Abstract 14971: Loss of ZBTB46 Decreases BCL2A Expression and Increases Endothelial Cell Apoptosis

Abstract

Introduction: Endothelial cell (EC) activation, injury, and apoptosis in areas of the vasculature experiencing low and oscillatory shear stress (or disturbed flow) play a key role in the development and pathogenesis of atherosclerosis. In in vivo and in vitro studies, we have demonstrated that ZBTB46, a member of the BTB-ZF protein family of transcription factors, is down-regulated in ECs exposed to disturbed flow when compared to cells experiencing unidirectional laminar shear stress. Additionally, we have observed that germline deletion of ZBTB46 leads to decreased BCL2A expression in mouse carotid arteries. Given that ZBTB46 down-regulation occurs in areas of the vasculature prone to increased EC apoptosis and loss of ZBTB46 in vivo is associated with down regulation of anti-apoptotic protein BCL2A, we explored the role of ZBTB46 in EC apoptosis. Hypothesis: Loss of ZBTB46 negatively regulates transcription of the anti-apoptotic mediator BCL2A and sensitizes EC to apoptosis. Methods: Human aortic endothelial cells (HAEC) were transfected with either ZBTB46-siRNA or control siRNA, grown to 2 days post-confluence to induce ZBTB46 expression and then harvested for RNA. Gene expression was assessed by quantitative real-time PCR. In assays to measure apoptosis, siZBTB46 transfected cells, and corresponding controls were treated with various concentrations (0-400 micro molar) of hydrogen peroxide (H 2 O 2 ) for 18 hours, stained for Annexin V and imaged by confocal microscopy Results: Down regulation of ZBTB46 using siRNA (siZBTB46) in HAEC led to a 78% ± 9% (SEM) reduction in BCL2A1 mRNA expression (n=4, p=0.004) when compared to controls (siCtrl). BCL2A1 downregulation also coincided with increased apoptosis in ZBTB46 deficient HAEC (siZBTB46 39% ± 2.9% v siCtrl 27% ± 3.5% n=6, p=0.026) as measured by Annexin V staining. Additionally, H 2 O 2 -induced apoptosis was exacerbated by loss of ZBTB46 in HAEC at 100 μM (siZBTB46 68% ± 2.1% v siCtrl 30% ± 3.1% n=5-6, p=0.001), 200 μM (siZBTB46 82% ± 1.2% v siCtrl 44% ± 3.2% n=4-5, p=0.001), and 400 μM H 2 O 2 (siZBTB46 88% ± 1.1% v siCtrl 58% ± 5.6% n=5-6, p=0.001). Conclusions: Loss of ZBTB46 in endothelial cells reduces BCL2A expression and increases the susceptibility of endothelial cells to apoptosis.