Subretinal Fibrosis Research Articles

Soluble very low-density lipoprotein receptor (sVLDLR) inhibits fibrosis in neovascular age-related macular degeneration.

Subretinal fibrosis is a key pathological feature in neovascular age‐related macular degeneration (nAMD). Previously, we identified soluble very low‐density lipoprotein receptor (sVLDLR) as an endogenous Wnt signaling inhibitor. This study investigates whether sVLDLR plays an anti‐fibrogenic role in nAMD models, including Vldlr −/− mice and laser‐induced choroidal neovascularization (CNV). We found that fibrosis factors including P‐Smad2/3, α‐SMA, and CTGF were upregulated in the subretinal area of Vldlr −/− mice and the laser‐induced CNV model. The antibody blocking Wnt co‐receptor LRP6 significantly attenuated the overexpression of fibrotic factors in these two models. Moreover, there was a significant reduction of sVLDLR in the interphotoreceptor matrix (IPM) in the laser‐induced CNV model. A transgenic strain (sVLDLR‐Tg) with sVLDLR overexpression in the IPM was generated. Overexpression of sVLDLR ameliorated the profibrotic changes in the subretinal area of the laser‐induced CNV model. In addition, Wnt and TGF‐β signaling synergistically promoted fibrogenesis in human primary retinal pigment epithelium (RPE) cells. CRISPR/Cas9‐mediated LRP6 gene knockout (KO) attenuated this synergistic effect. The disruption of VLDLR expression promoted, while the overexpression of sVLDLR inhibited TGF‐β‐induced fibrosis. These findings suggest that overactivated Wnt signaling enhances the TGF‐β pathway in subretinal fibrosis. sVLDLR confers an antifibrotic effect, at least partially, through the inhibition of Wnt signaling and thus, has therapeutic potential for fibrosis.

Three-Year Outcomes of Neovascular Age-Related Macular Degeneration in Eyes That Do Not Develop Macular Atrophy or Subretinal Fibrosis.

PurposeTo report the 36-month treatment outcomes of eyes with neovascular age-related macular degeneration (nAMD) receiving vascular endothelial growth factor (VEGF) inhibitors in daily practice who did not develop either subretinal fibrosis (SRFi) or macular atrophy (MA).MethodsThis is a retrospective analysis of data from the Fight Retinal Blindness registry. Treatment-naïve eyes starting intravitreal injection of VEGF inhibitors for nAMD from January 1, 2010, to September 1, 2017, and did not have SRFI and MA at baseline were tracked.ResultsWe identified 2478 eligible eyes, of which 1712 eyes did not develop SRFi or MA, 291 developed extrafoveal SRFI or MA, and 475 developed subfoveal SRFi or MA over 36 months. The estimated visual acuity stabilized from 6 months to 36 months in eyes that did not develop SRFI or MA with a mean (95% confidence interval [CI]) change in VA of −1 (−2, 0) letters, whereas eyes that developed extrafoveal (−3 [−5, −2] letters) or subfoveal (−10 [−11, −8] letters) SRFi or MA declined in vision in the same period. Eyes with no or extrafoveal SRFi or MA over 36 months were more likely to maintain their visual improvement from six months to 36 months (odds ratio [OR; 95% CI] = 2.3 [1.5, 3.3] for absence vs. subfoveal SRFi or MA, P ≤ 0.01 and OR = 2.0 [1.2, 3.4] for extrafoveal vs. subfoveal MA or SRFi, P = 0.01).ConclusionsTreatment-naïve nAMD eyes receiving VEGF inhibitors maintain their initial six-month visual improvement over three years if they do not develop SRFI or MA.Translational RelevanceThe nAMD is still a major cause of blindness despite antiangiogenic treatments. We found that eyes that did not develop subretinal fibrosis or macular atrophy maintained their initial vision improvement for at least three years, suggesting that identifying treatments for these complications is the final barrier to achieving excellent outcomes in nAMD.

MASSIVE ADVANCING NONEXUDATIVE TYPE 1 CHOROIDAL NEOVASCULARIZATION IN CTRP5 LATE-ONSET RETINAL DEGENERATION: Longitudinal Findings on Multimodal Imaging and Implications for Age-Related Macular Degeneration.

To describe longitudinal multimodal imaging findings of nonexudative choroidal neovascularization in CTRP5 late-onset retinal degeneration. Four patients with CTRP5-positive late-onset retinal degeneration underwent repeated ophthalmoscopic examination and multimodal imaging. All four patients (two siblings and their cousins, from a pedigree described previously) had the heterozygous S163R mutation. All four patients demonstrated large subretinal lesions in the mid-peripheral retina of both eyes. The lesions were characterized by confluent hypercyanescence with hypocyanescent borders on indocyanine green angiography, faintly visible branching vascular networks with absent/minimal leakage on fluorescein angiography, Type 1 neovascularization on optical coherence tomography angiography, and absent retinal fluid, consistent with nonexudative choroidal neovascularization. The neovascular membranes enlarged substantially over time and the birth of new membranes was observed, but all lesions remained nonexudative/minimally exudative. Without treatment, all involved retinal areas remained free of atrophy and subretinal fibrosis. We report the existence of massive advancing nonexudative Type 1 choroidal neovascularization in CTRP5 late-onset retinal degeneration. These findings have implications for age-related macular degeneration. They provide a monogenic model system for studying the mechanisms underlying the distinct events of choroidal neovascularization development, enlargement, progression to exudation, and atrophy in age-related macular degeneration. They suggest that choroidal hypoperfusion precedes neovascularization and that nonexudative neovascularization may protect against atrophy.

Associations and Outcomes of Patients with Submacular Hemorrhage Secondary to Age-related Macular Degeneration in the IVAN Trial

To compare demographics, visual acuity (VA) and retinal morphology between those with, and without baseline submacular hemorrhage (SMH) for patients enrolled in the Inhibit VEGF in Age-related Choroidal Neovascularization trial (IVAN). Secondary analyses of a randomized, controlled trial of image and clinical data. Setting; The IVAN trial collected data in 23 UK hospitals. Study population; IVAN study eyes (with untreated neovascular age-related macular degeneration at randomization) with at least 12 months of follow-up and adequate imaging. Intervention; Study eyes were randomly assigned between monthly ranibizumab, as-needed ranibizumab, monthly bevacizumab, or as-needed bevacizumab. Imaging at baseline was graded independently for the presence, type, position, and extent of SMH. Main outcome measures; The main outcome measures were VA (primary outcome), subretinal fibrosis, atrophic scarring, and retinal thickness outcomes at 12 and 24 months RESULTS: Of 605 IVAN trial participants, 535 were included in this analysis. Patients with SMH at baseline (286 [53%]) were older (P=.010) and affected eyes were more likely to have intraretinal fluid present (P=.038). The VA was significantly worse in those with baseline SMH at month 0 (P < .001; estimate of difference 6 letters; 95% CIs, 4-8 letters), but the difference decreased and was not significant at month 12 or 24. No significant association was found between baseline SMH and subretinal fibrosis, atrophic scarring, or central retinal thickness. The presence of SMH at baseline was associated with age, intraretinal fluid, and decreased baseline VA. By month 12, VA was no longer significantly different in those who presented with SMH at baseline.

Long-Term Outcomes of Bacillary Layer Detachment in Neovascular Age-Related Macular Degeneration

To evaluate the clinical characteristics, multimodal imaging features, and long-term treatment outcomes of eyes with neovascular age-related macular degeneration (nAMD) and bacillary layer detachment (BALAD) treated with intravitreal anti-VEGF therapy. Retrospective, longitudinal, case series. Treatment-naive patients with nAMD (n= 30) showing BALAD on OCT and undergoing anti-VEGF therapy. Clinical records and multimodal imaging results of up to 4 years after diagnosis were reviewed. Best-corrected visual acuity (BCVA) values were compared over time. The cumulative risk of and risk factors for subretinal fibrosis were assessed using Cox regression analyses, and adjusted hazard ratio (aHR) was computed. Thirty eyes of 30 patients were included. Macular neovascularization (MNV) subtypes were distributed as follows: type 1, 63%; type 2, 27%; mixed type 1 and 2, 3%; type 3, 3%; aneurysmal type 1, 3%. The BCVA significantly improved after anti-VEGF loading phase (Snellen equivalent, from 20 of 118 to 20 of 71, P= 0.03), but it returned to the baseline levels at 4 years (Snellen equivalent, 20 of 103, P= 0.6). The cumulative risk of subretinal fibrosis was 77% at 4 years. The risk factors associated with subretinal fibrosis included hemorrhagic BALAD (aHR, 2.02; 95% confidence interval [CI] 1.54-3.22; P < 0.01) and the presence of subretinal hyperreflective material (aHR, 1.83; 95% CI 1.35-3.14; P < 0.01). BALAD was found in association with all types of MNV in patients with nAMD. Long-term observation revealed poor functional outcomes related to the high risk of subretinal fibrosis.

Intravitreal injection of triptolide attenuates subretinal fibrosis in laser-induced murine model

BackgroundChoroidal neovascularization (CNV) is a common cause of irreversible blindness in elderly patients in developed countries, and subretinal fibrosis is an advanced stage of CNV. Currently, there is no effective clinical treatment for subretinal fibrosis. PurposeTo investigate whether intravitreal injection of triptolide (TP) could attenuate subretinal fibrosis and determine its underlying mechanisms. MethodsCNV was induced by laser photocoagulation in C57BL/6J mice. Immediately after laser photocoagulation, 1 μl of free TP (10 μg), TP-nanolip-PEG (TP-loaded PEGylated nanoliposomes containing 10 μg TP), or the same volume of phosphate-buffered saline (PBS) was intravitreally administered to each respective group. Areas and ratios of subretinal fibrosis were calculated seven days after laser injury. Additionally, expression levels of M2 macrophage-related markers, molecules of the transforming growth factor (TGF)-β1/Smad signaling pathway, and markers for epithelial-mesenchymal transition (EMT) and endothelial-to-mesenchymal transition (EndoMT) were detected both in vitro and in vivo. ResultsThe areas of subretinal fibrosis were significantly reduced in both the free TP (10993.87 ± 2416.90 μm2) and TP-nanolip-PEG (7695.32 ± 2121.91 μm2) groups when compared with the PBS group (15971.97 ± 3203.10 μm2) (p < 0.05, n = 6). The ratio of subretinal fibrosis in the free TP monomer (20.8 ± 4.2%) and TP-nanolip-PEG (12.5 ± 4.0%) groups was lower than that in the PBS control group (41.7 ± 5.3%) (p < 0.01, n = 6). Moreover, both TP and TP-nanolip-PEG suppressed the polarization of M2 macrophages and downregulated gene expressions of TGF-β1, Smad 2, Smad 3, α-SMA, and collagen I (p < 0.05), but upregulated the gene expression of E-cadherin (p < 0.05), thus reversing TGF-β1 induced EMT/EndoMT and attenuating subretinal fibrosis. ConclusionsTP could attenuate subretinal fibrosis by suppressing the polarization of M2 macrophages and TGF-β1 induced EMT/EndoMT. TP-nanolip-PEG enhanced the inhibitory effects of TP on subretinal fibrosis, suggesting its therapeutic potential for CNV-related subretinal fibrosis.

Bacillary layer detachment in acute nonpenetrating ocular trauma

ObjectiveTo study the clinical and visual outcomes of patients presenting with bacillary layer detachment (BLD) on optical coherence tomography (OCT) in blunt ocular trauma. DesignRetrospective observational study. ParticipantsClinical fundus photographs and OCT scans with Spectralis machine were reviewed to identify patients with blunt ocular trauma showing BLD. MethodsPatients were further analyzed for changes in their anatomic features such as subretinal hemorrhage, intrabacillary layer bleed. subretinal fibrosis and choroidal rupture and reattachment of BLD, and visual outcomes over subsequent follow-up visits. ResultsOf a total of 77 eyes with blunt ocular trauma, 6 (8%) eyes with BLD were identified. All patients were male with presenting visual acuity ranging from 6/9 to 2/60 (mean logMAR = 1.119; Snellen's equivalent = 20/263). The time interval between trauma and presentation ranged from 1 to 7 days. Subretinal hemorrhage and choroidal rupture were noted in all 6 eyes. On OCT, foveal involvement by the BLD was noted in 5 eyes. Intrabacillary layer hemorrhage was noted in all patients. Reattachment of the bacillary layer and visual acuity improvement were noted in all eyes at the final visit. The time interval for resolution of BLD ranged from 2 to 10 days. Visual acuity of 6/36 or less was secondary to subretinal fibrosis close to the fovea and subfoveal choroidal rupture. ConclusionBLD in blunt ocular trauma is a rare finding, shows complete resolution, and appears not to influence visual or anatomic outcome.

Identification and quantification of fibrotic areas in the human retina using polarization-sensitive OCT.

Subretinal fibrosis is one of the most prevalent causes of blindness in the elderly population, but a true gold standard to objectively diagnose fibrosis is still lacking. Since fibrotic tissue is birefringent, it can be detected by polarization-sensitive optical coherence tomography (PS-OCT). We present a new algorithm to automatically detect, segment, and quantify fibrotic lesions within 3D data sets recorded by PS-OCT. The algorithm first compensates for the birefringence of anterior ocular tissues and then uses the uniformity of the birefringent optic axis as an indicator to identify fibrotic tissue, which is then segmented and quantified. The algorithm was applied to 3D volumes recorded in 57 eyes of 57 patients with neovascular age-related macular degeneration using a spectral domain PS-OCT system. The results of fibrosis detection were compared to the clinical diagnosis based on color fundus photography (CFP), and the precision of fibrotic area measurement was assessed by three repeated measurements in a sub-set of 15 eyes. The average standard deviation of the fibrotic area obtained in eyes with a lesion area > 0.7 mm2 was 15%. Fibrosis detection by CFP and PS-OCT agreed in 48 cases, discrepancies were only observed in cases of lesion area < 0.7 mm2. These remaining discrepancies are discussed, and a new method to treat ambiguous cases is presented.

Double-layer sign in neovascular age-related macular degeneration - do we treat?

Neovascular age-related macular degeneration (nAMD) is managed medically with intravitreal anti-angiogenic therapy and monitored with optical coherence tomography (OCT) to detect exudation in retinal layers. One OCT feature suggesting early-stage macular neovascularization of choroidal origin (type 1 MNV) is two hyperreflective bands separated by a narrow hyporeflective band, called ‘double-layer sign’ (DLS). We report a clinicopathologic correlation of DLS. Comprehensive ophthalmologic examination and multimodal imaging were performed during a 9-year follow-up in the pseudophakic right eye of a woman presenting at age of 79 years with inactive subretinal fibrosis due to AMD and best-corrected visual acuity (BCVA) of 20/50. Two years later (7 years before death), BCVA was 20/200. Near-infrared reflectance (Figure 1A, left) with corresponding OCT B-scan through the fovea and optic nerve head (Figure 1A, right) shows a DLS nasal to a central fibrovascular scar (pink, orange and turquoise arrowheads) along with outer retinal tubulation (ORT, white arrowhead). These structures appear in a registered histologic section (Figure 1B). In the magnified OCT (Figure 1C), the upper hyperreflective band of the DLS (yellow arrowheads) corresponds to the RPE-basal lamina (RPE-BL) complex and is irregularly thickened. The lower hyperreflective band corresponds to Bruch’s membrane (BrM). Histology shows that the hyporeflective line separating these bands to create the DLS may correspond to type 1 MNV beneath basal laminar deposit (BLamD), a thickened extracellular matrix material (Figure D1; D2; E1 fuchsia asterisk, E2) or just thick BLamD without type 1 MNV (Figure 1D3, 1E2). With a 7-year interval between OCT and histology, BLamD was likely thinner at the time of imaging; however, a DLS was already visible at that early time-point. Our clinicopathologic correlation reinforces that while minimizing retinal exudation is critical to preserving photoreceptors, a DLS on OCT can indicate BLamD without type 1 MNV, which would not benefit from anti-angiogenic therapy.

Inhibition of epithelial\u2013mesenchymal transition in retinal pigment epithelial cells by a retinoic acid receptor-\u03b1 agonist

Epithelial–mesenchymal transition (EMT) in retinal pigment epithelial (RPE) cells plays a key role in proliferative retinal diseases such as age-related macular degeneration by contributing to subretinal fibrosis. To investigate the potential role of retinoic acid receptor-α (RAR-α) signaling in this process, we have now examined the effects of the RAR-α agonist Am580 on EMT induced by transforming growth factor-β2 (TGF-β2) in primary mouse RPE cells cultured in a three-dimensional type I collagen gel as well as on subretinal fibrosis in a mouse model. We found that Am580 inhibited TGF-β2-induced collagen gel contraction mediated by RPE cells. It also attenuated the TGF-β2-induced expression of the mesenchymal markers α-smooth muscle actin, fibronectin, and collagen type I; production of pro-matrix metalloproteinase 2 and interleukin-6; expression of the focal adhesion protein paxillin; and phosphorylation of SMAD2 in the cultured RPE cells. Finally, immunofluorescence analysis showed that Am580 suppressed both the TGF-β2-induced translocation of myocardin-related transcription factor-A (MRTF-A) from the cytoplasm to the nucleus of cultured RPE cells as well as subretinal fibrosis triggered by laser-induced photocoagulation in a mouse model. Our observations thus suggest that RAR-α signaling inhibits EMT in RPE cells and might attenuate the development of fibrosis associated with proliferative retinal diseases.

Long-Term Outcome of Pars Plana Vitrectomy for Retained Posterior Segment Intraocular Foreign Body Secondary to Gunshot Injury.

PurposeTo report the long-term outcome of pars plana vitrectomy (PPV) for management of retained posterior segment intraocular foreign body (IOFB) secondary to gunshot injury.MethodsThis is a retrospective interventional case series including consecutive patients who had PPV for retained posterior segment IOFB secondary to gunshot injury. Main outcome measures were final best-corrected visual acuity (BCVA), long-term globe survival and detection of complications. Spearman correlation analyzed relationships between numerical data. Kruskal–Wallis test compared differences in initial BCVA and final BCVA across variables. Categorical variables were tested using Chi square or Fisher’s exact test. P value is significant at 0.05.ResultsThe study included 103 eyes of 103 patients. Mean baseline BCVA was 0.01 decimal unit (2 logMAR). Mean duration from primary repair to PPV was 3 weeks. Mean duration of post-operative follow-up was 60 months. Mean final BCVA was 0.04 decimal unit (1.3 logMAR), p 0.001. Post-operatively, BCVA improved in 58.2% of patients. Nineteen patients (18%) gained ≥2 lines of vision, and 15 patients (14.5%) achieved final BCVA of 0.4 decimal unit (logMAR 0.4). All complications were related to the original injury. These included macular scar (19%), macular pucker (6%), recurrent retinal detachment (4%), subretinal fibrosis (3%), consecutive optic atrophy (3%), and PVR (3%). Phthisis bulbi or sympathetic ophthalmia did not develop in any case.ConclusionPPV for removal of IOFB caused by gunshot injury yielded long-term favorable functional outcome with excellent globe survival. Poor initial BCVA, location of IOFB in the posterior pole, associated lens injury and retinal detachment are significant adverse prognostic factors for final BCVA but not for globe survival.

Metabolic Reprogramming of the Retinal Pigment Epithelium Drives TGFβ2‐Induced Epithelial‐Mesenchymal Transition

Transforming growth factor-beta 2 (TGFβ2) is a key orchestrator of retinal wound healing through induction of epithelial-mesenchymal-transition (EMT) in retinal pigment epithelial cells (RPE). Here we describe a previously unrecognized function of TGFβ2 in modulating mitochondrial morphology and metabolic function in human RPE cells. Treating ARPE-19 (human RPE cell line) with TGFβ2 (10 ng/ml) induced defects in mitochondrial network integrity with increased sphericity and fragmentation. Correspondingly, TGFβ2 reduced expression of genes regulating mitochondrial dynamics, reduced citrate synthase activity and intracellular ATP content. High-resolution respirometry showed that TGFβ2 reduced mitochondrial oxidative phosphorylation (OXPHOS) levels consistent with reduced expression of NDUFB5, a key gene of Complex I of the electron transport chain. The reduced mitochondrial respiration was associated with a compensatory increase in gene expression of glycolytic enzymes (PFKFB3, PKM2, LDHA) and glycolytic reserve. TGFβ2 induced a severe suppression of PGC-1α gene expression and treatment with the selective small molecule activator of PGC-1α, ZLN005, blocked TGFβ2-induced upregulation of mesenchymal genes (αSMA, Snai1, CTGF, COL1A1) and TGFβ2-induced migration using the scratch wound assay. Our data show that EMT is accompanied by mitochondrial dysfunction and a profound metabolic shift towards reduced OXPHOS and increased glycolysis that may be driven by PGC-1α suppression. The PGC-1α promoter, ZLN005, effectively blocks EMT in RPE and thus serves as a novel therapeutic avenue for treatment for subretinal fibrosis.

Suppression of PGC-1α Drives Metabolic Dysfunction in TGFβ2-Induced EMT of Retinal Pigment Epithelial Cells.

PGC-1α, a key orchestrator of mitochondrial metabolism, plays a crucial role in governing the energetically demanding needs of retinal pigment epithelial cells (RPE). We previously showed that silencing PGC-1α induced RPE to undergo an epithelial-mesenchymal-transition (EMT). Here, we show that induction of EMT in RPE using transforming growth factor-beta 2 (TGFβ2) suppressed PGC-1α expression. Correspondingly, TGFβ2 induced defects in mitochondrial network integrity with increased sphericity and fragmentation. TGFβ2 reduced expression of genes regulating mitochondrial dynamics, reduced citrate synthase activity and intracellular ATP content. High-resolution respirometry showed that TGFβ2 reduced mitochondrial OXPHOS levels consistent with reduced expression of NDUFB5. The reduced mitochondrial respiration was associated with a compensatory increase in glycolytic reserve, glucose uptake and gene expression of glycolytic enzymes (PFKFB3, PKM2, LDHA). Treatment with ZLN005, a selective small molecule activator of PGC-1α, blocked TGFβ2-induced upregulation of mesenchymal genes (αSMA, Snai1, CTGF, COL1A1) and TGFβ2-induced migration using the scratch wound assay. Our data show that EMT is accompanied by mitochondrial dysfunction and a metabolic shift towards reduced OXPHOS and increased glycolysis that may be driven by PGC-1α suppression. ZLN005 effectively blocks EMT in RPE and thus serves as a novel therapeutic avenue for treatment of subretinal fibrosis.

Recognizable Patterns of Submacular Fibrosis in Enhanced S-Cone Syndrome

To highlight recognizable patterns of subretinal fibrosis in enhanced S-cone syndrome (ESCS). Retrospective case series. Forty-seven patients with subretinal fibrosis identified from 101 patients with clinically diagnosed ESCS, confirmed by full-field electroretinography (35/47), genetic testing (34/47), or both. Multimodal retinal imaging, electroretinography, and genetic analysis. Patterns of subretinal fibrosis with angiographic, OCT, and genetic correlations. Eighty-five eyes of 47 patients (24 male patients; 36 unrelated consanguineous families) had subretinal fibrosis. Mean age at presentation was 14 years. Best-corrected visual acuity ranged from 20/20 to hand movements. All 34 genetically tested patients were homozygous for pathogenic NR2E3 variants. Subretinal fibrosis was always in the macular area, although it extended beyond in some patients. Six recurrent patterns of submacular fibrosis were noted: central unifocal nodular, circumferential unifocal nodular, multifocal nodular, arcuate, helicoid, and thick geographic. Some patients showed a combination of patterns. Previous misdiagnosis as inflammatory disease was common. Fibrosis was fairly symmetrical in a given patient but not always present or identical in other affected individuals with a given homozygous mutation from the same or other families. These recognizable patterns of submacular fibrosis are part of the ESCS phenotypic spectrum and strongly suggest the disease. In addition to facilitating diagnosis, recognition of these patterns can spare patients unnecessary workup for an inflammatory cause.

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

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). RPE cells (ARPE-19 cell line) were treated with transforming growth factor-β1 (TGF-β1) to induce EMT. Changes of the mRNA and protein expression levels of LRG1 were tested in the TGF-β1 treated cells. The recombinant human LRG1 protein (rLRG1) and siRNA 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 mRNA and protein expression level of NOX4 were measured according to the above treatments. VAS2870 was used as a NOX4 inhibitor in rLRG1-treated cells. EMT-related markers were detected to verify the effect of NOX4 in the process of EMT. TGF-β1 promoted the expression of LRG1 at both the mRNA and protein levels during the process of EMT which showed the up-regulation of fibronectin and α-SMA, as well as the down-regulation of ZO-1. Furthermore, the rLRG1 promoted EMT of ARPE-19 cells, which manifested high levels of fibronectin and α-SMA and low level of ZO-1, whereas knockdown of LRG1 prevented EMT by decreasing the expressions of fibronectin and α-SMA and increasing the expression of ZO-1 in ARPE-19 cells. Besides, the rLRG1 activated and LRG1 siRNA suppressed NOX4 expression. EMT was inhibited when VAS2870 was used in the rLRG1-treated cells. 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.

Different Phenotypes in Pseudodominant Inherited Retinal Dystrophies.

Retinal dystrophies (RD) are a group of Mendelian disorders caused by rare genetic variations leading to blindness. A pathogenic variant may manifest in both dominant or recessive mode and clinical and genetic heterogeneity makes it difficult to establish a precise diagnosis. In this study, families with autosomal dominant RD in successive generations were identified, and we aimed to determine the disease's molecular origin in these consanguineous families. Whole exome sequencing was performed in the index patient of each family. The aim was to determine whether these cases truly represented examples of dominantly inherited RD, or whether another mode of inheritance might be applicable. Six potentially pathogenic variants in four genes were identified in four families. In index patient with enhanced S-cone syndrome in F1, we identified a new digenetic combination: a heterozygous variant p.[G51A];[=] in RHO and a homozygous pathogenic variant p.[R311Q];[R311Q] in NR2E3. Helicoid subretinal fibrosis associated with recessive NR2E3 variant p.[R311Q];[R311Q] was identified in F2. A new frameshift variant c.[105delG];[105delG] in RDH12 was found in F3 with cone-rod dystrophy. In F4, the compound heterozygous variants p.[R964*];[W758*] were observed in IMPG2 with a retinitis pigmentosa (RP) phenotype. We showed that both affected parents and the offspring, were homozygous for the same variants in all four families. Our results provide evidence that in consanguineous families, autosomal recessive can be transmitted as pseudodominant inheritance in RD patients, and further extend our knowledge of pathogenic variants in RD genes.

Fundus Autofluorescence in Neovascular Age-Related Macular Degeneration: A Clinicopathologic Correlation Relevant to Macular Atrophy

Macular atrophy (MA) of retinal pigment epithelium (RPE) and photoreceptors leads to vision loss in neovascular age-related macular degeneration (nAMD) despite successful treatment with antiangiogenic agents. To enhance understanding of MA, fortify the cellular basis of fundus autofluorescence (FAF) imaging, and inform management of nAMD, we performed histologic analysis of an eye with multimodal clinical imaging and apparent prior exudation due to nAMD. Case study and clinicopathologic correlation. A White woman in whom age-related macular degeneration (AMD) findings of inactive subretinal fibrosis (right eye) were followed for 9 years using FAF and OCT, with no detectable subretinal fluid or other recurrent exudation and no intravitreal injections before her death at age 90 years. The right eye was preserved 6.25 hours after death, postfixed in osmium tannic acid paraphenylenediamine, and prepared for submicrometer epoxy resin sections (n = 115), with 19 matched to clinical OCT B-scans. Light microscopic morphology of a hyperautofluorescent (hyperFAF) area due to prior exudation ("floodplain" hyperFAF), hypoautofluorescent (hypoFAF) spots of MA, and areas of unremarkable FAF. Floodplain hyperFAF was visible throughout the 9 years of follow-up, with several hypoFAF atrophic spots expanding within it over time. The hyperFAF pattern corresponded to outer retinal atrophy (ORA) on OCT and photoreceptor loss over dysmorphic yet continuous RPE in histology. The hypoFAF spots inside the floodplain corresponded to complete RPE and outer retinal atrophy (cRORA) on OCT and loss of both photoreceptors and RPE in histology. In contrast, areas of unremarkable FAF showed continuous RPE accompanied by full-length photoreceptors and a thick outer nuclear layer. This direct clinicopathologic correlation for FAF imaging is the first for nAMD. Fundus autofluorescence is a projection image that involves optical signal modulation by photoreceptors as well as emission signal sources in RPE. Hyperautofluorescence due to an exudative floodplain signifies loss of photoreceptors over continuous RPE. Hypoautofluorescence in MA signifies loss of both cell layers. For maximal value, fundus autofluorescence imaging should be interpreted with the multilayer perspective provided by OCT. Prevention of exudation in nAMD may preserve photoreceptors.

Strongest Correlation Between Contrast Sensitivity and Morphological Characteristics in Bilateral nAMD.

In patients with neovascular age-related macular degeneration (nAMD) there is often an inconsistency between their subjective visual impairment and a still relatively preserved standard Early Treatment of Diabetic Retinopathy Study (ETDRS) best corrected visual acuity. Therefore, in order to better capture the specific functional defects in nAMD, other tests need to be evaluated. In a previous study, we reported contrast sensitivity of the better eye to best correlate with near distance and distance vision related quality of life in patients with bilateral nAMD. Here, we evaluated Pelli-Robson contrast sensitivity, ETDRS visual acuity, low luminance visual acuity and Radner maximum reading speed and correlated them with several morphologic parameters as measured on fundus autofluorescence imaging, optical coherence tomography and optical tomography angiography in 54 patients. A multiple regression analysis was performed which correlated each visual function parameter with the anatomic features. The results showed the strongest correlations between the total area of macular geographic atrophy as well as the percentage of geographic atrophy in the central 1 mm and contrast sensitivity. Further, the regression model selected the total area of macular geographic atrophy, the photoreceptor inner and outer segments interface disruption score, the presence of subretinal fibrosis in the central 1 mm and the central retinal thickness as the variables that explained 71% of the variation in contrast sensitivity when including all eyes. Hence, our results suggest that among the evaluated measures of vision, contrast sensitivity is best correlated with the morphologic impairment in bilateral nAMD. Thus, contrast sensitivity may complement ETDRS visual acuity in clinical trials and serve as a standard diagnostic tool in clinical practice.

OUTER RETINAL TUBULATION MAY RESULT FROM FIBROSED TYPE 2 NEOVASCULARIZATION: Clinical Observations and Model of Pathogenesis.

To investigate the role of Type 2 macular neovascularization with subsequent subretinal fibrosis in the pathogenesis of outer retinal tubulation (ORT). We conducted a retrospective cohort study of patients with stabilized inactive exudative macular degeneration who had been treated with intravitreal injections of antivascular endothelial growth factor agents. Baseline fluorescein and optical coherence tomography images were included. Macular neovascularizations (MNVs) were classified by type and size. Consecutive optical coherence tomography images analyzed for ORT development. One hundred forty-four eyes of 134 patients were included in this study. Sixty eyes presented with pure Type 1 MNV. Eighty-four eyes presented with some Type 2 component of MNV. In total, evidence of ORT is shown in 55 (38%) eyes. In the Type 1 group, 6.7% developed ORT. Outer retinal tubulation developed in 61% of eyes with some Type 2 component of the MNV. Among eyes that developed ORT, 92.7% presented with some Type 2 component. In a multivariate analysis, Type 2 membranes on optical coherence tomography (22.2 [6.1-80.8]; P < 0.001), larger MNV size {>1 DA (5.1 [1.1-24.2]; P = 0.041) and >1.5 DA (9.0 [1.8-44.0]; P = 0.007)}, and presence of subretinal fibrovascular material (3.1 [1.1-8.5]; P = < 0.03) are associated with higher odds of ORT formation. Once the ORT is formed, fibrosis was observed directly underlying the ORT on SD-optical coherence tomography in 70.9% of cases. Type 2 membranes at presentation predict ORT formation. Fibrosis often underlies ORT. This suggests that contraction of Type 2 MNV-derived fibrosis may be important in ORT formation.

The HIF-1α/p53/miRNA-34a/Klotho axis in retinal pigment epithelial cells promotes subretinal fibrosis and exacerbates choroidal neovascularization.

Wet age‐related macular degeneration (wAMD), characterized by choroidal neovascularization (CNV), is a leading cause of irreversible vision loss among elderly people in developed nations. Subretinal fibrosis, mediated by epithelial‐mesenchymal transition (EMT) of retinal pigment epithelium (RPE) cells, leads to unsuccessful anti‐vascular endothelial growth factor (VEGF) agent treatments in CNV patients. Under hypoxic conditions, hypoxia‐inducible factor‐1α (HIF‐1α) increases the stability and activation of p53, which activates microRNA‐34a (miRNA‐34a) transcription to promote fibrosis. Additionally, Klotho is a target gene of miRNA‐34a that inhibits fibrosis. This study aimed to explore the role of the HIF‐1α/p53/miRNA‐34a/Klotho axis in subretinal fibrosis and CNV. Hypoxia‐induced HIF‐1α promoted p53 stability, phosphorylation and nuclear translocation in ARPE‐19 cells (a human RPE cell line). HIF‐1α‐dependent p53 activation up‐regulated miRNA‐34a expression in ARPE‐19 cells following hypoxia. Moreover, hypoxia‐induced p53‐dependent miRNA‐34a inhibited the expression of Klotho in ARPE‐19 cells. Additionally, the HIF‐1α/p53/miRNA‐34a/Klotho axis facilitated hypoxia‐induced EMT in ARPE‐19 cells. In vivo, blockade of the HIF‐1α/p53/miRNA‐34a/Klotho axis alleviated the formation of mouse laser‐induced CNV and subretinal fibrosis. In short, the HIF‐1α/p53/miRNA‐34a/Klotho axis in RPE cells promoted subretinal fibrosis, thus aggravating the formation of CNV.