Abstract 18406: Insulin-Like Growth Factor-1 Increases Synthesis of Mature Type 1 Collagen via Induction of LARP6 Expression and Binding to the 5’ Stem-Loop Structure of Collagen mRNA

Abstract

Structural integrity of the fibrous cap of atherosclerotic lesions is a major determinant of plaque stability. We have previously shown that either insulin-like growth factor-1 (IGF-1) -infusion or smooth muscle cell-specific IGF-1-overexpression increases collagen content in atherosclerotic plaque in apoe-/- mice. To identify the mechanism responsible for this increase in collagen, we investigated the effect of IGF-1 on the mRNA-binding protein, la-related protein 6 (LARP6). LARP6 has been shown to bind a conserved stem-loop secondary structure present in the 5’UTR of Col1a1 and Col1a2 mRNAs and to coordinate their efficient translation into the heterotrimer of type 1 collagen. Western blot from primary culture of human aortic smooth muscle cells (hASMCs) or mouse embryonic fibroblasts (mEFs) shows that IGF-1 increased expression of both LARP6 (~2.9-fold, p<0.001) and procollagen-1α (~2.4-fold, p<0.001) within 3 hours. Immunoprecipitation of LARP6, followed by measurement of Col1a1 and Col1a2 mRNA via qPCR indicates that IGF-1 increased association of LARP6 with Col1a1 and Col1a2 mRNA (p>0.01). Mutation of the 5’stem-loop structure of Col1a1 mRNA (SL-/-), which inhibits binding of LARP6 but does not affect the coding region of the Col1a1 gene, abolished the ability of IGF-1 to increase collagen-1α (p<0.001). Moreover, the SL-/- mutation blocked the ability of IGF-1 to increase the rate of pepsin-resistant collagen synthesis, measured via 3 H-proline incorporation assay (p<0.001). Furthermore, western blot of crude aortic lysate from apoe-/- mice suggests that IGF-1-infusion increases LARP6 expression in VSMCs in vivo . To assess collagen maturation in vivo , aortic valve cross-sections were stained with picro-sirius red and examined using a polarizing microscope. Apoe-/- mice with smooth muscle cell-specific IGF-1-overexpression showed a strikingly amplified ratio of thick mature collagen fibers to thin immature collagen fibers in plaque. In summary, we show that IGF-1 increases expression of LARP6 in vascular smooth muscle cells leading to increased binding of LARP6 to Col1a1 and Col1a2 mRNAs and enhanced synthesis of mature type 1 collagen. These findings uncover a critical cellular mechanism whereby IGF-1 promotes plaque stability.