Platelet-derived growth factor-BB induces apoptosis in cultured vascular smooth muscle cells derived from human saphenous vein.

Apoptosis is increasingly recognized as an important factor in vascular remodelling and disease [I]. Platelet-derived growth factor-BB (PDGF-BB) is a potent mitogen in many cell types, including human vascular smooth muscle cells [2], and has been implicated in the vascular response to injury and atherogenesis [3]. Induction of apoptosis is known to be influenced by the presence of survival factors such as basic fibroblast growth factor (bFGF) [4] and insulin-like growth factor (IGF-I) [5]. PDGF-BB has also been reported to act as a survival factor in human cultured aortic and coronary arterial smooth muscle cells [5]. The intracellular signals responsible for cell survival are incompletely understood, however the small GTP-ase Rho binds to PDGF-receptor upon stimulation [6] and is required for GI progression [7]. In addition inactivation of rho causes apoptosis in smooth muscle cells [8]. Lovastatin, an inhibitor of HMG CoA reductase prevents isoprenylation of small GTP-ases and disrupts their function [9]. In this study, we investigated the effect of lovastatin on apoptosis of human vascular smooth muscle cells stimulated by PDGF-BB. Primary human saphenous vein smooth muscle cells were isolated from explants of normal saphenous vein surplus to material used for coronary artery bypass grafts as previously described [lo]. The cells were routinely cultured in DMEM supplemented with sodium pyruvate (1.2mM). penicillin (120IU/ml), streptomycin sulphate (12hg/ml), gentamicin (3Opg/ml) and 15% (v/v) foetal calf serum (FCS). Cells were seeded at -14x10’ cells/cm2 in 9cm Petri dishes and growth-arrested for 7 days in NCTC-109 serum-free medium (pH 7.4), supplemented with L-glutamine (4mM), 15mM HEPES, and bovine serum albumin (2.5mdrnl). Cultures were incubated with PDGF-BB (20ng/ml) alone, lovastatin (10pM) alone, vehicle alone, or co-incubated with both PDGF-BB and lovastatin in serum-free medium for up to 48 hours. Following exposure, cells were harvested and processed using Cell TestTM Plus DNA reagent kit (Becton Dickinson, CA USA). Cell cycle phases and the hypodiploid, apoptotic (&) region were then quantified [ I l l . The presence of apoptotic cells was also confirmed with DAPI staining, and immunocytochemically using a terminal deoxynucleotide transferase-mediated dUTP-FITC end labelling (TUNEL) reagent (Boehringer Mannheim, Germany). Stimulation with PDGF-BB alone increased the proportion of cells in S-phase from 4.2i1% to S.OiI% ( n 4 ) at 24 hours (Fig. 1.). Levels of apoptosis also increased from 1 l i l % to 24*:1% (n=4) at 48h (Fig. 2). Lovastatin alone had no significant effect on either S-phase or apoptosis compared with vehicle (Fig. 1. & 2.). In the presence of lovastatin PDGF-BB had no significant effect on Sphase entry or apoptosis. (Fig. I & 2.). S-phase (Yo)