Abstract 892: The Forkhead Transcription Factor, FOXO3a, is a Negative Regulator of Angiogenic Immediate Early Gene, CYR61, Leading to Inhibition of Vascular Smooth Muscle Cells Proliferation and Neointimal Hyperplasia
Objective: Cysteine-rich angiogenic protein 61 (CYR61, CCN1) is an immediate early gene expressed in vascular smooth muscle cells (VSMCs) on growth factor stimulation and its expression has been associated with postangioplasty restenosis and atherosclerosis. The forkhead transcription factors are known to inhibit growth in a variety of cell types. We hypothesized that the forkhead transcription factor, FOXO3a may play a role in controlling CYR61 regulation in VSMCs.
Methods and Results: To evaluate FOXO3a effect on CYR61 expression, rat VSMCs were infected with adenoviral vectors expressing constitutively active FOXO3a (Ad-TM-FOXO3a). FOXO3a gene transduction suppressed CYR61 mRNA expression and decreased protein level. Transfection experiments with deletion constructs of the CYR61 promoter revealed the potential binding sites for forkhead transcription factor at the approximately 700-bp upstream of the TATA-box in the promoter region. Co-transfection of Ad-TM-FOXO3a with the reporter plasmid (pGL3-CYR61) resulted in a significant decrease in luciferase expression, confirming CYR61 regulation by FOXO3a. Ex vivo exposure of rat aortic rings to angiotensin II (10−9mol/L, 2 hours), a known inducer of CYR61, rapidly increased CYR61 in VSMCs, which was significantly reduced by Ad-TM-FOXO3a, whereas antisense FOXO3a transduction restored CYR61 expression. In rat balloon carotid arterial injury model, CYR61 expressed in VSMCs in early stage of injury and remained elevated until 14 days, which was suppressed by Ad-TM-FOXO3a. After 14 days, there was a significantly reduction in neointimal area by FOXO3a transduction (0.20±0.07 versus 0.06±0.02mm2, P<0.01) compared with control group. Such reduction of neointimal hyperplasia by Ad-TM-FOXO3a was reversed by co-transfection of adenoviral vector expressing CYR61.
Conclusion: These data suggest that FOXO3a is a negative transcription factor of CYR61 gene and that suppression of CYR61 is one of the several mechanisms by which FOXO3a inhibits VSMC proliferation and neointimal hyperplasia.