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Volume 15, Issue 3, Pages 157-166 (October 2008) Matrix metalloproteinase-9 in spontaneously hypertensive hyperlipidemic rats Yasuhiko Asano, Shinichi Iwai, Masako Okazaki, Toshio Kumai, Yasunari Munemasa, Shigeko Oonuma, Mamoru Tadokoro, Shinichi Kobayashi, Katsuji Oguchi Pathophysiology Volume 15, Issue 3, Pages 157-166 (October 2008) DOI: 10.1016/j.pathophys.2007.02.001 Copyright © 2008 Elsevier Ireland Ltd Terms and Conditions

Fig. 1 Histological changes of the aorta of SHHR and SD rats at 8 months of age. The aorta (aortic arch) were analysed with Sudan III in SD rat (A) and SHHR (B). Lipid depositions (neutral fat) are demonstrated by the orange colour (arrows) following Sudan III staining (B). Original magnification ×200. Pathophysiology 2008 15, 157-166DOI: (10.1016/j.pathophys.2007.02.001) Copyright © 2008 Elsevier Ireland Ltd Terms and Conditions

Fig. 2 Gelatin zymography of plasma of WKY rat, SD rat, SHR, HLR and SHHR. Samples were diluted 100-fold in ddH2O. The gelatinolytic activities are recognized as clear bands against a black background. MMP-2 are recognized at 72kDa. The graph shows values that represent the % increases in activities of MMP-2 in plasma relative to WKY rats determined by gelatin zymography. WKY: n=19; SD: n=8; SHR: n=5; HLR: n=5; SHHR: n=14. Each column represents means±S.E.M. *WKY vs. SHHR; P<0.001, #SD vs. SHHR; P<0.05. Pathophysiology 2008 15, 157-166DOI: (10.1016/j.pathophys.2007.02.001) Copyright © 2008 Elsevier Ireland Ltd Terms and Conditions

Fig. 3 Gelatin zymography of plasma of WKY rat, SD rat, SHR, HLR and SHHR. Samples were diluted 10-fold in ddH2O. The gelatinolytic activities are recognized as clear bands against a black background. MMP-2 and MMP-9 are recognized at 72kDa and 92kDa, respectively. The graph shows values that represent the % increases in activities of MMP-9 in plasma relative to WKY rats determined by gelatin zymography. WKY: n=19; SD: n=8; SHR: n=5; HLR: n=5; SHHR: n=14. Each column represents means±S.E.M. *WKY and SD vs. SHHR, WKY vs. HLR; P<0.001, ##SHR vs. HLR and SHHR, SD vs. HLR; P<0.01, #HLR vs. SHHR; P<0.05. Pathophysiology 2008 15, 157-166DOI: (10.1016/j.pathophys.2007.02.001) Copyright © 2008 Elsevier Ireland Ltd Terms and Conditions

Fig. 4 Western blot analysis of plasma of WKY rat, SD rat, SHR, HLR and SHHR using anti-MMP-9 monoclonal antibodies. MMP-9 immunoreactivity was revealed as bands of molecular mass 92kDa of the MMP-9 in plasma (A). The graph shows values that represent the % increases in activities of MMP-9 in plasma relative to WKY rats determined by Western blot analysis (B). Pathophysiology 2008 15, 157-166DOI: (10.1016/j.pathophys.2007.02.001) Copyright © 2008 Elsevier Ireland Ltd Terms and Conditions

Fig. 5 Gelatin zymography of vitreous of WKY rat, SD rat, SHR, HLR and SHHR. Samples were not diluted. The gelatinolytic activities are recognized as clear bands against a black background. MMP-2 and MMP-9 are recognized at 72kDa and 92kDa, respectively (A). The graph shows values that represent the % increases in activities of MMP-2 (B), MMP-9 (C) in vitreous relative to WKY rats determined by gelatin zymography. WKY: n=9; SD: n=7; SHR: n=6; HLR: n=7; SHHR: n=8. Each column represents means±S.E.M. *WKY vs. SHHR; P<0.001, #SD, SHR and HLR vs. SHHR; P<0.05. Pathophysiology 2008 15, 157-166DOI: (10.1016/j.pathophys.2007.02.001) Copyright © 2008 Elsevier Ireland Ltd Terms and Conditions

Fig. 6 A cross-section of retina showing immunostaining of MMP-9. The MMP-9 was stained brown. Arrows show staining of MMP-9. SD (A), and SHHR (B). Scale bar=50μm. Pathophysiology 2008 15, 157-166DOI: (10.1016/j.pathophys.2007.02.001) Copyright © 2008 Elsevier Ireland Ltd Terms and Conditions

Fig. 7 Values represent the % increases in expression of mRNA of MMP-2 (A), MMP-9 (B) and TIMP-3 (C) in retina relative to WKY rats determined by quantitative real-time PCR. WKY: n=9; SD: n=7; SHR: n=6; HLR: n=7; SHHR: n=8. Each column represents means±S.E.M. #SD vs. HLR; P<0.05. Pathophysiology 2008 15, 157-166DOI: (10.1016/j.pathophys.2007.02.001) Copyright © 2008 Elsevier Ireland Ltd Terms and Conditions