Pathogenic Role of the Wnt Signaling Pathway Activation In Laser-Induced Choroidal Neovascularization

Abstract

PURPOSE. Choroidal neovascularization (CNV) is a severe complication of AMD. The Wnt signaling pathway has been shown to mediate angiogenesis. The purpose of this study was to investigate the pathogenic role of the Wnt pathway in CNV and explore the therapeutic potential of a novel Wnt signaling inhibitor in CNV.METHODS. Adult rats and mice were photocoagulated using diode laser to induce CNV. On the same day, the animals were intravitreally injected with a monoclonal antibody (Mab2F1) blocking LRP6 or nonspecific mouse IgG. The Wnt signaling activation and target gene expression in the eyecup were determined by Western blot analysis. Fundus angiography was used to examine leakage from the laser lesion. CNV areas were measured on choroidal flatmount using FITC-dextran. The canonical wingless-type MMTV integration site (Wnt) signaling pathway plays a critical role in the regulation of inflammation and angiogenesis. 5,6 Wnt ligands are secreted, cysteine-rich glycosylated proteins, 3 which bind to frizzled (Fz) receptors or to the coreceptor complex of Fz and low-density lipoprotein receptor-related protein 5 or 6 (LRP5/6). RESULTS. Levels of 4-6Binding of Wnt ligands results in LRP6 phosphorylation and activation, 7,8 leading to dissociation of the kinase complex containing glycogen synthase kinase-3b (GSK3b), 9 axin and adenomatous polyposis.3 The GSK3b complex dissociation prevents transcription factor b-catenin from phosphorylation and degradation.10 Consequently, b-catenin is accumulated in the cytoplasm and translocated into the nucleus, complexes with TCF/LEF family transcription factors, 11 regulating expression of Wnt target genes including VEGF, which is the key pathogenic factor in CNV. [12][13][14][15] Although studies of the pathogenesis and treatment of AMD have been delayed by lacking of ideal animal models, 16 laserinduced CNV rodent models are commonly used to study CNV in wet AMD. [17][18][19][20] Moreover, our previous study has shown that the Wnt signaling pathway is activated in very low-density lipoprotein receptor (VLDLR) knockout (KO) mice, a genetic animal model of subretinal neovascularization (NV). 21 The present study investigated the role of the Wnt signaling pathway in laser-induced CNV and explored therapeutic potential of a blocker of Wnt signaling in laser-induced CNV and VLDLR KO models. METHODS AnimalsCare, use, and treatment of experimental animals were in strict agreement with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Male C57BL/6J mice and VLDLR KO mice (10 weeks old; The Jackson Laboratory, Bar Harbor, ME) and male Brown Norway rats (8-10 weeks old; Charles River, Wilmington, MA) were used in this study. In all procedures, animals were anesthetized by intramuscular injection of 50 mg/kg ketamine hydrochloride (Vedco, St. Joseph, MO) and 10 mg/kg xylazine (Vedco), and pupils were dilated with topical administration of 1% cyclopentolate (Wilson, Mustang, OK). CNV InductionLaser photocoagulation (532 nm, 150-250 mW, 0.01 second, 50 lm; model di...

Knockdown of dickkopf2 inhibits vascular endothelia growth factor expression through the Wnt/β-catenin signaling pathway in human retinal pigment epithelial cells under hypoxic conditions

“…Therefore, attenuation of the Wnt/β-catenin pathway may be a potential strategy for treating CNV. Monoclonal antibody (Mab)2F1, a novel inhibitor of the canonical Wnt pathway, has been demonstrated to suppress the hypoxia-induced activation of Wnt signaling in cultured RPE cells, thereby ameliorating CNV (27). Similar to the role of Mab2F1 in RPE cells under hypoxic conditions, in the present study, it was demonstrated that knockdown of DKK2 inhibited the hypoxia-induced expression of β-catenin in RPE cells.…”

“…A key regulator of this pathway is intracellular β-catenin, which is a transcription coactivator (26). It has been reported that the aberrant upregulation of the Wnt/β-catenin signaling pathway at multiple levels, including Wnt ligands, low-density lipoprotein receptor-related protein 6 and β-catenin, has been observed in laser-induced CNV models (27). In addition, nuclear β-catenin regulates gene transcription by interacting with Wnt target and activator genes (T-cell factor-1/lymphoid enhancer binding factor-1) and knockdown of HIF-1α inhibits the hypoxia-increased accumulation of β-catenin in the nucleus (28).…”

Endothelial microRNA-150 is an intrinsic suppressor of pathologic ocular neovascularization

“…All of these genes are potent proangiogenic genes associated with ocular angiogenesis. FZD4 is a receptor for Wnt signaling, which is a highly conserved angiogenic pathway activated in both OIR and laser-induced CNV models (16,33,34). On the other hand, Dll4/Notch signaling controls vessel sprouting and branching, and inhibition of DLL4 showed reduced pathological neovessels in OIR (35).…”

Wnt Modulators in the Aqueous Humor Are Associated With Outer Retinal Damage Severity in Patients With Neovascular Age-Related Macular Degeneration

“…[7][8][9][10][11] Recently, the Wnt pathway has been suggested to be a pathogenic mechanism of AMD, especially nAMD. 8 The therapeutic effects of Wnt inhibitors, such as monoclonal antibodies to Wnts, have been demonstrated in a mouse model of wet AMD. 8 However, others reported that increased Wnt signaling in light-induced retinal degeneration plays a role in retinal protection.…”

“…8 The therapeutic effects of Wnt inhibitors, such as monoclonal antibodies to Wnts, have been demonstrated in a mouse model of wet AMD. 8 However, others reported that increased Wnt signaling in light-induced retinal degeneration plays a role in retinal protection. 7,12 They suggested that upregulated Wnt signaling in Müller glia during retinal degeneration could be a protective mechanism to prevent further photoreceptor injury.…”