ABSTRACT
Age-related macular degeneration (AMD) is one of the major causes of blindness in the elderly worldwide. Anti-vascular endothelial growth factor (VEGF) drugs have been widely used to treat the neovascular type of AMD (nAMD). However, VEGF acts not only as a pro-angiogenic factor but also as an anti-apoptotic factor in the eyes. In this study, we found that anti-VEGF drugs, including bevacizumab (Bev), ranibizumab (Ran), and aflibercept (Afl), induced epithelial-mesenchymal transition (EMT) in ARPE-19 cells in vitro, accompanied by the induction of CCN2, a potent pro-fibrotic factor. Similarly, intravitreal injection of Afl into mouse eyes resulted in EMT in the retinal pigmented epithelium (RPE). Co-treatment with CCN5, an anti-fibrotic factor that down-regulates CCN2 expression, significantly attenuated the adverse effects of the anti-VEGF drugs both in vitro and in vivo. Inhibition of the VEGF signaling pathway with antagonists of VEGF receptors, SU5416 and ZM323881, induced EMT and up-regulated CCN2 in ARPE-19 cells. Additionally, knock-down of CCN2 with siRNA abolished the adverse effects of the anti-VEGF drugs in ARPE-19 cells. Collectively, these results suggest that anti-VEGF drugs induce EMT in RPE through the induction of CCN2 and that co-treatment with CCN5 attenuates the adverse effects of anti-VEGF drugs in mouse eyes.
Subject(s)
Epithelial-Mesenchymal Transition , Retinal Pigment Epithelium , Vascular Endothelial Growth Factor A , Epithelial-Mesenchymal Transition/drug effects , Retinal Pigment Epithelium/metabolism , Retinal Pigment Epithelium/drug effects , Retinal Pigment Epithelium/pathology , Animals , Humans , Mice , Vascular Endothelial Growth Factor A/metabolism , Macular Degeneration/metabolism , Macular Degeneration/pathology , Macular Degeneration/drug therapy , Macular Degeneration/chemically induced , Cell Line , Bevacizumab/pharmacology , CCN Intercellular Signaling Proteins/metabolism , CCN Intercellular Signaling Proteins/genetics , Angiogenesis Inhibitors/pharmacology , Ranibizumab/pharmacology , Recombinant Fusion Proteins/pharmacology , Signal Transduction/drug effects , Repressor Proteins , Receptors, Vascular Endothelial Growth FactorABSTRACT
[This corrects the article DOI: 10.1371/journal.pone.0269937.].
ABSTRACT
Choroidal neovascularization (CNV) is a defining characteristic feature of neovascular age-related macular degeneration (nAMD) that frequently results in irreversible vision loss. The current strategies for the treatment of nAMD are mainly based on neutralizing vascular endothelial growth factor (VEGF). However, anti-VEGF therapies are often associated with subretinal fibrosis that eventually leads to damages in macula. In this study, we tested whether an anti-fibrotic and anti-angiogenic protein CCN5 can potentially be an effective and safe therapeutic modality in a mouse model of CNV. Laser photocoagulation was utilized to induce CNV, which was followed by intravitreal injection of recombinant adeno-associated virus serotype 2 encoding CCN5 (rAAV2-CCN5). Our data demonstrated that rAAV2-CCN5, but not a control viral vector, rAAV2-VLP, prominently attenuated both CNV lesions and angiogenesis. Aflibercept, which was utilized as a positive control, exhibited similar effects on CNV lesions and angiogenesis in our experimental settings. Upon laser photocoagulation, retinal pigmented epithelium (RPE) cells underwent significant morphological changes including cellular enlargement and loss of hexagonality. rAAV2-CCN5 significantly normalized these morphological defects. Laser photocoagulation also led to fibrotic deformation in RPE cells through inducing epithelial-mesenchymal transition (EMT), which was completely blocked by rAAV2-CCN5. In a striking contrast, aflibercept as well as rAAV2-VLP failed to exhibit any effects on EMT. Collectively, this study suggest that CCN5 might provide a potential novel strategy for the treatment of nAMD with a capability to inhibit CNV and fibrosis simaultaneously.
Subject(s)
Choroidal Neovascularization , Parvovirinae , Animals , Choroidal Neovascularization/metabolism , Dependovirus/genetics , Disease Models, Animal , Epithelial-Mesenchymal Transition , Epithelium/metabolism , Fibrosis , Mice , Mice, Inbred C57BL , Parvovirinae/genetics , Vascular Endothelial Growth Factor AABSTRACT
Retinal pigment epithelium (RPE) plays an essential role in maintaining retinal function, and its defect is thought to be critically implicated in various ocular disorders. This study demonstrated that the matricellular protein CCN5 was down-regulated in ARPE-19 cells treated with the pro-fibrotic agent transforming growth factor (TGF)-ß. A recombinant adenovirus expressing CCN5 (AdCCN5) was used to restore the level of CCN5 in these cells. AdCCN5 prevented TGF-ß-induced fibrotic changes, including disruption of tight junctions, up-regulation of mesenchymal marker proteins, and down-regulation of epithelial marker proteins. In addition, AdCCN5 prevented TGF-ß-induced functional defects, including increased migratory activity and reduced phagocytic activity. Notably, AdCCN5 reversed morphological and functional defects pre-established by TGF-ß prior to viral infection. The CCN5 level was down-regulated in RPE of 18-month-old Ccl2-/- mice, which exhibited retinal defects. Restoration of the CCN5 level via intravitreal injection of a recombinant adeno-associated virus expressing CCN5 (AAV9-CCN5) normalized the altered expression of mesenchymal, epithelial, and functional marker proteins, as assessed by western blotting and immunohistochemistry. Taken together, these data suggest that down-regulation of CCN5 is associated with fibrotic deformation of RPE under pathological conditions and that restoration of the CCN5 level effectively promotes recovery of deformed RPE.
Subject(s)
Down-Regulation , Intracellular Signaling Peptides and Proteins , Retinal Diseases , Retinal Pigment Epithelium/metabolism , Animals , Cell Line , Dependovirus , Fibrosis , Intracellular Signaling Peptides and Proteins/biosynthesis , Intracellular Signaling Peptides and Proteins/genetics , Mice , Mice, Knockout , Retinal Diseases/genetics , Retinal Diseases/metabolism , Retinal Diseases/pathology , Retinal Pigment Epithelium/pathology , Transduction, Genetic , Transforming Growth Factor beta/biosynthesis , Transforming Growth Factor beta/geneticsABSTRACT
Age-related macular degeneration (AMD) is a leading cause of blindness in the elderly. The two types of AMD are: dry and wet AMD. While laser-induced choroidal neovascularization has been used extensively in the studies of wet AMD, there is no established mouse model that fully recapitulates the cardinal features of dry AMD. A lack of appropriate mouse model for dry AMD has hampered the translational research on the pathogenesis of the disease and the development of therapeutic agents. We hypothesized that 5XFAD mice, an animal model for the study of Alzheimer's disease, can be used as a mouse model for dry AMD with regard to the amyloid beta (Aß) related pathology. In this study, the ultrastructure of the retinal pigment epithelium (RPE) of 5XFAD mice was analyzed using transmission electron microscopy. Of importance, the aged 5XFAD mice show ultrastructural changes in the RPE and Bruch's membrane (BM) that are compatible with the cardinal features of human dry AMD, including a loss of apical microvilli and basal infolding of the RPE, increased BM thickness, basal laminar and linear deposits, and accumulation of lipofuscin granules and undigested photoreceptor outer segment-laden phagosomes. In microarray-based analysis, the RPE complex of the aged 5XFAD mice shows differential gene expression profiles consistent with dry AMD in the inflammation response, immune reaction pathway, and decreased retinol metabolism. Taken together, we suggest that aged 5XFAD mice can be used as a mouse model of dry AMD to study Aß related pathology and develop a new therapeutic approaches.
