Arterioscler Thromb Vasc Biol EGF-Like Domain of Tenascin-C Is Proapoptotic for Cultured Smooth Muscle Cells by Kurt Wallner, Chen Li, Prediman K. Shah, Kai-Jin Wu, Stephen M. Schwartz, and Behrooz G. Sharifi Arterioscler Thromb Vasc Biol Volume 24(8):1416-1421 August 1, 2004 Copyright © American Heart Association, Inc. All rights reserved.

Figure 1. Representative effect of Tn-C on the gelatinolytic proteins produced by SMCs. A, Human SMCs (passage 3) were cultured in DMEM and added to the 24-well plates precoated with the long isoform of Tn-C (Tn) in the defined media (insulin+transferrin+selenium-X; GIBCO-BRL) and diluted with serum-free DMEM to make defined media. Figure 1. Representative effect of Tn-C on the gelatinolytic proteins produced by SMCs. A, Human SMCs (passage 3) were cultured in DMEM and added to the 24-well plates precoated with the long isoform of Tn-C (Tn) in the defined media (insulin+transferrin+selenium-X; GIBCO-BRL) and diluted with serum-free DMEM to make defined media. Some wells were coated with collagen (Cgn) according to manufacturer’s (Cohesion Technologies) instruction for preparation of fibrillar collagen films. Media were collected 8 hours and 24 hours after plating the cells, and protein concentrations were measured. Equal amounts of total proteins were loaded on each lane of a zymogram. The results displayed here are representative of 3 experiments. Kurt Wallner et al. Arterioscler Thromb Vasc Biol. 2004;24:1416-1421 Copyright © American Heart Association, Inc. All rights reserved.

Figure 2. Degradation of Tn-C by cultured human SMCs Figure 2. Degradation of Tn-C by cultured human SMCs. Human SMCs were added to Tn-C–coated dishes in the presence of serum (S) or in defined media for 10 hours10 or 24 hours.24 Purified large Tn-C isoform was used as a control (C). Figure 2. Degradation of Tn-C by cultured human SMCs. Human SMCs were added to Tn-C–coated dishes in the presence of serum (S) or in defined media for 10 hours10 or 24 hours.24 Purified large Tn-C isoform was used as a control (C). The coated Tn-C was solubilized with 5% SDS, electrophoresed on 5% SDS-PAGE, and transferred to a polyvinylidene diflouride membrane. Three blots were generated that were incubated with 1:10 000 dilution of antihuman fibronectin-like domain antibody (A) or 1:1000 dilution of monoclonal anti-EGF-like antibody (B). C, The anti-EGF-like monoclonal antibody was preincubated with a 0.1 μmol/L solution of recombinant fibronectin-like domain for 30 minutes at 37°C before addition to the blot. D, SMCs were cultured onto Tn-C–coated dishes in the presence of 25 μmol/L GM6001, a metalloproteinase inhibitor, followed by Tn-C extraction and analysis by Western blot using anti-EGF-l monoclonal antibody as described for (B). Kurt Wallner et al. Arterioscler Thromb Vasc Biol. 2004;24:1416-1421 Copyright © American Heart Association, Inc. All rights reserved.

Figure 3. Fragmentation of Tn-C in human carotid endarterectomy specimens. Figure 3. Fragmentation of Tn-C in human carotid endarterectomy specimens. Human carotid endarterectomy specimens were obtained after surgery and immediately snap-frozen with liquid nitrogen. The frozen samples were pulverized and then extracted with a buffer (0.1 mol/L CAPS, 0.15 mol/L NaCl, pH 11) known to be effective in extracting Tn-C.32 After centrifugation, protein concentrations of the supernatants were determined, and 1 μg from each sample was analyzed by SDS-PAGE and Western blot as described using anti-human EGF-like domain monoclonal antibody (sigma). Kurt Wallner et al. Arterioscler Thromb Vasc Biol. 2004;24:1416-1421 Copyright © American Heart Association, Inc. All rights reserved.

Figure 4. Effect of plasma protease inhibitors on EGF-like domain-induced SMC apoptosis. Figure 4. Effect of plasma protease inhibitors on EGF-like domain-induced SMC apoptosis. SMCs were plated in 96-well microtiter plates in DMEM +10% serum, and then the media were replaced with the DMEM defined in the absence (C) or presence of 0.01 μmol/L EGF-like domain supplemented with increasing concentrations of α-macroglobulin or chymostatin. After 48 hours, cell viability was determined by MTT assay. Each data point represents the average of 3 separate experiments. Kurt Wallner et al. Arterioscler Thromb Vasc Biol. 2004;24:1416-1421 Copyright © American Heart Association, Inc. All rights reserved.

Figure 5. Protease inhibitors block caspase-3 activation. Figure 5. Protease inhibitors block caspase-3 activation. SMCs were treated with the recombinant EGF-like domain in the presence of the indicated concentrations of chymotrypsin inhibitor (Chystatin; μM), hirudin, a thrombin inhibitor (Thn I; U/mL), and plasmin inhibitor (Pln I; μg/mL). In another set of experiments, cells were treated with 1 μmol/L starosporine (Starpn) or 250 ng/mL actinomycin D (Actin D) in the presence and absence of 4 μmol/L chymostatin. Cell extracts were prepared and caspase-3 activation was measured as described. Kurt Wallner et al. Arterioscler Thromb Vasc Biol. 2004;24:1416-1421 Copyright © American Heart Association, Inc. All rights reserved.