1. Progression of aortic valve stenosis: TGF-β1 is present in calcified aortic valve cusps and promotes aortic valve interstitial cell calcification via apoptosis 
Bo Jian, MD, PhD, Navneet Narula, MD, Quan-yi Li, PhD, Emile R Mohler, MD, Robert J Levy, MD 
The Annals of Thoracic Surgery 
Volume 75, Issue 2, Pages (February 2003)
DOI: /S (02)

2. Fig 1
The presence of tumor growth factor-β1 (TGF-β1) in human calcified aortic valves and noncalcified control valves. (A) In calcified valves, intense immunopositive TGF-β1 staining (brown) was observed both in the calcified sites (* denotes calcified region) and in the surrounding noncalcified extracellular matrix and occasional cells. (B) Normal noncalcified valves show no extracellular matrix staining, but occasional cell oriented immunostaining. Nonspecific rabbit IgG shows an absence of peroxidase reactivity (data not shown). Peroxidase immunohistochemistry. (Original magnification, ×200.)
The Annals of Thoracic Surgery  , DOI: ( /S (02) )

3. Fig 2
Tumor growth factor-β1 (TGF-β1) causes sheep aortic valve interstitial cell aggregation and calcification, as demonstrated in (A) nodules that have formed after 72 hrs in response to TGF-β1 compared to (B) a comparable culture without TGF-β1 treatment showing no nodule formation. (C) Increased 45Ca accumulation in TGF-β1 treated cultures compared to control cultures in the absence of TGF-β1 (*p < 0.05, **p < 0.001, student’s t-test). (D) After 14 days in culture in the presence of TGF-β1. All nodules are intensely positive for Coomassie Blue (A and B) and Alizarin Red S (D). Arrows indicate nodules. (Original magnification, ×100.)
The Annals of Thoracic Surgery  , DOI: ( /S (02) )

4. Fig 3
Increased sheep aortic valve interstitial cell alkaline phosphatase (arrows) per histochemistry, and apoptosis, following tumor growth factor-β1 (TGF-β1) treatment, demonstrating nodule formation after 72 hrs associated with apoptosis. (A) Alkaline phosphatase positive staining (deep purple) in the nodules after 72 hrs, compared to (B) cultures not treated with TGF-β1 that reveal isolated cells that are positive for alkaline phosphatase. (C and D) Annexin V positive (FITC) cells localized in DAPI-stained nodules after 3 days (C) and 7 days (D) in culture in the presence of TGF-β1. (E) Hoechst positive cells in control cultures after 72 hrs (apoptotic nuclei indicated by arrows). (A and B: alkaline phosphatase histochemistry, counterstained with eosin, ×100; C and D: Annexin V-FITC, mounted with Vectashield mounting medium with DAPI, ×200; E: Hoechst 33258, ×200.)
The Annals of Thoracic Surgery  , DOI: ( /S (02) )

5. Fig 4
ZVAD, a caspase inhibitor that thereby inhibits apoptosis, prevented sheep aortic valve interstitial cell calcification and cell death induced by tumor growth factor-β1 (TGF-β1). (A) Apoptotic index (measured by counting apoptotic nuclei stained with Hoechst and expressed as percentage of the total cell number counted) showing ZVAD inhibited TGF-β1 induced apoptosis at 72 hrs compared to control (**p < 0.01 TGF-β1 treated versus control; *p < 0.01 TGF-β1 treated versus ZVAD treated, ANOVA). (B) 45Ca assay showed decreased 45Ca accumulation following ZVAD treatment (p < 0.01, ANOVA). (C–E) Live/dead assay revealed nodule formation with TGF-β1 plus caspase inhibitor, but qualitatively fewer dead cells following ZVAD treatment in 7 days culture, in which (C) is control, (D) is TGF-β1 treated, and (E) is TGF-β1 plus ZVAD treated cultures. (C–E) Green fluorescence represents live cells and red fluorescence represents dead cells, with intense overlap indicated by yellow (live/dead assay, ×100).
The Annals of Thoracic Surgery  , DOI: ( /S (02) )

6. Fig 5
Cytochalasin D (cytoD) prevented sheep aortic valve interstitial cell aggregation induced by tumor growth factor-β1 (TGF-β1), but potentiated TGF-β1 induced cell-oriented calcification and cell death. (A) 45Ca accumulation was significantly increased following cytochalasin D treatment alone (p < 0.01), and even more so in the presence of TGF-β1 (p < 0.01, ANOVA). Live/dead assay showed scattered red fluorescent dead cells in cytochalasin D treated culture (B) as compared to an increased proportion of dead cells following cytochalasin D treatment in the presence of TGF-β1 (C) (live/dead assay, ×100).
The Annals of Thoracic Surgery  , DOI: ( /S (02) )