Inhibition of hypoxia-inducible factors suppresses subretinal fibrosis.

Age-related macular degeneration (AMD) is a common cause of vision loss. The aggressive form of AMD is associated with ocular neovascularization and subretinal fibrosis, representing a responsive outcome against neovascularization mediated by epithelial-mesenchymal transition of retinal pigment epithelium (RPE) cells. A failure of the current treatment (anti-vascular endothelial growth factor therapy) has also been attributed to the progression of subretinal fibrosis. Hypoxia-inducible factors (HIFs) increase gene expressions to promote fibrosis and neovascularization. HIFs act as a central pathway in the pathogenesis of AMD. HIF inhibitors may suppress ocular neovascularization. Nonetheless, further investigation is required to unravel the aspects of subretinal fibrosis. In this study, we used RPE-specific HIFs or von Hippel-Lindau (VHL, a regulator of HIFs) conditional knockout (cKO) mice, along with pharmacological HIF inhibitors, to demonstrate the suppression of subretinal fibrosis. Fibrosis was suppressed by treatments of HIF inhibitors, and similar suppressive effects were detected in RPE-specific Hif1a/Hif2a- and Hif1a-cKO mice. Promotive effects were observed in RPE-specific Vhl-cKO mice, where fibrosis-mediated pathologic processes were evident. Marine products' extracts and their component taurine suppressed fibrosis as HIF inhibitors. Our study shows critical roles of HIFs in the progression of fibrosis, linking them to the potential development of therapeutics for AMD.

Subretinal Fibrosis Developing 10 Years After First Presentation with Chronic Ocular Sarcoidosis in a Child.

PURPOSE: To provide a comprehensive overview of the diagnostic and therapeutic journey of a pediatric patient with persistent sarcoid-associated panuveitis over a 10-year period, who ultimately developed bilateral macular subretinal fibrosis and visual loss. METHODS: Retrospective case report. RESULTS: The patient was diagnosed with sarcoidosis after undergoing a transbronchial biopsy. She was followed up because of granulomatous panuveitis, multifocal choroiditis, and papillitis bilaterally. She maintained a stable condition, and visual acuity was 0.3 RE and 0.5 LE. Immunomodulatory therapy included prednisone, methotrexate, and adalimumab. The patient was lost to follow-up for 20 months because of the COVID-19 pandemic. She was represented with active uveitis and was not responding to TNF-ɑ inhibitors (adalimumab and infliximab). Ultimately, the patient's intraocular inflammation was successfully controlled by using intravitreal steroids (Triamcinolone and Fluocinolone acetonide implant). However, the visual outcome was guarded because of bilateral subretinal fibrosis. CONCLUSION: 10% of patients with sarcoidosis-associated uveitis risk blindness in one eye. The index case progressed to sight-robbing bilateral subretinal fibrosis, a rare complication of ocular sarcoidosis despite a combination of conventional and biologic anti-inflammatory therapies. There is a pressing need to develop new treatment agents for refractory non-infectious uveitis.

Anti-angiogenic biomolecules in neovascular age-related macular degeneration; therapeutics and drug delivery systems.

Blindness in the elderly is often caused by age-related macular degeneration (AMD). The advanced type of AMD known as neovascular AMD (nAMD) has been linked to being the predominant cause of visual impairment in these people. Multiple neovascular structures including choroidal neovascular (CNV) membranes, fluid exudation, hemorrhages, and subretinal fibrosis, are diagnostic of nAMD. These pathological alterations ultimately lead to anatomical and visual loss. It is known that vascular endothelial growth factor (VEGF), a type of proangiogenic factor, mediates the pathological process underlying nAMD. Therefore, various therapies have evolved to directly target the disease. In this review article, an attempt has been made to discuss general explanations about this disease, all common treatment methods based on anti-VEGF drugs, and the use of drug delivery systems in the treatment of AMD. Initially, the pathophysiology, angiogenesis, and different types of AMD were described. Then we described current treatments and future treatment prospects for AMD and outlined the advantages and disadvantages of each. In this context, we first examined the types of therapeutic biomolecules and anti-VEGF drugs that are used in the treatment of AMD. These biomolecules include aptamers, monoclonal antibodies, small interfering RNAs, microRNAs, peptides, fusion proteins, nanobodies, and other therapeutic biomolecules. Finally, we described drug delivery systems based on liposomes, nanomicelles, nanoemulsions, nanoparticles, cyclodextrin, dendrimers, and composite vehicles that are used in AMD therapy.

Role of microglia/macrophage polarisation in intraocular diseases (Review).

Macrophages form a crucial component of the innate immune system, and their activation is indispensable for various aspects of immune and inflammatory processes, tissue repair, and maintenance of the balance of the body's state. Macrophages are found in all ocular tissues, spanning from the front surface, including the cornea, to the posterior pole, represented by the choroid/sclera. The neural retina is also populated by specialised resident macrophages called microglia. The plasticity of microglia/macrophages allows them to adopt different activation states in response to changes in the tissue microenvironment. When exposed to various factors, microglia/macrophages polarise into distinct phenotypes, each exhibiting unique characteristics and roles. Furthermore, extensive research has indicated a close association between microglia/macrophage polarisation and the development and reversal of various intraocular diseases. The present article provides a review of the recent findings on the association between microglia/macrophage polarisation and ocular pathological processes (including autoimmune uveitis, optic neuritis, sympathetic ophthalmia, retinitis pigmentosa, glaucoma, proliferative vitreoretinopathy, subretinal fibrosis, uveal melanoma, ischaemic optic neuropathy, retinopathy of prematurity and choroidal neovascularization). The paradoxical role of microglia/macrophage polarisation in retinopathy of prematurity is also discussed. Several studies have shown that microglia/macrophages are involved in the pathology of ocular diseases. However, it is required to further explore the relevant mechanisms and regulatory processes. The relationship between the functional diversity displayed by microglia/macrophage polarisation and intraocular diseases may provide a new direction for the treatment of intraocular diseases.

Wnt5a/ß-catenin-mediated epithelial-mesenchymal transition: a key driver of subretinal fibrosis in neovascular age-related macular degeneration.

BACKGROUND: Neovascular age-related macular degeneration (nAMD), accounts for up to 90% of AMD-associated vision loss, ultimately resulting in the formation of fibrotic scar in the macular region. The pathogenesis of subretinal fibrosis in nAMD involves the process of epithelial-mesenchymal transition (EMT) occurring in retinal pigment epithelium (RPE). Here, we aim to investigate the underlying mechanisms involved in the Wnt signaling during the EMT of RPE cells and in the pathological process of subretinal fibrosis secondary to nAMD. METHODS: In vivo, the induction of subretinal fibrosis was performed in male C57BL/6J mice through laser photocoagulation. Either FH535 (a ß-catenin inhibitor) or Box5 (a Wnt5a inhibitor) was intravitreally administered on the same day or 14 days following laser induction. The RPE-Bruch's membrane-choriocapillaris complex (RBCC) tissues were collected and subjected to Western blot analysis and immunofluorescence to examine fibrovascular and Wnt-related markers. In vitro, transforming growth factor beta 1 (TGFß1)-treated ARPE-19 cells were co-incubated with or without FH535, Foxy-5 (a Wnt5a-mimicking peptide), Box5, or Wnt5a shRNA, respectively. The changes in EMT- and Wnt-related signaling molecules, as well as cell functions were assessed using qRT-PCR, nuclear-cytoplasmic fractionation assay, Western blot, immunofluorescence, scratch assay or transwell migration assay. The cell viability of ARPE-19 cells was determined using Cell Counting Kit (CCK)-8. RESULTS: The in vivo analysis demonstrated Wnt5a/ROR1, but not Wnt3a, was upregulated in the RBCCs of the laser-induced CNV mice compared to the normal control group. Intravitreal injection of FH535 effectively reduced Wnt5a protein expression. Both FH535 and Box5 effectively attenuated subretinal fibrosis and EMT, as well as the activation of ß-catenin in laser-induced CNV mice, as evidenced by the significant reduction in areas positive for fibronectin, alpha-smooth muscle actin (α-SMA), collagen I, and active ß-catenin labeling. In vitro, Wnt5a/ROR1, active ß-catenin, and some other Wnt signaling molecules were upregulated in the TGFß1-induced EMT cell model using ARPE-19 cells. Co-treatment with FH535, Box5, or Wnt5a shRNA markedly suppressed the activation of Wnt5a, nuclear translocation of active ß-catenin, as well as the EMT in TGFß1-treated ARPE-19 cells. Conversely, treatment with Foxy-5 independently resulted in the activation of abovementioned molecules and subsequent induction of EMT in ARPE-19 cells. CONCLUSIONS: Our study reveals a reciprocal activation between Wnt5a and ß-catenin to mediate EMT as a pivotal driver of subretinal fibrosis in nAMD. This positive feedback loop provides valuable insights into potential therapeutic strategies to treat subretinal fibrosis in nAMD patients.

Metformin regulates the LIN28B­mediated JNK/STAT3 signaling pathway through miR­140­3p in subretinal fibrosis.

Subretinal fibrosis (SF) is an important cause of submacular neovascularization that leads to permanent vision loss, but has no effective clinical treatment. The present study examined the influence of metformin on SF, and investigated whether the mechanism involves the microRNA (miR)-140-3p/LIN28B/JNK/STAT3-mediated regulation of oxidative stress, angiogenesis and fibrosis-associated indicators. A mouse model of laser-induced SF was established. In addition, an ARPE-19 fibrotic cell model was established using TGF-ß1. A Cell Counting Kit-8 assay was used to examine cell viability. Flow cytometry was used to measure reactive oxygen species levels, and western blotting was used to detect the levels of proteins associated with epithelial-mesenchymal transition (EMT), signaling and fibrosis. The levels of superoxide dismutase, malondialdehyde, glutathione-peroxidase and catalase were measured using kits. Scratch assays and Transwell assays were used to assess cell migration and invasion, respectively, and reverse transcription-quantitative PCR was used to determine the levels of miR-140-3p and LIN28B. Dual-luciferase assays were used to verify the targeting relationship between miR-140-3p and LIN28B, and coimmunoprecipitation was used to confirm the interaction between LIN28B and JNK. Masson staining and hematoxylin and eosin staining were used to examine collagenous fibers and the histopathology of eye tissue. In ARPE-19 cells induced by TGF-ß1, metformin promoted miR-140-3p expression and inhibited LIN28B expression and JNK/STAT3 pathway activation, thereby inhibiting oxidative stress, EMT and fibrosis in ARPE-19 cells. The overexpression of LIN28B or treatment with the JNK/STAT3 agonist anisomycin partially reversed the inhibitory effect of metformin on oxidative stress and fibrosis in ARPE-19 cells. The dual-luciferase reporter assay and coimmunoprecipitation assay showed that miR-140-3p targeted the 3' untranslated region of LIN28B mRNA and inhibited LIN28B expression. LIN28B targeted and bound to JNK and regulated the JNK/STAT3 pathway. Therefore, it may be concluded that metformin can promote miR-140-3p expression, inhibit LIN28B and then inhibit the JNK/STAT3 pathway to alleviate SF.

Molecular pathogenesis of subretinal fibrosis in neovascular AMD focusing on epithelial-mesenchymal transformation of retinal pigment epithelium.

Age-related macular degeneration (AMD) is a leading cause of vision loss among elderly people in developed countries. Neovascular AMD (nAMD) accounts for more than 90% of AMD-related vision loss. At present, intravitreal injection of anti-vascular endothelial growth factor (anti-VEGF) is widely used as the first-line therapy to decrease the choroidal and retinal neovascularizations, and thus to improve or maintain the visual acuity of the patients with nAMD. However, about 1/3 patients still progress to irreversible visual impairment due to subretinal fibrosis even with adequate anti-VEGF treatment. Extensive literatures support the critical role of epithelial-mesenchymal transformation (EMT) of retinal pigment epithelium (RPE) in the pathogenesis of subretinal fibrosis in nAMD, but the underlying mechanisms still remain largely unknown. This review summarized the molecular pathogenesis of subretinal fibrosis in nAMD, especially focusing on the transforming growth factor-ß (TGF-ß)-induced EMT pathways. It was also discussed how these pathways crosstalk and respond to signals from the microenvironment to mediate EMT and contribute to the progression of nAMD-related subretinal fibrosis. Targeting EMT signaling pathways might provide a promising and effective therapeutic strategy to treat subretinal fibrosis secondary to nAMD.

Luteolin inhibits subretinal fibrosis and epithelial-mesenchymal transition in laser-induced mouse model via suppression of Smad2/3 and YAP signaling.

BACKGROUND: Subretinal fibrosis (SF) accounts for vision loss in patients with neovascular age-related macular degeneration (nAMD) even treated with adequate intravitreal injection of anti-vascular endothelial growth factor (anti-VEGF) drugs. Currently, there is no treatment available to prevent or treat SF caused by nAMD. PURPOSE: This study aims to investigate the potential effects of luteolin on SF and epithelial-mesenchymal transition (EMT) as well as the underlying molecular pathways both in vivo and in vitro. METHODS: Seven-week-old male C57BL/6J mice were employed to establish laser-induced choroidal neovascularization (CNV) and SF. One day after the laser induction, luteolin was administered intravitreally. SF and CNV were assessed with the immunolabeling of collagen type I (collagen I) and isolectin B4 (IB4), respectively. RPE65 and α-SMA colocalization in the lesions was used to evaluate the extent of EMT in retinal pigment epithelial (RPE) cells by using immunofluorescence. In vitro, luteolin was administered to TGFß1-treated primary human RPE (phRPE) cells. RT-qPCR, Western blot and immunofluorescence were employed to evaluate the change of EMT-related molecules, epithelial markers, and relevant signaling pathways. The functional changes associated with EMT were investigated using the scratch assay, Transwell migration assay, and collagen gel contraction assay. CCK-8 was used to determine the cell viability of phRPE cells. RESULTS: On day 7 and 14 after laser induction in mice, intravitreal injection of luteolin dramatically decreased the immunolabeled sizes of both collagen I and IB4, as well as the amount of colocalized double immunostaining of α-SMA and RPE65 in laser-induced SF lesions. In vitro, TGFß1-treated phRPE cells demonstrated increased cell migration and contraction capacity, accompanied with considerable overexpression of fibronectin, α-SMA, N-cadherin and vimentin, as well as downregulation of E-cadherin and ZO-1. The above changes were largely inhibited by luteolin co-incubation. Mechanistically, luteolin could evidently decrease the phosphorylation of Smad2/3, whereas increase the phosphorylation of YAP in TGFß1-treated phRPE cells. CONCLUSION: This study demonstrates that luteolin exhibits the anti-fibrotic effect in a laser-induced mouse model by inhibiting EMT of RPE cells via deactivating Smad2/3 and YAP signaling, which provides a potential natural compound for the prevention and treatment of SF and fibrosis-related diseases.

METTL3-mediated m6A 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. N6-Methyladenosine (m6A) modification has been implicated in the EMT process and multiple fibrotic diseases. The role of m6A 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 m6A epitranscriptomic microarray and further verification, high-mobility group AT-hook 2 (HMGA2) was identified as the key 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-m6A-HMGA2 in subretinal fibrosis and EMT of RPE cells, providing a novel therapeutic target for subretinal fibrosis secondary to nAMD.

Animal model of subretinal fibrosis without active choroidal neovascularization.

Subretinal fibrosis can occur during neovascular age-related macular degeneration (nAMD) and consequently provokes progressing deterioration of AMD patient's vision. Intravitreal anti-vascular endothelial growth factor (VEGF) injections decrease choroidal neovascularization (CNV), however, subretinal fibrosis remains principally unaffected. So far, no successful treatment nor established animal model for subretinal fibrosis exists. In order to investigate the impact of anti-fibrotic compounds on solely fibrosis, we refined a time-dependent animal model of subretinal fibrosis without active choroidal neovascularization (CNV). To induce CNV-related fibrosis, wild-type (WT) mice underwent laser photocoagulation of the retina with rupture of Bruch's membrane. The lesions volume was assessed with optical coherence tomography (OCT). CNV (Isolectin B4) and fibrosis (type 1 collagen) were separately quantified with confocal microscopy of choroidal whole-mounts at every time point post laser induction (day 7-49). In addition, OCT, autofluorescence and fluorescence angiography were carried out at designated timepoints (day 7, 14, 21, 28, 35, 42, 49) to monitor CNV and fibrosis transformation over time. From 21 to 49 days post laser lesion leakage in the fluorescence angiography decreased. Correspondingly, Isolectin B4 decreased in lesions of choroidal flat mounts and type 1 collagen increased. Fibrosis markers, namely vimentin, fibronectin, alpha-smooth muscle actin (α-SMA) and type 1 collagen were detected at different timepoints of tissue repair in choroids and retinas post laser. These results prove that the late phase of the CNV-related fibrosis model enables screening of anti-fibrotic compounds to accelerate the therapeutic advancement for the prevention, reduction, or inhibition of subretinal fibrosis.

Subretinal fluid may protect against macular atrophy in neovascular age-related macular degeneration: 5 years of follow-up from Fight Retinal Blindness registry.

PURPOSE: The purpose of the study was to assess the association of macular atrophy (MA) according to the activity of macular neovascularization (MNV) (inactive, only subretinal fluid [SRFL], or active, i.e. including intraretinal fluid [IRFL]) using optical coherence tomography (OCT) in patients with neovascular age-related macular degeneration (nAMD). METHODS: Multicentric observational study. Treatment-naïve nAMD eyes without subfoveal MA or subretinal fibrosis (SF) at baseline were included since 1st January 2010 and 30th September 2016 to allow up to 5 years of treatment follow-up. Eyes were grouped based on their predominant activity status as: (1) mostly inactive, (2) mostly active non-SRFL only [IRFL] or (3) mostly active-SRFL only [onlySRFL]. Kaplan-Meier survival curves estimated the time to development of MA or SF. Cox proportional hazards models evaluated predictors of developing subfoveal MA or SF. The main outcome measure was the risk of developing MA according to predominant MNV activity. RESULTS: A total of 973 eyes were eligible for analysis. OnlySRFL eyes had lower risk of developing subfoveal MA (HR [95% CI]: 0.56 [0.36, 0.88]; p = 0.024) and extrafoveal MA (HR [95% CI]: 0.41 [0.27, 0.61]; p < 0.001) than IRFL eyes. IRFL eyes had lower visual acuity (VA) (54.5 letters) and the highest proportion of eyes with vision ≤35 letters (25%) at 5 years while onlySRFL eyes had comparable 5-year VA (63.7 letters) to inactive eyes (63.7 letters). CONCLUSION: Subretinal fluid appears to protect against MA. Distinguishing the compartment of retinal fluid and understanding its relationship with MA and SF can guide the management of nAMD.

Peptidyl arginine deiminase 4 deficiency protects against subretinal fibrosis by inhibiting Müller glial hypercitrullination.

Retinal scarring with vision loss continues to be an enigma in individuals with advanced age-related macular degeneration (AMD). Müller glial cells are believed to initiate and perpetuate scarring in retinal degeneration as these glial cells participate in reactive gliosis and undergo hypertrophy. We previously showed in the murine laser-induced model of choroidal neovascularization that models wet-AMD that glial fibrillary acidic protein (GFAP) expression, an early marker of reactive gliosis, increases along with its posttranslational modification citrullination. This was related to increased co-expression of the citrullination enzyme peptidyl arginine deiminase-4 (PAD4), which also colocalizes to GFAP filaments. However, whether such hypercitrullination in Müller glial drives fibrotic pathology has remained understudied. Here, using male and female C57Bl6 mice subjected to laser injury, we investigated in a temporal study how citrullination impacts GFAP and PAD4 dynamics. We found that high molecular weight citrullinated species that accumulate in Müller glia corresponded with dynamic changes in GFAP and PAD4 showing their temporal redistribution from polymeric cytoskeletal to soluble protein fractions using immunostaining and western blot analysis. In conditional glial-specific PAD4 knockout (PAD4cKO) mice subjected to laser injury, there was a stark reduction of citrullination and of polymerized GFAP filaments. These injured PAD4cKO retinas showed improved lesion healing, as well as reduced fibronectin deposition in the subretinal space at 30 days. Taken together, these findings reveal that pathologically overexpressed PAD4 in reactive Müller glia governs GFAP filament dynamics and alters their stability, suggesting chronic PAD4-driven hypercitrullination may be a target for retinal fibrosis.

A Case of Bullous Central Serous Chorioretinopathy Treated with Surgical Removal of Submacular Fibrin and Subsequent Photodynamic Therapy under Silicone Oil.

Bullous retinal detachment is a rare complication in the chronic phase of central serous chorioretinopathy (CSC). Only a small subset of eyes with chronic CSC develops into the bullous variant of CSC (bCSC). In patients with bCSC, the elevated concentration of fibrin in the subretinal space leads to persistent retinal detachment and eventually, severe vision loss. We experienced a case of unilateral bCSC with a massive accumulation of subretinal fibrin. Multiple leakage points and dilated choroidal veins were also observed. The patient underwent surgical removal of subretinal fibrin and silicone oil injection followed by photodynamic therapy (PDT). After this treatment, the retina was successfully reattached, and the affected eye was free from recurrent exudative changes for more than 18 months. Massive subretinal fibrin could be surgically removed to prevent the formation of subretinal fibrosis and retinal fold, and PDT under silicone oil can control the underlying exudative changes in bCSC.

Complement activation contributes to subretinal fibrosis through the induction of epithelial-to-mesenchymal transition (EMT) in retinal pigment epithelial cells.

BACKGROUND: We previously reported higher plasma levels of complement fragments C3a and C5a in neovascular Age-related Macular Degeneration (nAMD) patients with macular fibrosis. This study aimed to understand whether complement activation contributes to the development of macular fibrosis and the underlying mechanisms involved. METHODS: Complement activation was blocked using a C5 neutralizing antibody (BB5.1) in C57BL/6J mice after induction of subretinal fibrosis using the two-stage laser protocol. Fibrotic lesions were examined 10 days after the 2nd laser through fundus examination and immunohistochemistry. The expression of C5aR in fibrotic lesions and retinal pigment epithelial (RPE) cultures were examined by confocal microscopy. Primary murine RPE cells were treated with C3a or C5a (10-100 ng/mL) or TGF-ß2 (10 ng/mL). Epithelial-to-mesenchymal transition (EMT) was assessed through various readouts. The expression of E-cadherin, vimentin, fibronectin, α-SMA, Slug, ERK/AKT and pSMAD2/3 were determined by Western blot and immunocytochemistry. Collagen contraction and wound-healing assays were used as functional readouts of EMT. The production of IL-6, TGF-ß1, TGF-ß2 and VEGF by RPE cells were determined by ELISA. PMX53 was used to block C5aR in RPE cultures and in vivo in mice with subretinal fibrosis. RESULTS: Extensive C5b-9 deposition was detected at the site of subretinal fibrosis. BB5.1 treatment completely abrogated complement activation and significantly reduced subretinal fibrosis. C5aR was detected in RPE and infiltrating MHC-II+ cells in subretinal fibrosis. In vitro, RPE cells constitutively express C5/C5a and C5aR, and their expression was increased by TGF-ß2 treatment. C5a but not C3a increased fibronectin, α-SMA, vimentin and Slug expression, and decreased E-cadherin expression in RPE cells. C5a treatment also increased the contractility and migration of RPE cells and enhanced the production of VEGF and TGF-ß1/2. C5a treatment induced pSmad2/3 and pERK1/2 expression in RPE cells and this was blocked by PMX53. PMX53 treatment significantly reduced sodium fluorescein leakage in the subretinal fibrosis model, while collagen-I+ lesions only mildly reduced. CONCLUSIONS: Complement activation is critically involved in the development of subretinal fibrosis, partially through C5a-C5aR-mediated EMT in RPE cells. Targeting complement activation rather than C5a may be a novel approach for the management of macular fibrosis.

YAP is critical to inflammation, endothelial-mesenchymal transition and subretinal fibrosis in experimental choroidal neovascularization.

Subretinal fibrosis causes local damage to the retina and irreversible vision loss, as the final stage of neovascular age-related macular degeneration (nAMD). More recently, the endothelial-to-mesenchymal transition (EndoMT) has been considered one of the most significant sources of myofibroblasts in subretinal fibrosis, though the underpinning molecular mechanisms remain unclear. In this study, a series of experiments were performed to test the hypothesis that Yes-associated protein (YAP) may be involved in EndoMT and subretinal fibrosis. We demonstrated that transforming growth factor (TGF)-ß2 stimulation induces YAP dephosphorylation (activated) and nuclear transcription in human umbilical vein endothelial cells (HUVECs) by increasing reactive oxygen species (ROS) levels. Moreover, TGF-ß2-mediated EndoMT and proinflammatory cytokine production in HUVECs were reduced by ROS clearance or YAP knockdown. Furthermore, the severity of subretinal fibrosis was markedly relieved by intravitreal administration of a small interfering RNA targeting YAP in the mouse laser-induced choroidal neovascularization (CNV) model. Our findings provide novel insights into a previously unknown effect of YAP on the EndoMT process and reveal YAP as a potential target for suppressing CNV-related subretinal fibrosis and protect vision.

Neovascular Remodeling and Subretinal Fibrosis as Biomarkers for Predicting Incomplete Response to Anti-VEGF Therapy in Neovascular Age-Related Macular Degeneration.

PURPOSE: To compare the progression of neovascular remodeling and subretinal fibrosis in neovascular age-related macular degeneration (NVAMD) after anti-vascular endothelial growth factor (VEGF) therapy. METHODS: Twenty eyes from 20 patients with subretinal fibrosis complicating NVAMD were retrospectively reviewed. All patients complied with at least three consecutive monthly intravitreal treatments and final follow-up visit at 12 months after the initial anti-VEGF treatment of aflibercept or ranibizumab. Using optical coherence tomography angiography (OCTA), the central macular thickness (CMT), microvascular density in the superficial capillary plexus (SCP), deep capillary plexus (DCP), choroidal neovascularization (CNV) lesions, as well as subretinal fibrotic lesions were compared between baseline and final visit. RESULTS: The mean number for anti-VEGF injections was 4.40 ± 0.88 during the 12 months of follow-up. There was no significant difference in best-corrected visual acuity (BCVA) and vascular density in SCP and DCP (p > 0.05) between baseline and final follow-up. The CMT decreased from 434.95 ± 87.62 µm at baseline to 365.15 ± 78.92 µm at final visit (p = 0.02). Compared with the baseline, the fine vessels, such as capillary tufts, regressed and the relative density of CNV lesion decreased by 19.12% (p = 0.01), while the relative density of the subretinal fibrosis increased approximately 1.21-fold (p = 0.03) at the final follow-up. CONCLUSIONS: The progression of neovascular remodeling and subretinal fibrosis may serve as biomarkers to predict incomplete response to anti-VEGF therapy in patients with NVAMD. Subretinal fibrosis complicating NVAMD remains a major obstacle for the management of NVAMD, and anti-VEGF treatment is a potential therapeutic strategy to target neovascular remodeling and subretinal fibrosis as either an additive or alternative therapeutic approach for NVAMD.

Prevalence, multimodal imaging and genotype-phenotype assessment of trauma related subretinal fibrosis in stargardt disease.

BACKGROUND AND OBJECTIVES: Stargardt disease produces lipofuscin accumulation predisposing to subretinal fibrosis (SRFib) after ocular trauma. Noninvasive imaging techniques allow in vivo assessment. The purpose of this study is to determine the prevalence of SRFib in a cohort of Stargardt patients, the presence of history of ocular trauma, the clinical features and possible genotype-phenotype associations in Stargardt patients with SRFib. METHODS: We evaluated retrospectively 106 Stargardt patients and analysed the multimodal imaging and the genotype of patients with SRFib. RESULTS: Six patients exhibited SRFib, three of them with history of ocular trauma. Multimodal imaging showed extensive SRFib principally in the temporal midperipheral retina with no fluid associated. SRFib was better defined by short wavelength autofluorescence and spectral domain optical coherence tomography and appeared clinically stable over time. There was no particular genotype associated to SRFib. CONCLUSION: SRFib occurs in a significant percentage of patients with Stargardt disease and can be diagnosed through multimodal imaging regardless the history of trauma, further sustaining the importance of an appropriate imaging in such patients. No genotype-phenotype association has been established, supporting the traumatic etiology in half of cases. The remaining cases may be classified as idiopathic or have a minimal trauma occurring early in life that may be not recalled by the patients.

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.

Mechanisms of RPE senescence and potential role of αB crystallin peptide as a senolytic agent in experimental AMD.

Oxidative stress in the retinal pigment epithelium (RPE) can cause mitochondrial dysfunction and is likely a causative factor in the pathogenesis of age-related macular degeneration (AMD). Under oxidative stress conditions, some of the RPE cells become senescent and a contributory role for RPE senescence in AMD pathology has been proposed. The purpose of this study is to 1) characterize senescence in human RPE; 2) investigate the effect of an αB Crystallin chaperone peptide (mini Cry) in controlling senescence, in particular by regulating mitochondrial function and senescence-associated secretory phenotype (SASP) production and 3) develop mouse models for studying the role of RPE senescence in dry and nAMD. Senescence was induced in human RPE cells in two ways. First, subconfluent cells were treated with 0.2 µg/ml doxorubicin (DOX); second, subconfluent cells were treated with 500 µM H2O2. Senescence biomarkers (senescence-associated beta-galactosidase (SA-ßgal), p21, p16) and mitochondrial proteins (Fis1, DRP1, MFN2, PGC1-α, mtTFA) were analyzed in control and experimental groups. The effect of mini Cry on mitochondrial bioenergetics, glycolysis and SASP was determined. In vivo, retinal degeneration was induced by intravenous injection of NaIO3 (20 mg/kg) and subretinal fibrosis by laser-induced choroidal neovascularization. Increased SA-ßgal staining and p16 and p21 expression was observed after DOX- or H2O2-induced senescence and mini Cry significantly decreased senescence-positive cells. The expression of mitochondrial biogenesis proteins PGC-1 and mTFA increased with senescence, and mini Cry reduced expression significantly. Senescent RPE cells were metabolically active, as evidenced by significantly enhanced oxidative phosphorylation and anaerobic glycolysis, mini Cry markedly reduced rates of respiration and glycolysis. Senescent RPE cells maintain a proinflammatory phenotype characterized by significantly increased production of cytokines (IFN-Ë , TNF-α, IL1-α IL1-ß, IL-6, IL-8, IL-10), and VEGF-A; mini Cry significantly inhibited their secretion. We identified and localized senescent RPE cells for the first time in NaIO3-induced retinal degeneration and laser-induced subretinal fibrosis mouse models. We conclude that mini Cry significantly impairs stress-induced senescence by modulating mitochondrial biogenesis and fission proteins in RPE cells. Characterization of senescence could provide further understanding of the metabolic changes that accompany the senescent phenotype in ocular disease. Future studies in vivo may better define the role of senescence in AMD and the therapeutic potential of mini Cry as a senotherapeutic.

Risk factors for subretinal fibrosis in patients with Vogt Koyanagi Harada syndrome.

PURPOSE: To identify the risk factors for the development of subretinal fibrosis (SRF) among patients with Vogt-Koyanagi-Harada (VKH) syndrome. METHODS: In this case-control study, electronic clinical records from patients diagnosed with VKH syndrome who attended the Inflammatory Eye Disease Clinic at a tertiary care ophthalmology reference center were assessed to identify risk factors from demographic, clinical, and epidemiological variables. Cases were defined as SRF and VKH, whereas the controls were VKH patients without SRF. RESULTS: A total of 150 electronic charts were reviewed, 92 patients with a follow-up longer than 12 months were included; 39 cases and 53 controls. A multivariate analysis found bullous serous retinal detachment as a significant risk factor for SRF (adjusted OR 8.93, 95% CI 1.94-41.1). CONCLUSION: Patients with VKH syndrome who develop a bullous retinal detachment have an 8 times higher risk of developing SRF in the long term.