Dll4/notch Signaling Regulate The Formation Of Choroidal Neovascularization

BackgroundChoroidal neovascularization (CNV) , including angiogenesis and vasculogenesis, is known to occur as a final common pathway encountered in a variety of chorioretinal diseases causing severe and irreversible vision loss. In the recent decades, the studies on pathophysiologic mechanisms and therapeutic strategy of CNV have been made a lot of achievements. However, the development of CNV is a very complicated dynamic process. The specific mechanism underlying is still poorly known.Delta-like ligand 4 (Dll4) is one of the Notch ligands in mammalian cells, which is specifically expressed in physiological and pathological vasculature. Remarkably, deletion of a single Dll4 allele resulted in early embryonic lethality due to the failure to form a functional vasculature. So far, there have been only two genes, Dll4 and vascular endothelial growth factor (VEGF), whose haploinsufficiency results in embryonic lethality, indicating their crucial role in the vascular development.Previous study had found that hypoxia inducible factor 1 (HIF-1) plays an important role on retinal pigment epithelium RPE cell to initiate the CNV responding to hypoxia stimuli in the microenvironment of retina, Bruch's memberane and choriocapillaris. Under hypoxia, RPE cells can influence choroidal microvascular endothelial cells (CECs) functions via HIF-1-VEGF pathway. In addition, some reports found that Dll4 was a hypoxia-regulated gene, which not only received signals from HIF-1 in endothelial cells (ECs), but also HIF-1-VEGF signaling by the hypoxia pathway.Combining these results, we hypothesized that in the pathologic retinachoroidal diseases, Dll4/Notch signaling may participate in HIF-1α-VEGF pathway to regulate angiogenesis of CNV. Our previeous study found that disruption of the Notch signaling resulted in a more severe neovascularization at the site of laser-induced CNV. However, the spacific mechanism of Dll4/Notch signaling effecting on CNV has not yet been reported.PurposeThe aim of this study was to observe expressions of Dll4 in ocular cells, and to investigate the influence of Dll4 on CNV angiogenesis via HIF-1α-VEGF signaling. Co-cultrue systems were used to imitate the natural relationship between RPE cells and CECs to implore the underlying mechanism by which RPE cells regulate the function of CECs under hypoxic conditions.Methods1. In the present study, RF/6A cells, an immortalized choroid-retinal endothelial cell line, were employed to represent CECs because of the convenience for experimental manipulation and the highly conserved evolutionary derivation from human. We established the chemical hypoxia model on RF/6A cells and RPE cells. RPE-RF/6A contact/separated co-culture systems were used to investigate the influence of Dll4/Notch signaling in HIF-1α-VEGF pathway to regulate the key step involved in CNV.2. Real time RT-PCR and western blot were used to analyze the expression changes of Dll4, VEGF, and HIF-1αin hypoxic RF/6A cells. RNA interference, immunofluorescence staining were used to confirm the relationship among these molecules. RF/6A cells were transfected with full-length human Dll4 or Small interfering RNA 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 signaling. Then RF/6A cells with different Dll4/Notch expression levels were co-cultured with RPE cells under hypoxic condition to investigate the functional changes of RF/6A cells responded to RPE cells stimuli.3. Real time RT-PCR and western blot were used to analyze the expression changes of Dll4 in hypoxic RPE cells. In vitro transfection techniques were used to modify the different expression status of Dll4 in the RPE cells to observe the effects of Dll4 on RPE cells proliferation, migration and invasion functions.Results1. 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 of co-culture system showed that enhancement of Dll4 in RF/6A led to the significant faster proliferation and stronger tube formation ability, but the inhibition of invasion. Moreover, the silence of Dll4 by siRNA caused the opposite effect. Pharmacological disruption of Notch signaling using GSI produced similar but not the same effect caused by Dll4 siRNA. Dll4 inhibition abrogated the invasion of RF/6A cells across RPE monolayer cells under hypoxia environment. In addition, we found that the expression of several key molecules involved in angiogenesis of CNV, including VEGFR1, VEGFR2, EphrinB2 and EphB4, altered in RF/6A cells with constitutively active Dll4 expression.2. The Dll4 mRNA and protein expressions increased significantly in a time-dependent manner responded to CoCl2 induced hypoxia in RPE cells. Enhancement of Dll4 in RPE cells led to the significant faster proliferation, but the inhibition of migration and invasion. Moreover, the silence of Dll4 by siRNA caused the opposite effect.ConclusionsDll4 is an important vascular regulator involved in CNV angiogenesis,which could be regulated by HIF-1αand VEGF during the CNV progression under hypoxia condition. Targeting Dll4/Notch signaling might be an innovative treatment strategy for anti-angiogenesis diseases in ocular.