The Iroquois homeobox gene (Irx5) is essential in the embryonic development and cardiac electrophysiology. Although recent studies have reported that IRX5 is involved in the regulation of cell cycle and apoptosis in prostate cancer cells, little is known about the role of IRX5 in the adult vasculature. Here we report novel observations on the role of IRX5 in adult vascular smooth muscle cells (VSMCs) during proliferation in vitro and in vivo. Comparative studies to determine relative expression of Irx5 expression in the peripheral vasculature using primary human endothelial cells (HUVECs), VSMCs, and intact carotid arteries demonstrate significantly higher expression in VSMCs. Sprague-Dawley rat carotid arteries were subjected to balloon catherization and the presence of IRX5 protein was examined by immunohistochemistry after two weeks. Results indicate markedly elevated IRX5 signal at 14 days compared to uninjured controls Total RNA was isolated from injured and uninjured arteries and Irx5 expression was measured by RT-PCR. Results demonstrate a significant increase in Irx5 expression from 3-14days post-injury when compared to controls. Irx5 genetic gain- and loss-of-function studies resulted in modulation of DNA synthesis, using thymidine and BrdU incorporation assays in primary rat aortic VSMCs (RASMCs). Quantitative RT-PCR results revealed modulation of p27kip1, E2f1, and Pcna expression in Irx5 transduced VSMCs when compared to control conditions. Subsequently, apoptosis was observed and confirmed by morphological observation, caspase-3 cleavage, and enzymatic activation when compared to control conditions. Taken together, these results indicate that Irx5 plays an important role in VSMC G1/S cell cycle checkpoint control and apoptosis.
- DNA Synthesis
- Cell Cycle
- Copyright © 2016, American Journal of Physiology - Cell Physiology