Background & Aim: Cysteine-rich angiogenic protein 61 (CYR61) 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 regulating CYR61 expression in VSMCs.
Methods and Results: 1) 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, which corresponded to decreased protein synthesis.
2) 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 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.
3) Ex vivo exposure of rat aortic rings to angiotensin II (10^-9mol/L, 2 hours) rapidly induced CYR61 in VSMCs, which was significantly reduced by Ad-TM-FOXO3a and recovered by antisense FOXO3a transduction.
4) 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 substantial reduction in neointimal area (0.20짹0.07 versus 0.06짹0.02mm짼, n=12, P<0.01) compared with the control group. This reduction in neointimal hyperplasia by FOXO3a was reversed by co-transfection of adenoviral vectors expressing CYR61 (Ad-CYR61).
Conclusion: These data suggest that FOXO3a inhibits VSMC proliferation via downregulation of CYR61 and that this signaling axis may represent a therapeutic target in controlling atherosclerosis or vascular proliferative diseases.