Subretinal fibrosis secondary to neovascular age-related macular degeneration:mechanisms and potential therapeutic targets

Subretinal fibrosis is the end-stage sequelae of neovascular age-related macular degeneration.It causes local damage to photoreceptors,retinal pigment epithelium,and choroidal vessels,which leads to permanent central vision loss of patients with neovascular age-related macular degeneration.The pathogenesis of subretinal fibrosis is complex,and the underlying mechanisms are largely unknown.Therefore,there are no effective treatment options.A thorough understanding of the pathogenesis of subretinal fibrosis and its related mechanisms is important to elucidate its complications and explore potential treatments.The current article reviews several aspects of subretinal fibrosis,including the current understanding on the relationship between neovascular age-related macular degeneration and subretinal fibrosis;multimodal imaging techniques for subretinal fibrosis;animal models for studying subretinal fibrosis;cellular and non-cellular constituents of subretinal fibrosis;pathophysiological mechanisms involved in subretinal fibrosis,such as aging,infiltration of macrophages,different sources of mesenchymal transition to myofibroblast,and activation of complement system and immune cells;and several key molecules and signaling pathways participating in the pathogenesis of subretinal fibrosis,such as vascular endothelial growth factor,connective tissue growth factor,fibroblast growth factor 2,platelet-derived growth factor and platelet-derived growth factor receptor-β,transforming growth factor-βsignaling pathway,Wnt signaling pathway,and the axis of heat shock protein 70-Toll-like receptors 2/4-interleukin-10.This review will improve the understanding of the pathogenesis of subretinal fibrosis,allow the discovery of molecular targets,and explore potential treatments for the management of subretinal fibrosis.

Transforming growth factor -β neutralizing antibodies inhibit subretinal fibrosis in a mouse model

AIM:To determine the involvement of the transforming growth factor(TGF)-β with the development of experimental subretinal fibrosis in a mouse model.· METHODS:Subretinal fibrosis was induced by subretinal injection of macrophage-rich peritoneal exudate cells(PECs) and the local expression of TGF-β isoforms was assessed by quantitative real-time reverse transcription-polymerase chain reaction(RT-PCR) and enzyme-linked immunosorbent assay(ELISA) at various time points.In addition,we investigated the effect of TFG-β-neutralizing antibodies(TGF-β NAb) on subretinal fibrosis development.· RESULTS:TGF-β1 and TGF-β2 mRNA level was significantly elevated at day 2 after subretinal fibrosis induction and increased further to 5 and 6.5-fold respectively at day 5,reaching the peak.TGF-β3 mRNA was not detected in the present study.The result of ELSIA showed that active TGF-β1 and TGF-β2 levels were upregulated to 10-fold approximately,while total TGF-β1 and TGF-β2 levels were even upregulated more than 10-fold and more than 20-fold respectively in subretinal fibrosis mice in comparison with na?觙ve mice at day 5.TGF-β NAb resulted in a reduced subretinal fibrosis areas by 65% compared to animals from control group at day 7.· CONCLUSION:Our results indicate that TGF-β signaling may contribute to the pathogenesis of subretinal fibrogenesis and TGF-β inhibition may provide an effective,novel treatment of advanced and late-stage neovascular age-related macular degeneration.·

Interleukin-6 receptor blockade suppresses subretinal fibrosis in a mouse model

AIM:To determine the involvement of the interleukin(IL)-6 with the development of experimental subretinal fibrosis in a mouse model.METHODS:Subretinal fibrosis was induced by subretinal injection of macrophage-rich peritoneal exudate cells and the local expression of IL-6 was assessed by quantitative real-time reverse transcriptionpolymerase chain reaction(RT-PCR)and enzyme-linked immunosorbent assay(ELISA)at various time points.In addition,we investigated the effect of IL-6 receptor(IL-6R)monoclonal antibody(MR16-1)on subretinal fibrosis development.RESULTS:IL-6 mRNA level was significantly elevated at 1d after subretinal fibrosis induction and increased further to about 12-fold at 2d,reaching the peak.The result of ELISA showed that IL-6 protein was not detected in naive mice.At 2d after subretinal fibrosis induction,IL-6 protein level was upregulated to 67.33±14.96 pg/mg in subretinal fibrosis mice.MR16-1treatment resulted in a reduced subretinal fibrosis area by 48%compared to animals from control group at 7d.CONCLUSION:Our results indicated that IL-6 signaling may contribute to the pathogenesis of subretinal fibrogenesis and IL-6R inhibition may provide an effective,novel treatment of advanced and late-stage neovascular age-related macular degeneration.

Inhibitory effect on subretinal fibrosis by anti-placental growth factor treatment in a laser-induced choroidal neovascularization model in mice

AIM:To investigate whether anti-placental growth factor(PGF) can inhibit subretinal fibrosis and whether this effect is mediated by the inhibitory effect of PGF on epithelial-mesenchymal transition(EMT) of retinal pigment epithelial(RPE) cells.METHODS:Subretinal fibrosis model was established in laser induced choroidal neovascularization(CNV) mice on day 21 after laser photocoagulation.Immunofluorescence staining(IFS) of cryosections and enzyme-linked immunosorbent assay(ELISA) were used to detect the expression of PGF.IFS of whole choroidal flat-mounts was used to detect the degree of subretinal fibrosis.IFS of cryosections and ELISA were used to detect the expression of EMT related indicators in subretinal fibrosis lesions.RESULTS:The expression of PGF protein in subretinal fibrosis lesions was significantly up-regulated(P<0.05),and mainly co-stained with pan-cytokeratin labeled RPE cells.Intravitreal injection of anti-PGF neutralizing antibody reduced the area of subretinal fibrosis and the ratio of fibrotic/angiogenic area significantly at the concentrations of 0.25,0.5,1.0,and 2.0 μg/μL(all P<0.05).The expression of E-cadherin in the local RPE cells decreased,while α-SMA increased significantly in subretinal fibrosis lesions,and the application of anti-PGF neutralizing antibody could reverse these changes(P<0.05).CONCLUSION:The expression of PGF is up-regulated in the lesion site of subretinal fibrosis and mainly expressed in RPE cells.Intravitreal injection of anti-PGF neutralizing antibody can significantly inhibit the degree of subretinal fibrosis in CNV mice,and this effect may be mediated by the inhibition of PGF on EMT of RPE cells.

METTL3-mediated m^(6)A modification of HMGA2 mRNA promotes subretinal fibrosis and epithelial-mesenchymal transition

Subretinal fibrosis is a major cause of the poor visual prognosis for patients with neovascular age-related macular degeneration(nAMD).Myofibroblasts originated from retinal pigment epithelial(RPE)cells through epithelial-mesenchymal transition(EMT)contribute to the fibrosis formation.N^(6)-Methyladenosine(m^(6)A)modification has been implicated in the EMT process and multiple fibrotic diseases.The role of m^(6)A modification in EMT-related subretinal fibrosis has not yet been elucidated.In this study,we found that during subretinal fibrosis in the mouse model of laser-induced choroidal neovascularization,METTL3 was upregulated in RPE cells.Through m^(6)A epitranscriptomic microarray and further verification,high-mobility group AT-hook 2(HMGA2)was identified as thekey downstream target of METTL3,subsequently activating potent EMT-inducing transcription factor SNAIL.Finally,by subretinal injections of adeno-associated virus vectors,we confirmed that METTL3 deficiency in RPE cells could efficiently attenuate subretinal fibrosis in vivo.In conclusion,our present research identified an epigenetic mechanism of METTL3-m^(6)A-HMGA2 in subretinal fibrosis and EMT of RPE cells,providing a novel therapeutic target for subretinal fibrosis secondary to nAMD.

Spectral Domain Optical Coherence Tomography of Vogt-Koyanagi-Harada Disease: Novel Findings and New Insights into the Pathogenesis

Objective To provide novel spectral domain optical coherence tomography (SD OCT) findings of Vogt-Koyanagi-Harada (VKH) disease as well as new insights into the pathogenesis of this disease. Methods Detailed SD OCT and fluorescein angiography (FA) findings of 18 consecutive VKH patients (11 women and 7 men) from December 2007 to April 2009 who were in acute uveitic stage at presentation were reviewed. All the patients had been followed up for at least 6 months with reevaluation(s) of SD OCT performed in 10 patients. Results Intraretinal cysts were found to be located in various layers of the outer retina. In addition to the photoreceptor layer, they could also be found between the outer plexiform layer and the outer nuclear layer, or spanning the external limiting membrane. On FA, intraretinal cysts could be hypofluorescent, normofluorescent, or hyperfluorescent. Some intraretinal cysts had a characteristic FA pattern, in which a small round hypofluorescent area was surrounded by a ring of hyperfluorescence (donut-shaped dye pooling). Subretinal fibrinoid deposit appeared in acute uveitic stage in two severe VKH patients and seemed to develop from subretinal exudates and evolved into typical subretinal fibrosis. Gradual transfiguration/migration and progressive proliferation/pigmentation of the subretinal fibrinoid deposit/subretinal fibrosis was observed in one patient. Conclusions Intraretinal cysts could form in various layers of the outer retina and may result from extension of choroidal inflammation. Subretinal fibrosis may develop from subretinal exudates in VKH patients and may cause substantial visual impairment.

LRG1 promotes epithelial-mesenchymal transition of retinal pigment epithelium cells by activating NOX4

AIM:To investigate the effect of leucine-rich-alpha-2-glycoprotein 1(LRG1)on epithelial-mesenchymal transition(EMT)in retinal pigment epithelium(RPE)cells,and to explore the role of NADPH oxidase 4(NOX4).METHODS:RPE cells(ARPE-19 cell line)were treated with transforming growth factor-β1(TGF-β1)to induce EMT.Changes of the m RNA and protein expression levels of LRG1 were tested in the TGF-β1 treated cells.The recombinant human LRG1 protein(r LRG1)and si RNA of LRG1 were used to establish accumulation of exogenous LRG1 model and the down-regulation of LRG1 model in ARPE-19 cells respectively,and to detect EMT-related markers including fibronectin,α-smooth muscle actin(α-SMA)and zonula occludens-1(ZO-1).The m RNA and protein expression level of NOX4 were measured according to the above treatments.VAS2870 was used as a NOX4 inhibitor in r LRG1-treated cells.EMT-related markers were detected to verify the effect of NOX4 in the process of EMT.RESULTS:TGF-β1 promoted the expression of LRG1 at both the m RNA and protein levels during the process of EMT which showed the up-regulation of fibronectin andα-SMA,as well as the down-regulation of ZO-1.Furthermore,the r LRG1 promoted EMT of ARPE-19 cells,which manifested high levels of fibronectin andα-SMA and low level of ZO-1,whereas knockdown of LRG1 prevented EMT by decreasing the expressions of fibronectin andα-SMA and increasing the expression of ZO-1 in ARPE-19 cells.Besides,the r LRG1 activated and LRG1 si RNA suppressed NOX4 expression.EMT was inhibited when VAS2870 was used in the r LRG1-treated cells.CONCLUSION:These results for the first time demonstrate that LRG1 promotes EMT of RPE cells by activating NOX4,which may provide a novel direction to explore the mechanisms of subretinal fibrosis.