Abstract 46: Differential Gene and Transcription Factor Binding Site Expression Between the Intima and Media Drives Intimal Hyperplasia

Abstract

Background: Vein grafts (VG) fail due to rapid intimal expansion in the weeks to months following implantation. Histologically, this results in a metabolically active intima juxtaposed to a quiescent medial layer. In this study, we leverage this dichotomy to identify the key genes that control intimal growth and the critical transcription factors (TF) that drive their activation.

Methods: Microsurgical techniques were used to separate the intima and media of rabbit carotid VGs harvested at 1 (n=4), 3 (n=6), and 6 (n=4) months (m) following implantation. Each layer was independently analyzed for differential mRNA expression using a whole-genome microarray. rVista and TRANSFAC were used to identify TF binding sites within 5000 bp of differentially expressed genes.

Results: Early growth of the intima was driven by an enhanced proliferative response and an accumulation of cell mass (Fig). During this early hyperplastic phase, substantial differences in intimal and medial gene expression were observed (197 genes at 1m and 346 at 3m, p<0.001) that resolved by 6m (8 genes). Genes involved in cell cycle regulation and cellular movement dominated the 1 and 3m differences (Fig). TF binding site analysis of up-regulated intimal genes at 1 month revealed enrichment of ETS and HOX, while CREB and bHLH-ZIP were the most over-represented among up-regulated 3m intimal genes. In the medial layer, RXR and zinc finger TF binding sites were most prominent among up-regulated genes at 1 and 3m, respectively.

Conclusion: Corresponding to the most active phase of intimal growth, smooth muscle cells demonstrated marked up-regulation of an array of genes that modulate cell proliferation and migration, with these genes under the control of a discrete set of TFs. While previous attempts at modifying TF binding (via an E2F decoy) in the initial days following VG implantation have been unsuccessful, manipulation of a broader set of TFs in the time frame of weeks to months may prove a more promising strategy.