Inhibition of Vascular Smooth Muscle Cell Growth Through Antisense Transcription of a Rat Insulin-Like Growth Factor I Receptor cDNA

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Abstract
Insulin-like growth factor I (IGF I) is an autocrine/paracrine growth factor that is produced in multiple tissues and is essential for normal developmental growth. Its effects are mediated by activation of a membrane-bound tyrosine kinase receptor, IGF IR. On the basis of the partial rat IGF IR alpha-chain cDNA sequence previously reported, we cloned cDNA encoding the full-length rat IGF IR. The deduced amino acid sequence predicts a 1370-amino acid receptor precursor, which includes signal sequence, a 707-amino acid alpha-chain, a 4-Arg cleavage site, and a 629-amino acid beta-chain. Overall, similarity to human IGF IR is 89% and 98% at the nucleotide and amino acid levels, respectively. Antisense IGF IR expression constructs in vectors incorporating Epstein-Barr virus replicative signals and the cytomegalovirus promoter/enhancer or the inducible human metallothionein IIa promoter/enhancer were assembled and stably transfected into cultured rat aortic smooth muscle cells. Clone CA9 (constitutively expressing abundant antisense IGF IR transcripts), clones MA5 and MA7 (expressing antisense IGF IR transcripts inducibly), and clones ME8 and ME10 (expressing vector alone) were characterized. There was a 57% reduction in IGF IR mRNA levels in clone CA9 after confluence compared with clone ME10. This resulted in a 51% decrease in IGF I binding sites in clone CA9, without a change in binding affinity (Kd), and a 55% and 57% reduction in DNA synthesis rates, basally and in response to 10 ng/mL IGF I, respectively. Clones MA5/MA7 similarly showed a 54% reduction in IGF IR number after confluence following exposure to 100 mumol/L ZnSO4 and a 44% and 58% reduction in DNA synthesis, basally and in response to 10 ng/mL IGF I, respectively. Growth curves indicated that proliferation of clone CA9 in the presence of 10% serum was reduced by 60% compared with clone ME10. Thus, cloning of cDNA encoding the full-length rat IGF IR indicates that this receptor is highly conserved. Antisense targeting of this receptor in vascular smooth muscle cells (VSMCs) demonstrates that a decrease in IGF IR density results in marked inhibition of VSMC proliferation. These findings indicate an important role for this ligand-receptor system in regulating VSMC growth. Specifically, they suggest that modulation of VSMC IGF IR density may be an important mechanism whereby growth of these cells is controlled.