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1.
Figure 2

Figure 2. Trans-ECM migration of macrophages, but not neutrophils, is blocked in peritoneal tissue of Plg–/– mice. . From: Inflammatory macrophage migration requires MMP-9 activation by plasminogen in mice.

Peritoneal tissue was dissected and examined after thioglycollate treatment. (A) Macrophage (Mac-3 antibody). Original magnification, ×100; insets, ×400. Arrowheads indicate mesothelial layer; asterisk indicates macrophage accumulation area. (B) Neutrophil (neutrophil antibody). Original magnification, ×200. (C and D) Macrophage and neutrophil distribution (expressed as percentage of tissue area) in peritoneal tissue (n = 5–6). *P < 0.05, **P < 0.01.

Yanqing Gong, et al. J Clin Invest. 2008 September 2;118(9):3012-3024.
2.
Figure 1

Figure 1. Plg activation is required for macrophage recruitment.. From: Inflammatory macrophage migration requires MMP-9 activation by plasminogen in mice.

(A) Macrophage recruitment in Plg+/+ and Plg–/– mice after thioglycollate injection (n = 3–7). (B) Recruitment of macrophages is suppressed in Plg–/– mice and enhanced in PAI1–/– mice (n = 9–22 mice per group). (C) Plg in plasma of Plg+/+ mice (n = 3) is unaffected by thioglycollate treatment. (D) Plasmin (83 kDa) activity in PLF was measured by casein zymography. (E) Intensity of plasmin bands in Plg+/+ PLF (3 independent assays). (F) Aprotinin and tranexamic acid (TA) reduce thioglycollate-induced macrophage recruitment in Plg+/+ mice (n = 8–10). *P < 0.05, **P < 0.01.

Yanqing Gong, et al. J Clin Invest. 2008 September 2;118(9):3012-3024.
3.
Figure 7

Figure 7. Prevention of AAA by Plg deficiency.. From: Inflammatory macrophage migration requires MMP-9 activation by plasminogen in mice.

Three weeks after treatment (CaCl2 or NaCl), abdominal aorta was dissected and examined (n = 6–8). (A) Aortic diameter as measured before and after treatment (left). Representative photograph (right). (B) Aorta stained with EVG for elastic lamella (top row) and H&E for inflammatory cells (bottom row). Original magnification, ×400. (C) Macrophages (Mac-3 antibody) in aorta. EL, elastic lamellae. Original magnification, ×200; inset, ×400. (D) Macrophage distribution expressed as percentage of tissue area in aortic tissue (n = 4–5). *P < 0.05, **P < 0.01.

Yanqing Gong, et al. J Clin Invest. 2008 September 2;118(9):3012-3024.
4.
Figure 3

Figure 3. Collagen content is altered in thioglycollate-injected Plg–/– mice. . From: Inflammatory macrophage migration requires MMP-9 activation by plasminogen in mice.

(AC) Peritoneal tissue from Plg+/+ and Plg–/– mice was stained after thioglycollate treatment. (A) Collagen stained blue by Masson’s trichrome. Original magnification, ×200; insets, ×400. Arrowheads indicate mesothelial layer. (B) Total collagen as percentage of total section area (n = 4–6). **P < 0.01. (C) Collagen IV and laminin. Arrowheads indicate mesothelial layer; asterisk indicates macrophage accumulation area (n = 4–6). Original magnification, ×200; insets, ×400. (D) Collagen IV degradation. PLF of mice was isolated after thioglycollate injection, and soluble collagen IV degradation products were quantified by ELISA (n = 4). *P < 0.05.

Yanqing Gong, et al. J Clin Invest. 2008 September 2;118(9):3012-3024.
5.
Figure 5

Figure 5. MMP-9 activation is required for Plg-induced macrophage migration in vitro.. From: Inflammatory macrophage migration requires MMP-9 activation by plasminogen in mice.

(A and B) Macrophage migration across Matrigel or collagen IV with serum as the chemoattractant was assayed. (A) MMP-9 neutralization blocks Plg-mediated macrophage migration. Plg+/+ macrophage migration was determined in the presence or absence of Plg, with MMP-9 antibody or control IgG. Inset: Gelatin zymography assay for culture medium of peritoneal macrophages treated with PBS, Plg plus IgG, or Plg plus MMP-9 antibody. (B) MMP-9 deficiency blocks Plg-mediated macrophage migration. Migration of Mmp9+/+ or Mmp9–/– macrophages was determined with or without Plg treatment. (C) MMP-9 knockout blocks Plg-mediated macrophage migration across Matrigel with MCP-1 as the chemoattractant. (D) Reconstitution of actMMP-9 restores macrophage migration in response to PLF from Plg–/– mice. The migration of Plg+/+ macrophages was determined in response to PLF (chemoattractant) collected from Plg+/+ and Plg–/– mice with or without actMMP-9. In AD, “Control” indicates wells with cells and medium only. Migrated cells were counted by microscopy in 4 high-power fields for each insert (3 separate experiments performed in triplicate). **P < 0.01.

Yanqing Gong, et al. J Clin Invest. 2008 September 2;118(9):3012-3024.
6.
Figure 8

Figure 8. Plg-mediated AAA formation requires MMP-9.. From: Inflammatory macrophage migration requires MMP-9 activation by plasminogen in mice.

(AC) MMP activity in aorta 1 week after CaCl2 or NaCl (Sham) treatment. (DG) CaCl2-treated mice were injected with proMMP-9, actMMP-9, or PBS (n = 5–7), and 1 week after treatment, the abdominal aortas were examined. (A) Extracted aorta tissue (5 μg protein) was analyzed by gelatin zymography. (B) Intensity of actMMP-9 bands in zymogram assays (3 independent assays). (C) Intensity ratio (actMMP-9/proMMP-9) of zymogram assays. (D) Aortic diameter before and after treatment (left). Representative aortas are shown (right). (E) Aorta sections stained for elastic lamellae (EVG) and inflammatory cells (H&E). Original magnification, ×400. (F) Macrophages (Mac-3 antibody). Original magnification, ×200; insets, ×400. (G) Macrophage distribution expressed as percentage of total sample area in aorta (n = 4–5). *P < 0.05, **P < 0.01.

Yanqing Gong, et al. J Clin Invest. 2008 September 2;118(9):3012-3024.
7.
Figure 6

Figure 6. Macrophages require MMP-9 in vivo to migrate across peritoneal tissue, and actMMP-9 rescues impaired macrophage migration in Plg–/– mice. . From: Inflammatory macrophage migration requires MMP-9 activation by plasminogen in mice.

(A) Macrophage migration in Mmp9+/+ or Mmp9–/– mice and in Plg+/+ mice treated with control IgG or anti–MMP-9 antibody was measured 72 hours after thioglycollate injection (n = 5–7). Inset, gelatin zymograph of PLF from Plg+/+ mice treated with IgG or MMP-9 antibody shows that the MMP-9 neutralization abolishes MMP-9 activation. (BJ) Before thioglycollate injection, mice were treated with PBS, proMMP-9, and actMMP-9, and 48 hours after thioglycollate injection, peritoneal lavage and tissue were collected. Results are from 3 independent assays (n = 5–7). (B) actMMP-9 restores the suppressed macrophage recruitment to the peritoneal cavity in Plg–/– mice. (CH) Collagen IV (collagen IV antibody) and macrophage (Mac-3 antibody) immunostaining in Plg–/– mice. Arrowheads indicate mesothelial layer; asterisk indicates macrophage accumulation area. Original magnification (C and D), ×100; insets, ×200. Original magnification (EH), ×200; insets, ×400. (I) Macrophage distribution expressed as percentage of total sample area in peritoneal tissue. (J) Soluble collagen IV degradation products in PLF of Plg–/– mice. *P < 0.05, **P < 0.01.

Yanqing Gong, et al. J Clin Invest. 2008 September 2;118(9):3012-3024.
8.
Figure 4

Figure 4. MMP-9 is activated by Plg in vivo and in vitro.. From: Inflammatory macrophage migration requires MMP-9 activation by plasminogen in mice.

(AD) MMP–9 activity. PLF was collected after thioglycollate treatment at different time points and subjected to MMP-9 activity analysis. proMMP-9 (105 kDa) and actMMP-9 (95 kDa, 88 kDa), as well as proMMP-2 (72 kDa and 69 kDa), were identified by molecular weight relative to markers and purified protein. (A) Gelatin zymography. (B and C) Quantitation of the intensity of actMMP-9 bands and the ratio of actMMP-9 to proMMP-9 bands from zymogram results (n = 3). (D) MMP-9 immunoblot. PLF was purified by gelatin-agarose and detected with MMP-9 antibody. (E and F) Peritoneal tissue extracted and analyzed by gelatin zymography. (E) Gelatin zymography. (F) Intensity of actMMP-9 bands in peritoneal tissue (n = 3). (G) Colocalization of macrophages and MMP-9 in peritoneal tissue. Tissue sections, 72 hours after thioglycollate treatment, double immunofluorescently stained for macrophages (Mac-3 antibody, green) and MMP-9 (MMP-9 antibody, red). Original magnification, ×100. Representative images taken from 3 experiments. (H) Peritoneal macrophages derived from Plg+/+ mice were isolated and cultured, which was followed by treatment with or without Plg (10 μg/ml) for 24 or 48 hours. The culture medium was collected and subjected to gelatin zymography. (I) Intensity of actMMP-9 bands in the culture medium of macrophages (3 independent assays).*P < 0.05, **P < 0.01.

Yanqing Gong, et al. J Clin Invest. 2008 September 2;118(9):3012-3024.

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