Abstract
Purpose: :
Delta-like ligand 4 (Dll4) plays a crucial role in angiogenesis. However, the specific effect of Dll4 on choroidal neovascularization (CNV) is unclear. The aim of this study was to investigate the influence of Dll4 on CNV angiogenesis via HIF-1α-VEGF signaling.
Methods: :
RF/6A cells, an immortalized choroid-retinal endothelial cell line, were used to establish a chemically-induced hypoxia model. Real-time RT-PCR and Western blotting were used to analyze changes in the expression of Dll4, VEGF and HIF-1α in hypoxic RF/6A cells. RNA interference, immunofluorescence staining, a laser-induced CNV model and intravitreal injection techniques were used to confirm the relationships among these molecules. RF/6A cells were transfected with full-length human Dll4 or a small interfering RNA (SiRNA) targeting Dll4 to modify the different expression status of Dll4. In addition, the Notch inhibitor, gamma secretase inhibitor (GSI), was used to block total Notch-mediated signaling. Finally, RF/6A cells, expressing different levels of Dll4/Notch, were co-cultured with retinal pigment epithelial (RPE) cells under hypoxic conditions to investigate the functional changes in RF/6A cells in response to RPE cell stimuli.
Results: :
The Dll4 mRNA and protein expressions increased significantly in a time-dependent manner responded to CoCl2 induced hypoxia in RF/6A cells, whose upregulation was mediated by HIF-1α-VEGF signaling. Results from the co-culture system showed that the enhancement of Dll4 expression in RF/6A cells led to significantly faster proliferation and stronger tube forming ability, but inhibited cell invasion, while siRNA-mediated Dll4 silencing had the opposite effect. Pharmacological disruption of Notch signaling using GSI produced a similar, but not identical effect, to that caused by the Dll4 siRNA. Dll4 inhibition abolished the invasion of RF/6A cells across a monolayer of RPE cells in a hypoxic environment. In addition, the expression of several key molecules involved in the angiogenesis of CNV, including VEGFR1, VEGFR2, EphrinB2 and EphB4, was altered in RF/6A cells showing constitutively active Dll4 expression.
Conclusions: :
Dll4 is an important vascular regulator in CNV angiogenesis, which appears to be regulated by HIF-1α and VEGF during the progression of CNV under hypoxic conditions. Targeting Dll4/Notch signaling could be an innovative treatment strategy for anti-angiogenesis in ocular diseases.
Keywords: choroid: neovascularization • signal transduction