Transforming Growth Factor-β1 Mechanisms in Aortic Valve Calcification: Increased Alkaline Phosphatase and Related Events Jocelyn N. Clark-Greuel, PhD,

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Transforming Growth Factor-β1 Mechanisms in Aortic Valve Calcification: Increased Alkaline Phosphatase and Related Events Jocelyn N. Clark-Greuel, PhD, Jeanne M. Connolly, MS, Elizabeth Sorichillo, BS, Navneet R. Narula, MD, H. Scott Rapoport, PhD, Emile R. Mohler, MD, Joseph H. Gorman, MD, Robert C. Gorman, MD, Robert J. Levy, MD The Annals of Thoracic Surgery Volume 83, Issue 3, Pages 946-953 (March 2007) DOI: 10.1016/j.athoracsur.2006.10.026 Copyright © 2007 The Society of Thoracic Surgeons Terms and Conditions

Fig 1 Progression of sheep aortic valve interstitial cell (SAVIC) calcification without and with transforming growth factor-beta 1 (TGF-β1) treatment. Panels A, B, and C show quantitation of alizarin red staining of SAVIC at days 3, 7, and 14, respectively, of treatment without (black) or with (white) 10 ng/mL TGF-β1. Data are expressed as relative intensity in arbitrary units (AU) (see Methods) and are derived from four representative fields analyzed from each of three cell culture experiments. Representative alizarin red stained culture results (calcifications stained red, magnification 100×) are shown above each quantitative panel (calcification increases with TGF-β1 treatment, p = 0.049; *p < 0.001 vs without TGF-β1 at the same time point). The Annals of Thoracic Surgery 2007 83, 946-953DOI: (10.1016/j.athoracsur.2006.10.026) Copyright © 2007 The Society of Thoracic Surgeons Terms and Conditions

Fig 2 Sheep aortic valve interstitial cells (SAVIC) alkaline phosphatase (ALP) activity without and with transforming growth factor-beta 1 (TGF-β1) treatment over time. Panels A, B, and C demonstrate the quantitation of ALP staining of SAVIC at days 3, 7, and 14, respectively, of treatment without (black) or with (white) 10 ng/mL TGF-β1. The ALP-stained culture results (nitroblue tetrazolium positive, blue-black, magnification 100×) are shown as inserts above each quantitative panel, which are derived from five representative fields from each of three cell culture experiments. Data are shown as arbitrary units (AU). The ALP activity increases with TGF-β1 treatment, p < 0.001; *p < 0.001 vs without TGF-β1 at the same time point. The Annals of Thoracic Surgery 2007 83, 946-953DOI: (10.1016/j.athoracsur.2006.10.026) Copyright © 2007 The Society of Thoracic Surgeons Terms and Conditions

Fig 3 Apoptosis of sheep aortic valve interstitial cells (SAVIC) without and with transforming growth factor-beta 1 (TGF-β1) treatment. Panels A, B, and C show annexin V staining quantitated for SAVIC at days 3, 7, and 14, respectively, of treatment without (black) or with (white) 10 ng/mL TGF-β1. Results expressed as arbitrary units (AU). Representative annexin V (green, fluorescein isothiocyanate) fluorescent micrographs from SAVIC culture results with 4,6-diamidino-2-phenylindole (blue) stained nuclei (magnification 100×) are shown as insets above each quantitative panel (apoptosis increases with TGF-β1 treatment, p = 0.009, which is derived from four representative fields from each of three cell culture experiments. The Annals of Thoracic Surgery 2007 83, 946-953DOI: (10.1016/j.athoracsur.2006.10.026) Copyright © 2007 The Society of Thoracic Surgeons Terms and Conditions

Fig 4 Matrix metalloproteinases (MMP9 and MMP2) activity in sheep aortic valve interstitial cells (SAVIC) cultures without and with transforming growth factor-beta 1 (TGF-β1) treatment. Gel zymogram bands demonstrate MMP activity in SAVIC cultures at 3, 7, and 14 days, respectively, of treatment without (black) or with (white) 10 ng/mL TGF-β1. Representative zymograms of conditioned medium from culture are shown as insets above each quantitative panel. Data are shown as optical density (OD) of scans from three individual cell culture experiments. Pro-MMP9 secretion is virtually absent without TGF-β1 treatment, and increased over 13-fold with TGF-β1 treatment (p = 0.008; *p = 0.029 and *p = 0.021 vs without TGF-β1 at 7 and 14 days, respectively). Active MMP2 secretion likewise increased over time with TGF-β1 treatment (p = 0.01), but only threefold. The Annals of Thoracic Surgery 2007 83, 946-953DOI: (10.1016/j.athoracsur.2006.10.026) Copyright © 2007 The Society of Thoracic Surgeons Terms and Conditions