatory Response and Endothelial Leakage in Acute Lung Injury

doi: 10.2147/JIR.S331939

Figure Lengend Snippet: Scheme showing that β5 integrin/MMP-9 axis-mediated acute lung injury by promoting pulmonary inflammatory disorders and endothelial permeability.

Article Snippet: Murine recombinant MMP-9 (R&D, 909-MM), MMP-9 ELISA kits (MMPT90), IL-6 ELISA kits (M6000B), TNF-α ELISA kits (DY410), CXCL-1 ELISA kits (MKC00B), and MPO ELISA kits (DY3667) were from R&D Systems.

Techniques: Permeability

Journal:

Article Title: Macrophage expression of active MMP-9 induces acute plaque disruption in apoE-deficient mice

doi: 10.1172/JCI25074

Figure Lengend Snippet: Design and validation of experimental approach. (A) Schematic representation of the HSC transplantation experimental design. Bone marrow cells were harvested from 6- to 8-week-old apoE–/– donor mice and were transduced with macrophage-specific retroviral vectors encoding HA epitope–tagged EGFP (CD68S-HA-EGFP) or MMP-9 (CD68S–MMP-9) (29). After transduction, cells were injected intravenously into 35- to 45-week-old recipient apoE–/– mice. Mice were sacrificed 12 weeks after transplant, and lesion characteristics in the brachiocephalic artery were analyzed. (B–E) Analysis of retroviral gene expression in preestablished atherosclerotic lesions. Immunohistochemistry of similar lesions from apoE–/– mice transplanted with HSCs transduced with CD68S-HA-EGFP (B and C) or CD68S–MMP-9 (D and E) retroviruses reveals comparable levels of macrophage staining with an antibody recognizing MAC-2 (B and D). Only a subset of macrophages in lesions from mice receiving CD68S-HA-EGFP–transduced HSCs show reactivity with an antibody against the HA epitope (C and E). Original magnification, ×10 (B–E).

Article Snippet: Immunohistochemical characterization of the lesions was performed using antibodies recognizing HA-epitope (Zymed Laboratories Inc.), murine MAC-2 (ATCC; monoclonal supernatant diluted 1:4), murine MMP-9 (R&D Systems), and human fibrin/fibrinogen (DakoCytomation) using a 3-step protocol with 3,3′-diaminobenzidine as chromagen, as described previously ( 29 ).

Techniques: Transplantation Assay, Transduction, Injection, Expressing, Immunohistochemistry, Staining

Journal:

Article Title: Macrophage expression of active MMP-9 induces acute plaque disruption in apoE-deficient mice

doi: 10.1172/JCI25074

Figure Lengend Snippet: CD68S–MMP-9 drives overexpression of MMP-9 in isolated and lesional macrophages. (A) Expression of HA-EGFP and MMP-9 by thioglycollate-elicited macrophages from apoE–/– mice transplanted with HSCs transduced with either CD68S-HA-EGFP or CD68S–MMP-9 retroviruses was assessed by immunoblotting of lysates (α-HA) or gelatin zymography of supernatants (ZYMO), respectively, from macrophages cultured ex vivo for 24 hours. A similar blot probed with an antibody against macrosialin (α-mCD68) served as loading control. Each lane represents samples from macrophages from an individual mouse in each group. Pro, proform. (B–E) Analysis of MMP-9 expression in advanced atherosclerotic lesions. Immunohistochemistry of similar lesions from apoE–/– mice transplanted with HSCs transduced with CD68S-HA-EGFP (B and C) or CD68S–MMP-9 (D and E) retroviruses reveals comparable levels of macrophage staining with an antibody recognizing MAC-2 (B and D). Macrophages in lesions from mice receiving CD68S–MMP-9–transduced HSCs show significantly increased levels of MMP-9 expression compared with lesions from mice receiving CD68S-HA-EGFP–transduced HSCs (C and E). Original magnification, ×10 (B–E).

Article Snippet: Immunohistochemical characterization of the lesions was performed using antibodies recognizing HA-epitope (Zymed Laboratories Inc.), murine MAC-2 (ATCC; monoclonal supernatant diluted 1:4), murine MMP-9 (R&D Systems), and human fibrin/fibrinogen (DakoCytomation) using a 3-step protocol with 3,3′-diaminobenzidine as chromagen, as described previously ( 29 ).

Techniques: Over Expression, Isolation, Expressing, Transduction, Western Blot, Zymography, Cell Culture, Ex Vivo, Immunohistochemistry, Staining

Journal:

Article Title: Macrophage expression of active MMP-9 induces acute plaque disruption in apoE-deficient mice

doi: 10.1172/JCI25074

Figure Lengend Snippet: Macrophage overexpression of MMP-9 does not alter lesion matrix composition. Representative shoulder regions from lesions from apoE–/– mice transplanted with HSCs transduced with CD68S-HA-EGFP (A–D) or CD68S–MMP-9 (E–H) retroviruses showed similar macrophage content as assessed by staining with an antibody recognizing MAC-2 (A and E) but significant overexpression of MMP-9 in the group receiving CD68S–MMP-9–transduced HSCs (B and F). Collagen content, as measured by staining with Sirius red, was similar in lesions from both groups of mice (C and G), and Verhoeff–Van Gieson (VVG) staining revealed no differences in intimal elastin content or the integrity of the elastin lamelli in the medial arterial wall (D and H). Original magnification, ×20 (A–H).

Article Snippet: Immunohistochemical characterization of the lesions was performed using antibodies recognizing HA-epitope (Zymed Laboratories Inc.), murine MAC-2 (ATCC; monoclonal supernatant diluted 1:4), murine MMP-9 (R&D Systems), and human fibrin/fibrinogen (DakoCytomation) using a 3-step protocol with 3,3′-diaminobenzidine as chromagen, as described previously ( 29 ).

Techniques: Over Expression, Transduction, Staining

Journal:

Article Title: Macrophage expression of active MMP-9 induces acute plaque disruption in apoE-deficient mice

doi: 10.1172/JCI25074

Figure Lengend Snippet: Enhanced macrophage expression of MMP-9 does not induce plaque rupture in apoE–/– mice

Article Snippet: Immunohistochemical characterization of the lesions was performed using antibodies recognizing HA-epitope (Zymed Laboratories Inc.), murine MAC-2 (ATCC; monoclonal supernatant diluted 1:4), murine MMP-9 (R&D Systems), and human fibrin/fibrinogen (DakoCytomation) using a 3-step protocol with 3,3′-diaminobenzidine as chromagen, as described previously ( 29 ).

Techniques: Expressing

Journal:

Article Title: Macrophage expression of active MMP-9 induces acute plaque disruption in apoE-deficient mice

doi: 10.1172/JCI25074

Figure Lengend Snippet: Activation of MMP-9 is required in macrophages for elastin degrading capacity. (A) RAW-264.7 cells transduced with pBM-IRES-PURO retroviral vectors encoding MMP-9, MMP-12, MMP-13, or an empty vector control (WT) were incubated with 3H-labeled insoluble elastin for 48 hours. Elastin degradation was quantified by scintillation counting of solubilized 3H-elastin in cleared cell supernatants. Bars represent mean ± SD from triplicate samples of the quantity of elastin degraded (μg) calculated from the specific activity and are representative of triplicate experiments. Only the expression of native MMP-12 induces significant degradation of elastin. (B) Supernatants from RAW-264.7 cells transduced with pBM-IRES-PURO retroviral vectors encoding MMP-9, MMP-9 G100L mutant, or an empty vector control were analyzed by gelatin zymography. Supernatants from cells expressing MMP-9 were incubated in the presence (+) or absence (–) of 4-aminophenylmercuric acetate to induce activation via the autolytic removal of the prodomain. (C) RAW-264.7 cells described in B were analyzed for their ability to degrade 3H-labeled insoluble elastin as described in A. Representative data from triplicate experiments are represented as in A and show that expression of the autoactivating G100L MMP-9 mutant significantly enhances elastin degrading capacity.

Article Snippet: Immunohistochemical characterization of the lesions was performed using antibodies recognizing HA-epitope (Zymed Laboratories Inc.), murine MAC-2 (ATCC; monoclonal supernatant diluted 1:4), murine MMP-9 (R&D Systems), and human fibrin/fibrinogen (DakoCytomation) using a 3-step protocol with 3,3′-diaminobenzidine as chromagen, as described previously ( 29 ).

Techniques: Activation Assay, Transduction, Plasmid Preparation, Incubation, Labeling, Activity Assay, Expressing, Mutagenesis, Zymography

Journal:

Article Title: Macrophage expression of active MMP-9 induces acute plaque disruption in apoE-deficient mice

doi: 10.1172/JCI25074

Figure Lengend Snippet: CD68S vectors drive the overexpression of MMP-9 in macrophages and atherosclerotic lesions in apoE–/– mice. MMP-9 expression in thioglycollate-elicited peritoneal macrophages (Thio) and aortic arch lysates from apoE–/– mice transplanted with HSCs transduced with CD68S–MMP-9 or CD68S–MMP-9 G100L retroviruses was analyzed by gelatin zymography as described in the Figure ​Figure2A2A legend. Immunoblotting with an antibody against macrosialin (α-mCD68) served as loading control for the isolated macrophages and a measure of macrophage content of lesions in the aortic arch. The proforms and active forms of MMP-9 and MMP-2 are highlighted.

Article Snippet: Immunohistochemical characterization of the lesions was performed using antibodies recognizing HA-epitope (Zymed Laboratories Inc.), murine MAC-2 (ATCC; monoclonal supernatant diluted 1:4), murine MMP-9 (R&D Systems), and human fibrin/fibrinogen (DakoCytomation) using a 3-step protocol with 3,3′-diaminobenzidine as chromagen, as described previously ( 29 ).

Techniques: Over Expression, Expressing, Transduction, Zymography, Western Blot, Isolation

Journal:

Article Title: Macrophage expression of active MMP-9 induces acute plaque disruption in apoE-deficient mice

doi: 10.1172/JCI25074

Figure Lengend Snippet: Enhanced macrophage expression of active MMP-9 induces multiple features of ruptured plaques in the brachiocephalic arteries of apoE–/– mice

Article Snippet: Immunohistochemical characterization of the lesions was performed using antibodies recognizing HA-epitope (Zymed Laboratories Inc.), murine MAC-2 (ATCC; monoclonal supernatant diluted 1:4), murine MMP-9 (R&D Systems), and human fibrin/fibrinogen (DakoCytomation) using a 3-step protocol with 3,3′-diaminobenzidine as chromagen, as described previously ( 29 ).

Techniques: Expressing

Journal:

Article Title: Macrophage expression of active MMP-9 induces acute plaque disruption in apoE-deficient mice

doi: 10.1172/JCI25074

Figure Lengend Snippet: Representative lesions from apoE–/– mice with macrophage overexpression of MMP-9 G100L demonstrate multiple features of ruptured plaques. Representative brachiocephalic lesions from apoE–/– mice transplanted with HSCs transduced with CD68S–MMP-9 G100L (A–K) or CD68S–MMP-9 (L–O) were stained with the following: Carstairs stain (A–C, F–H, L, and M), Gomori aldehyde fuchsin (GAF) (J, K, and O); a fibrin/fibrinogen antibody (D, I, and N); and control IgG (E). Original magnification, ×10 (A, F, J, L, and O); ×20 (B, D, E, G, K, M, and N); ×40 (C, H, and I). (A–E) The lesion shoulder (A, inset) from a mouse receiving HSCs transduced with CD68S–MMP-9 G100L shows fibrin deposition with Carstairs staining (B and C, black arrowheads), distinct from SMCs (B and C, white arrowheads). Fibrin deposition was confirmed by positive staining with anti-fibrin/fibrinogen antibody (D) but not with nonimmune control IgG (E). The staining with the anti-fibrin/fibrinogen antibody was more extensive than Carstairs-positive fibrin areas, but this is consistent with the antibody recognizing multiple forms of the protein (B–D). (F–J) A lesion from a different mouse transplanted with HSCs transduced with CD68S–MMP-9 G100L demonstrates rupture of the thinned fibrous cap (F, inset; J, arrowhead) with rbcs within the lesion (G, arrowheads). It also shows modest Carstairs staining of mature thrombin deposits (H, arrowheads), which were confirmed by fibrin/fibrinogen staining (I, arrowheads). Areas of fibrous cap disruption are apparent with GAF (J, yellow asterisks), but no evidence of elastin destruction in the medial layers is observed (K). (L–O) Lesion from a mouse transplanted with MMP-9–transduced HSCs shows Carstairs staining of SMCs (M, white arrowheads) but no evidence of fibrin deposits, although positive areas are seen with the antibody recognizing fibrin/fibrinogen (N). GAF staining of this lesion (O) shows elastin (arrowhead) covering the shoulder.

Article Snippet: Immunohistochemical characterization of the lesions was performed using antibodies recognizing HA-epitope (Zymed Laboratories Inc.), murine MAC-2 (ATCC; monoclonal supernatant diluted 1:4), murine MMP-9 (R&D Systems), and human fibrin/fibrinogen (DakoCytomation) using a 3-step protocol with 3,3′-diaminobenzidine as chromagen, as described previously ( 29 ).

Techniques: Over Expression, Transduction, Staining, Disruption

Journal:

Article Title: Macrophage expression of active MMP-9 induces acute plaque disruption in apoE-deficient mice

doi: 10.1172/JCI25074

Figure Lengend Snippet: Comparison of brachiocephalic lesion parameters in apoE–/– mice expressing MMP-9 and active MMP-9 in their macrophages

Article Snippet: Immunohistochemical characterization of the lesions was performed using antibodies recognizing HA-epitope (Zymed Laboratories Inc.), murine MAC-2 (ATCC; monoclonal supernatant diluted 1:4), murine MMP-9 (R&D Systems), and human fibrin/fibrinogen (DakoCytomation) using a 3-step protocol with 3,3′-diaminobenzidine as chromagen, as described previously ( 29 ).

Techniques: Comparison, Expressing

Journal:

Article Title: Macrophage expression of active MMP-9 induces acute plaque disruption in apoE-deficient mice

doi: 10.1172/JCI25074

Figure Lengend Snippet: Characteristics of plaque rupture are frequently observed in the aortae of apoE–/– mice with enhanced macrophage expression of active MMP-9

Article Snippet: Immunohistochemical characterization of the lesions was performed using antibodies recognizing HA-epitope (Zymed Laboratories Inc.), murine MAC-2 (ATCC; monoclonal supernatant diluted 1:4), murine MMP-9 (R&D Systems), and human fibrin/fibrinogen (DakoCytomation) using a 3-step protocol with 3,3′-diaminobenzidine as chromagen, as described previously ( 29 ).

Techniques: Expressing

Journal:

Article Title: Macrophage expression of active MMP-9 induces acute plaque disruption in apoE-deficient mice

doi: 10.1172/JCI25074

Figure Lengend Snippet: Multiple sites with characteristics of plaque rupture in the aortae of mice with macrophages expressing MMP-9 G100L. Longitudinal sections of the aortic arch, its branches (minus the brachiocephalic artery), and thoracic aortae stained with Carstairs were evaluated in apoE–/– mice transplanted with HSCs transduced with CD68S–MMP-9 G100L (A–F) or CD68S–MMP-9 (G–J). These lesions represent the most severely affected mice in each group. Original magnification, ×10 (A, B, and G); ×20 (H–J); ×40 (C–F). (A–F) A mouse receiving HSCs transduced with CD68S–MMP-9 G100L had at least 13 different regions in which lesions showed evidence of previous plaque rupture (A, asterisks). Rupture was most apparent and frequent at the downstream side of the lesion in the lesser curvature as illustrated at higher magnifications (B–F). rbcs are embedded in a fibrin-rich matrix detected by Carstairs stain (C) and confirmed by anti-fibrin/fibrinogen immunohistochemistry (D), while other areas are primarily fibrin rich (E, Carstairs stain; F, fibrin/fibrinogen immunostaining). Features of plaque rupture were observed in all mice in this group (n = 7), with at least 5 regions in the lesions in each aorta and its branches. (G–J) This aorta was from 1 of only 2 of the 8 total mice receiving HSCs transduced with CD68S–MMP-9 that showed any areas with features of plaque rupture in longitudinal sections. The 2 regions boxed in G with characteristics of plaque rupture are shown at higher power (H–J). rbcs and a fibrin deposit are seen in H (black arrowhead), while the lesion in I and J shows fibrin deposition by Carstairs (I) that is confirmed by anti-fibrin/fibrinogen staining (J).

Article Snippet: Immunohistochemical characterization of the lesions was performed using antibodies recognizing HA-epitope (Zymed Laboratories Inc.), murine MAC-2 (ATCC; monoclonal supernatant diluted 1:4), murine MMP-9 (R&D Systems), and human fibrin/fibrinogen (DakoCytomation) using a 3-step protocol with 3,3′-diaminobenzidine as chromagen, as described previously ( 29 ).

Techniques: Expressing, Staining, Transduction, Immunohistochemistry, Immunostaining