ABSTRACT
The standard-of-care therapeutics for the treatment of ocular neovascular diseases like wet age-related macular degeneration (AMD) are biologics targeting vascular endothelial growth factor signaling. There are currently no FDA approved small molecules for treating these blinding eye diseases. Therefore, therapeutic agents with novel mechanisms are critical to complement or combine with existing approaches. Here, we identified soluble epoxide hydrolase (sEH), a key enzyme for epoxy fatty acid metabolism, as a target of an antiangiogenic homoisoflavonoid, SH-11037. SH-11037 inhibits sEH in vitro and in vivo and docks to the substrate binding cleft in the sEH hydrolase domain. sEH levels and activity are up-regulated in the eyes of a choroidal neovascularization (CNV) mouse model. sEH is overexpressed in human wet AMD eyes, suggesting that sEH is relevant to neovascularization. Known sEH inhibitors delivered intraocularly suppressed CNV. Thus, by dissecting a bioactive compound's mechanism, we identified a new chemotype for sEH inhibition and characterized sEH as a target for blocking the CNV that underlies wet AMD.
Subject(s)
Chromones/pharmacology , Epoxide Hydrolases/antagonists & inhibitors , Epoxide Hydrolases/metabolism , Molecular Targeted Therapy/methods , Phenylalanine/analogs & derivatives , Wet Macular Degeneration/metabolism , Angiogenesis Inhibitors/pharmacology , Animals , Binding Sites , Choroidal Neovascularization/drug therapy , Choroidal Neovascularization/metabolism , Chromones/chemistry , Disease Models, Animal , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Epoxide Hydrolases/chemistry , Fatty Acids, Unsaturated/metabolism , Humans , Mice, Inbred C57BL , Molecular Docking Simulation , Phenylalanine/chemistry , Phenylalanine/pharmacology , Protein Domains , Proteomics/methods , Wet Macular Degeneration/pathologyABSTRACT
Ocular neovascularization underlies major blinding eye diseases such as "wet" age-related macular degeneration (AMD). Despite the successes of treatments targeting the vascular endothelial growth factor (VEGF) pathway, resistant and refractory patient populations necessitate discovery of new therapeutic targets. Using a forward chemical genetic approach, we identified the heme synthesis enzyme ferrochelatase (FECH) as necessary for angiogenesis in vitro and in vivo FECH is overexpressed in wet AMD eyes and murine choroidal neovascularization; siRNA knockdown of Fech or partial loss of enzymatic function in the Fechm1Pas mouse model reduces choroidal neovascularization. FECH depletion modulates endothelial nitric oxide synthase function and VEGF receptor 2 levels. FECH is inhibited by the oral antifungal drug griseofulvin, and this compound ameliorates choroidal neovascularization in mice when delivered intravitreally or orally. Thus, FECH inhibition could be used therapeutically to block ocular neovascularization.
Subject(s)
Ferrochelatase/metabolism , Macular Degeneration/pathology , Neovascularization, Pathologic/physiopathology , Retinal Neovascularization/physiopathology , Animals , Humans , MiceABSTRACT
Retinal angiogenesis in diabetes may lead to visual impairment and even irreversible blindness in people of working age group worldwide. The main pathological feature of proliferative diabetic retinopathy (PDR) is hypoxia, and overproduction of growth factors like vascular endothelial growth factor (VEGF) and erythropoietin (Epo). This results in pathological proliferation of retinal endothelial cells (RECs), leading to new vessel formation (angiogenesis). Inhibition of angiogenesis is a promising strategy for treatment of PDR and other retinal neovascular disorders. Pigment epithelium-derived factor (PEDF), a 50-kDa protein secreted by retinal pigment epithelium, inhibits the growth of new blood vessel induced in the eye in a variety of ways with a yet elusive mechanism. Here, we investigated the possible mechanism by which PEDF inhibits VEGF- and Epo-induced angiogenic effects in RECs is mediated through PI3K/Akt pathway. PEDF treatment induced the apoptosis in RECs by activating caspase-3 and DNA fragmentation. We found a dose-dependent increase in cell survival with VEGF or Epo, which was attenuated in the presence of PEDF. In addition, PEDF significantly (P < 0.05) inhibited migration and in vitro tube formation in RECs in the presence of VEGF as like PI3K/Akt inhibitor. Of interest, PEDF effectively abrogated VEGF-mediated phosphorylation of PI3K/Akt. Further studies using RECs transfected with constitutively active and dominant-negative forms of Akt suggest that PEDF could inhibit VEGF- and also Epo-induced angiogenesis by disruption of PI3K/Akt signaling.