Vascular smooth muscle cells (SMC) synthesize insulin-like growth factor-I (ILF-I), which is a mitogen for this cell type in vitro. Since IGF binding proteins (IGFBP) modulate IGF bioactivity, we determined which IGFBPs were secreted by porcine SMC. Porcine SMC secreted 34,000 and 24,000 M, forms of IGFBPs which were identified as IGFBP-2 and ICFBP-4, respectively, by immunoblotting. Northern blot analysis showed single transcripts of l . G kb and 2.4 kb for ICFBP-2 and IGFBP-4, respectively. Secretion of IGFBP-2 was not regulated to a significant degree, with insulin, IGF-II, IGF-I, forskolin, and dibutyryl cyclic adenosine monophosphate (CAMP) inducing minimal changes in IGFBP-2 secretion of less than 30% by radioimmunoassay (RIA). Insulin increased (2.8 i 0.1-fold) the abundance of IGFBP-4 protein in conditioned media (CM) and increased IGFBP-4 mRNA levels. Growth factors for SMC such as platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), and transforming growth factor beta-1 (TGFP-1) were without effect on either IGFBP-2 or -4. IGF-I treatment decreased the amount of IGFBP-4 present in CM, but a corresponding decrease in IGFBP-4 mRNA levels was not observed. In order to determine if IGFBP-4 could modulate IGF-I bioactivity, IGFBP-4 was added to pSMCs with and without IGF-I. IGF-I alone (20 ng/ml) induced a 1.6 to threefold increase in -'H-thymidine incorporation. Addition of IGFBP-4 (between 50 and 250 ng/ml) to cultures containing IGF-I (20 ng/ml) had no effect on DNA synthesis compared to that observed with IGF-I alone, while 500 ngiml consistently caused a small decrease (1 5 k 5%; mean 2 SE). lmmunoblotting of the C M obtained at the end of the 'H-thymidine assay showed a loss of intact IGFBP-4 in the cultures containing IGF-I. This corresponded with an increase in the abundance of a 16,000 M, immunoreactive fragment that did not bind IGF-I. Coincubation with insulin had no effect on the amount of IGFBP-4 that was converted to fragment, suggesting that the reaction was dependent upon IGF-I binding to IGFBP-4. In contrast, addition of ICFBP-4 (500 ng/ml) to human fibroblast cultures with ICF-l (20 n g h l ) almost completely inhibited the stimulatory effect of IGF-I on DNA synthesis and no increase in fragment was detected in the CM. In summary, SMC secrete IGFBP-2 and IGFBP-4, both of which have been shown to regulate IGF-mediated DNA synthesis. The factors that regulate the abundance of the two forms of IGFBPs differ suggesting that differential regulation of these substances may be an important mechanism by which SMC growth is regulated. o 1993 WiIcy-Liss, ill( T h e smooth muscle cell (SMC) i s t h e m a j o r constitue n t cell t y p e o f t h e atherosclerotic plaque (Geer e t al., 1961), therefore i d e n t i f i c a t i o n o f factors that control SMC replication may provide i m p o r t a n t insights i n t o t h e atherosclerotic process. Several mitogens for vascular SMC h a v e been identified, i n c l u d i n g t h e i n s u l i nl i k e g r o w t h factors (IGFs) (Clemmons, 1985; P f e i f l e e t al., 1987). SMCs in c u l t u r e h a v e been shown t o express IGF-I mRNA and t o synthesize I G F -I -l i k e peptides (Bornfeldt e t al., 1990; Clemmons, 1985; Delafontaine e t al. , 1991). M o r e i m p o r t a n t l y , IGF-I mRNA expression i s increased ninefold in t h e n e o i n t i m a l cells during 0 1993 WILEY-LISS, INC t h e development o f atherosclerotic lesions following balloon denudation injury. T h i s increase precedes an associated increase in SMC proliferation (Cercek e t al., 1990). T h e exact cell t y p e that expresses IGF-I mRNA in t h i s injury model h a s n o t been d e f i n i t i v e l y determined, however, it h a s been determined in other injury