The Fumagillin Analogue TNP-470 Inhibits DNA Synthesis of Vascular Smooth Muscle Cells Stimulated by Platelet-Derived Growth Factor and Insulin-like Growth Factor-I by Hidenori Koyama, Yoshiki Nishizawa, Masayuki Hosoi, Shinya Fukumoto, Kyoko Kogawa, Atsushi Shioi, and Hirotoshi Morii Circulation Research Volume 79(4):757-764 October 1, 1996 Copyright © American Heart Association, Inc. All rights reserved.

DNA synthesis in bovine SMCs DNA synthesis in bovine SMCs. A, Both PDGF-BB and IGF-I stimulate increase in a dose-dependent DNA synthesis in bovine SMCs. Subconfluent cells were incubated in serum-free DMEM supplemented with 0.1% BSA, 2.5 μg/mL holotransferrin, and 35 μg/mL ascorbic acid for 72 hours before addition of growth factors. DNA synthesis in bovine SMCs. A, Both PDGF-BB and IGF-I stimulate increase in a dose-dependent DNA synthesis in bovine SMCs. Subconfluent cells were incubated in serum-free DMEM supplemented with 0.1% BSA, 2.5 μg/mL holotransferrin, and 35 μg/mL ascorbic acid for 72 hours before addition of growth factors. Thereafter, cells were incubated for 20 hours in the presence of indicated concentrations of PDGF-BB or IGF-I followed by labeling with 2 μCi/mL [3H]thymidine for 2 hours. DNA synthesis was measured as TCA-insoluble radioactivity. •, PDGF-stimulated and ○, IGF-I–stimulated DNA synthesis. Values are presented as mean±SD of quadruplicate samples. *P<.05 vs 0 ng/mL, multiple comparison (Scheffé's type). B, The kinetics of DNA synthesis induced by PDGF-BB and IGF-I are comparable in bovine SMCs. Quiescent bovine SMCs were treated with 10 ng/mL PDGF-BB or 100 ng/mL IGF-I for the indicated time. DNA synthesis was determined as described above. •, PDGF-stimulated and ○, IGF-I–stimulated DNA synthesis. Values are presented as mean±SD of quadruplicate samples. C and D, TNP-470 dose dependently inhibits basal as well as PDGF- and IGF-I–stimulated DNA synthesis. Quiescent bovine SMCs were treated for 21 hours with vehicle, 10 ng/mL PDGF-BB, or 100 ng/mL IGF-I in the presence or absence of indicated concentrations of TNP-470. DNA synthesis was determined as described above. Values represented on graphs are mean±SD. *P<.05 vs TNP-470 (−), multiple comparison (Scheffé's type). E, TNP-470 inhibits cell number increase induced by PDGF-BB and IGF-I. Quiescent SMCs were treated with vehicle, 10 ng/mL PDGF-BB, or 100 ng/mL IGF-I alone or in combination with 10 ng/mL TNP-470. Cell numbers were counted after 72 hours. Values are mean±SD of triplicate samples. *P<.05 vs control; **P<.05 vs TNP-470 (−). F, TNP-470 inhibition of FCS-stimulated DNA synthesis is less marked than that observed with PDGF or IGF-I. Quiescent bovine SMCs were treated for 21 hours with 10% FCS alone or in combination with the indicated concentrations of TNP-470. DNA synthesis was determined as described above. Values are mean±SD of quadruplicate samples. *P<.05 vs TNP-470 (−), multiple comparison (Scheffé's type). Each experiment was repeated at least twice, with identical results. Hidenori Koyama et al. Circ Res. 1996;79:757-764 Copyright © American Heart Association, Inc. All rights reserved.

Protein tyrosine phosphorylation in bovine SMCs Protein tyrosine phosphorylation in bovine SMCs. Subconfluent bovine SMCs were incubated in serum-free DMEM containing 0.1% BSA, 2.5 μg/mL holotransferrin, and 35 μg/mL ascorbic acid for 3 days to make the cells quiescent. Protein tyrosine phosphorylation in bovine SMCs. Subconfluent bovine SMCs were incubated in serum-free DMEM containing 0.1% BSA, 2.5 μg/mL holotransferrin, and 35 μg/mL ascorbic acid for 3 days to make the cells quiescent. After the cells were pretreated with or without 10 ng/mL TNP-470 for 1 hour, they were stimulated for 10 minutes with vehicle, 10 ng/mL PDGF-BB, or 100 ng/mL IGF-I in the presence (+) or absence (−) of 10 ng/mL TNP-470 at 37°C. Cell lysates were fractionated in 8.0% SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted with antiphosphotyrosine antibody. Hidenori Koyama et al. Circ Res. 1996;79:757-764 Copyright © American Heart Association, Inc. All rights reserved.

Hidenori Koyama et al. Circ Res. 1996;79:757-764 Copyright © American Heart Association, Inc. All rights reserved.

Hidenori Koyama et al. Circ Res. 1996;79:757-764 Copyright © American Heart Association, Inc. All rights reserved.

Immediate early gene expression in bovine SMCs Immediate early gene expression in bovine SMCs. After quiescent SMCs were pretreated for 1 hour with (+) or without (−) TNP-470, the cells were stimulated for 60 minutes with vehicle, 10 ng/mL PDGF, or 100 ng/mL IGF-I, and the cells were harvested for RNA isolation. Immediate early gene expression in bovine SMCs. After quiescent SMCs were pretreated for 1 hour with (+) or without (−) TNP-470, the cells were stimulated for 60 minutes with vehicle, 10 ng/mL PDGF, or 100 ng/mL IGF-I, and the cells were harvested for RNA isolation. Total RNA (20 μg per lane) was separated on 1.0% agarose gel, blotted onto a nylon membrane, and hybridized with c-fos and c-myc cDNAs. The right panel shows the summary of the results of densitometric analysis for c-fos and c-myc mRNA levels corrected for rRNA, obtained from three individual experiments. Hidenori Koyama et al. Circ Res. 1996;79:757-764 Copyright © American Heart Association, Inc. All rights reserved.

Hidenori Koyama et al. Circ Res. 1996;79:757-764 Copyright © American Heart Association, Inc. All rights reserved.

Hidenori Koyama et al. Circ Res. 1996;79:757-764 Copyright © American Heart Association, Inc. All rights reserved.

Hidenori Koyama et al. Circ Res. 1996;79:757-764 Copyright © American Heart Association, Inc. All rights reserved.

Hidenori Koyama et al. Circ Res. 1996;79:757-764 Copyright © American Heart Association, Inc. All rights reserved.

Hidenori Koyama et al. Circ Res. 1996;79:757-764 Copyright © American Heart Association, Inc. All rights reserved.