Matrix metalloproteinases and their tissue inhibitors in pressure-overloaded human myocardium during heart failure progression
Victoria Polyakova, PhD*,*,
Stefan Hein, MD ,
Sawa Kostin, MD*,
Tibor Ziegelhoeffer, MD* and
Jutta Schaper, MD*
* Max-Planck-Institute
Kerckhoff-Clinic, Bad Nauheim, Germany
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Figure 1 Expression and quantitative analysis of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinase (TIMPs). Confocal micrographs showing typical staining patterns of MMPs, an example is MMP-1 fluorescence (green) in control (A) and diseased myocardium (B and C). Note that in the control myocardium, MMP-1 is localized mainly in the interstitial cells and signal occupies restricted areas of the extracellular matrix whereas diseased myocardium showed larger areas of MMP-1. Nuclei are stained blue with TOTO3. (D) Quantitative immunofluorescent data of MMPs and TIMPs are expressed as percent increase in aortic stenosis groups compared to controls. All analyzed MMPs and TIMPs demonstrate a trend to be increased with hypertrophy progression.
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Figure 2 Representative gelatin zymography and quantitative analysis. Gelatinolytic activities were identified as clear bands against the dark background. Zymographic activity of matrix metalloproteinase-2 is significantly enhanced in aortic stenosis patients as compared with control.
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Figure 3 Representative Western blots and quantitative results of MMPs (A to F) and TIMPs (G to I) expression normalized to actin content (42 kDa, shown in panel C) during the transition from compensated hypertrophy to heart failure. Abbreviations as in Figure 1.
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Figure 4 Immunostaining (serial sections) for collagen I (A), MMP-1 (B), and TIMP-1 (C) in myocardium of patients with the lowest ejection fraction demonstrating that the amount of TIMP-1 is obviously less in comparison with collagen I and MMP-1. Collagen I is red, MMP-1 and TIMP-1 are green, nuclei are blue. (D) Quantitative analysis of collagen I per tissue area demonstrates significantly increased levels of fibrosis in patients with decompensated hypertrophy. Abbreviations as in Figure 1.
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Figure 5 Immunostaining (serial sections) for MMP-9 (A), CD31 and vimentin (B), MMP-1 (C), alpha-smooth muscle actin and vimentin (D) showing absence of colocalization of MMPs in both endothelial cells and smooth muscle cells. Matrix metalloproteinases, endothelial cells, and smooth muscle cells are green; vimentin is red; and nuclei are blue. Abbreviations as in Figure 1.
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Figure 6 Cellular sources of MMP and TIMP (A to F). Double labeling for MMP-1 (green) (A) and vimentin (red) (B) showing that fibroblasts are the majority of cells in the MMP occupied area. Serial sections stained for MMP-2 (green) (C) and vimentin (red), CD68 (green) (D). Note: most of the cells in the MMP containing regions are fibroblasts and only a few macrophages are present (arrows). Sequential staining for TIMP (TIMP-1 shown as an example) (green) (E) and vimentin (red) (F) demonstrating fibroblasts in TIMP-positive areas. Nuclei are blue. Abbreviations as in Figure 1.
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Figure 7 Proliferating fibroblasts and presence of myofibroblasts in patients with decompensated hypertrophy (group III). (A) Cell-proliferation-associated antigen-Ki67 (green) is confined to fibroblasts labeled for vimentin (red); (B) quantification of Ki-67-positive fibroblasts. (C and D) Myofibroblasts (arrows) in the myocardial ECM expressing alpha-smooth muscle actin (green) and vimentin (red). (C) Myocytes are red with phalloidin. Nuclei are blue.
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