Evidence for an altered balance between matrix metalloproteinase-9 and its inhibitors in calcific aortic stenosis Jari Satta, MD, PhD, Jani Oiva, MS,

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Evidence for an altered balance between matrix metalloproteinase-9 and its inhibitors in calcific aortic stenosis Jari Satta, MD, PhD, Jani Oiva, MS, Tuula Salo, MD, PhD, Heidi Eriksen, MD, Pasi Ohtonen, MS, Fausto Biancari, MD, PhD, Tatu S Juvonen, MD, PhD, Ylermi Soini, MD, PhD The Annals of Thoracic Surgery Volume 76, Issue 3, Pages 681-688 (September 2003) DOI: 10.1016/S0003-4975(03)00529-0

Fig 1 During the progression of aortic stenosis (mild, moderate, and severe), in situ hybridization revealed imbalance in MMP9 (black bars), TIMP1 (grey bars), and TIMP2 (striped bars) production. The x-axis shows semiquantitatively scaled transcript intensity (0 = no signals, + = only weak signals, ++ = moderate signals, and +++ = strong signals), and the y-axis shows the proportional percentage of valves in each classified expression level. The Annals of Thoracic Surgery 2003 76, 681-688DOI: (10.1016/S0003-4975(03)00529-0)

Fig 2 Hematoxylin & eosin–stained calcified aortic valve samples shown by in situ hybridization strong signals to MMP-9 transcripts in the fusiform myofibroblast-like cells (antibody to α-smooth muscle actin) (arrows) (A), contrary to randomly seen signals for TIMP-1 (and much less for TIMP-2) (arrow) (D). Noncalcified valves expressed “steady-state” milieu by no signals for MMP-9 or its tissue inhibitors (B, C). BA-4 immunostaining by the avidin-biotin method expressed elastin in noncalcified valves as an intense and thin lamellar (arrow) zone beneath the surface endothelial cells (E), whereas diseased valves expressed scattered and nonuniform BA-4 immunopositivity (arrow) beneath the basement membrane and in the stroma adjacent to a calcific deposit (F). Magnification: A, D = ×160; B, C, E, F = ×110. The Annals of Thoracic Surgery 2003 76, 681-688DOI: (10.1016/S0003-4975(03)00529-0)

Fig 3 During mild to severe progression of aortic stenosis, the production of MMP9 (black bars) correlated positively with valve calcification (grey bars). In regard to BA4 (striped bars) immunopositivity (elastin content), the correlation was negative. On the x-axis are presented semiquantitatively determined MMP9 expression level (0 = no, + = weak, ++ = moderate, +++ = strong signals), extent of calcification (0 no calcification, + if < 25%, ++ if 25% to 50%, +++ if > 50% of the valvular area contained calcific tissues), and intensity of elastin immunoreactivity (+++ = normal elastin content, ++ = moderate elastin content, + = mild elastin content). The y-axis shows the proportional percentage of valves in each determined group. The Annals of Thoracic Surgery 2003 76, 681-688DOI: (10.1016/S0003-4975(03)00529-0)

Fig 4 MMP-9 and MMP-2 in tissue extracts of stenotic (S) and control (C) aortic valve pools analyzed by zymography. The stenotic valves contained both pro- and active MMP-9 forms (92 and 82 kDa, respectively), and pro-MMP-2 (72 kDa). Controls had only a faint pro-MMP-9 and a clear pro-MMP-2 band. Pro-MMP-9, active MMP-9, and pro-MMP-2 are marked by arrows on the right side of the figure (A). By reverse zymography, calcified valves contained only TIMP-1, whereas control valves produced both TIMP-1 and TIMP-2 proteins. TIMP-1 (28.5 kDa) and TIMP-2 (21 kDa) are marked by arrows on the right side of the figure (B). Molecular mass (in kDa) standards are marked on the left. The Annals of Thoracic Surgery 2003 76, 681-688DOI: (10.1016/S0003-4975(03)00529-0)