sphingosine-1-phosphate and Retinal-Neovascularization

Sphingosine-1-phosphate (S1P), the circulating HDL-bound lipid mediator that acts via S1P receptors (S1PR), is required for normal vascular development. The role of this signaling axis in vascular retinopathies is unclear. Here, we show in a mouse model of oxygen-induced retinopathy (OIR) that endothelial overexpression of S1pr1 suppresses while endothelial knockout of S1pr1 worsens neovascular tuft formation. Furthermore, neovascular tufts are increased in Apom

Topics: Animals; Lipoproteins, HDL; Lysophospholipids; Mice; Receptors, Lysosphingolipid; Retinal Neovascularization; Sphingosine; Sphingosine-1-Phosphate Receptors

Neovascularization is a major cause of blindness in various ocular diseases. Bioactive sphingosine 1-phosphate (S1P), synthesized by two sphingosine kinases (Sphk1, Sphk2), emerged as a key player in a multitude of cellular processes, including cell survival, proliferation, inflammation, migration, and angiogenesis. We investigated the role of Sphk2, S1P, and S1P receptors (S1PR) during retinal neovascularization using the oxygen-induced retinopathy mouse model (OIR).. Sphk2 overexpressing (tgSphk2) and Sphk2 knockout (Sphk2-/-) mice were used in the OIR model, exposed to 75% O2 over 5 days from postnatal day (P)7 to 12 to initiate vessel regression. After returning to room air, these mice developed a marked neovascularization. Retinae recovered from untreated and treated eyes at P7, P12, P14, and P17 were used for lectin-stained retinal whole mounts, mass spectrometry, and quantitative real-time PCR.. tgSphk2 mice showed higher retinal S1P concentrations, accelerated retinal angiogenesis, and increased neovascularization. Expression of S1PR, vascular endothelial growth factor α (VEGFα), and angiopoietin 1 and 2 was differentially regulated during the course of OIR in the different genotypes. Sphk2-/- displayed a markedly reduced retinal angiogenesis and neovascularization as well as decreased VEGFα and angiopoietin expression.. Using genetic models of Sphk2 overexpression or deletion we demonstrate a strong impact of Sphk2/S1P on retinal vasculopathy and expression of vascular growth factors like VEGF and angiopoietin in the retina. Consequently, Sphk2, S1P, and S1PR may offer attractive novel therapeutic targets for ischemic retinopathies.

Topics: Angiopoietin-1; Angiopoietin-2; Animals; Chromatography, Liquid; Disease Models, Animal; Lysophospholipids; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Oxygen; Phosphotransferases (Alcohol Group Acceptor); Real-Time Polymerase Chain Reaction; Receptors, Lysosphingolipid; Retina; Retinal Neovascularization; Retinopathy of Prematurity; Sphingosine; Tandem Mass Spectrometry; Vascular Endothelial Growth Factor A

Angiogenesis contributes to physiological and pathological conditions, including atherosclerosis. The Rap1 small G protein regulates vascular integrity and angiogenesis. However, little is known about the effectors of Rap1 involved in angiogenesis. It is not known whether afadin, an adherens junction protein that connects immunoglobulin-like adhesion molecule nectins to the actin cytoskeleton and binds activated Rap1, plays a role in angiogenesis.. We investigated the role of endothelial afadin in angiogenesis and attempted to clarify the underlying molecular mechanism.. Treatment of human umbilical vein endothelial cells (HUVECs) with vascular endothelial growth factor (VEGF) and sphingosine 1-phosphate (S1P) induced the activation of Rap1. Activated Rap1 regulated intracellular localization of afadin. Knockdown of Rap1 or afadin by small interfering RNA inhibited the VEGF- and S1P-induced capillary-like network formation, migration, and proliferation, and increased the serum deprivation-induced apoptosis of HUVECs. Knockdown of Rap1 or afadin decreased the accumulation of adherens and tight junction proteins to the cell-cell contact sites. Rap1 regulated the interaction between afadin and phosphatidylinositol 3-kinase (PI3K), recruitment of the afadin-PI3K complex to the leading edge, and the activation of Akt, indicating the involvement of Rap1 and afadin in the PI3K-Akt signaling pathway. Binding of afadin to Rap1 regulated the activity of Rap1 in a positive-feedback manner. In vivo, conditional deletion of afadin in mouse vascular endothelium using a Cre-loxP system impaired the VEGF- and S1P-induced angiogenesis.. These results demonstrate a novel molecular mechanism by which Rap1 and afadin regulate the VEGF- and S1P-induced angiogenesis.

Topics: Animals; Apoptosis; Cell Movement; Cell Proliferation; Cells, Cultured; Disease Models, Animal; Endothelial Cells; Hindlimb; Humans; Intercellular Junctions; Ischemia; Lysophospholipids; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Microfilament Proteins; Muscle, Skeletal; Neovascularization, Physiologic; Phosphatidylinositol 3-Kinases; Protein Transport; Proto-Oncogene Proteins c-akt; rac1 GTP-Binding Protein; rap1 GTP-Binding Proteins; Rats; Recombinant Fusion Proteins; Retinal Neovascularization; RNA Interference; Signal Transduction; Sphingosine; Time Factors; Vascular Endothelial Growth Factor A

Sphingosine-1-phosphate (S1P) is a bioactive lipid molecule that stimulates endothelial cell migration, proliferation, and survival in vitro, and tumor angiogenesis in vivo. In this study, we used a humanized monoclonal antibody (sonepcizumab) that selectively binds S1P to investigate its role in retinal and choroidal neovascularization (NV). Intraocular injection of sonepcizumab significantly reduced macrophage influx into ischemic retina and strongly suppressed retinal NV in mice with oxygen-induced ischemic retinopathy. In mice with laser-induced rupture sites in Bruch's membrane, intraocular injection of sonepcizumab significantly reduced the area of choroidal NV and concomitantly reduced fluorescein leakage from the remaining choroidal NV. Four weeks after intraocular injection of up to 1.8 mg of the sonepcizumab in non-human primates, electroretinograms and fluorescein angiograms were normal, and light microscopy of ocular sections showed no evidence of structural damage. These data show for the first time that S1P stimulates both choroidal and retinal NV and suggest that sonepcizumab could be considered for evaluation in patients with choroidal or retinal NV.

Topics: Animals; Antibodies, Monoclonal; Choroidal Neovascularization; Female; Humans; Ischemia; Lysophospholipids; Macaca fascicularis; Macrophages; Male; Mice; Mice, Inbred C57BL; Retinal Neovascularization; Retinal Vessels; Sphingosine

Vascular endothelial growth factor (VEGF) and insulin-like growth factor-1 (IGF-1) have been implicated as important stimulatory factors for retinal neovascularization. In this study, we used intraocular gene transfer with gutless adenoviral (AGV) vectors to determine the effect of increased intraocular expression of VEGF, IGF-1, or sphingosine kinase (SPK), which produces sphingosine-1-phosphate, another angiogenic factor. Retinal neovascularization did not occur from intravitreous AGV-vectored VEGF, IGF-1, SPK, or combined VEGF and IGF-1, except occasionally adjacent to the retinal penetration site from the injection. However, corneal and iris neovascularization occurred after 2 weeks in all eyes injected with AGV.VEGF, but not those injected with only AGV.IGF-1 or AGV.SPK. These data suggest that the superficial capillary bed of the retina is relatively insensitive to VEGF, IGF-1, or SPK in adult mice, except when combined with retinal trauma. However, AGV-vectored VEGF is sufficient to consistently cause severe corneal and iris neovascularization. This provides a model for anterior segment neovascularization, which unlike previous models is relatively inexpensive and is not plagued by spontaneous regression, and therefore, may be useful for identification of new treatments.

Topics: Adenoviridae; Animals; DNA Primers; Eye; Genetic Vectors; Humans; Image Processing, Computer-Assisted; Immunohistochemistry; Insulin-Like Growth Factor I; Lac Operon; Lysophospholipids; Mice; Neovascularization, Physiologic; Phosphotransferases (Alcohol Group Acceptor); Retinal Neovascularization; Reverse Transcriptase Polymerase Chain Reaction; Sphingosine; Transduction, Genetic; Vascular Endothelial Growth Factor A