Exosomal lncRNA-MIAT promotes neovascularization via the miR-133a-3p/MMP-X1 axis in diabetic retinopathy.

Diabetic retinopathy (DR), a most common microangiopathy of diabetes, causes vision loss and even blindness. The mechanisms of exosomal lncRNA remain unclear in the development of DR. Here, we first identifed the pro-angiogenic effect of exosomes derived from vitreous humor of proliferative diabetic retinopathy patients, where lncRNA-MIAT was enriched inside. Secondly, lncRNA-MIAT was demonstrated significantly increased in exosomes from high glucose induced human retinal vascular endothelial cell, and can regulate tube formation, migration and proliferation ability to promote angiogenesis in vitro and in vivo. Mechanistically, the pro-angiogenic effect of lncRNA-MIAT was via the lncRNA-MIAT/miR-133a-3p/MMP-X1 axis. The reduced level of lncRNA-MIAT in this axis mitigated the generation of retinal neovascular in mouse model of oxygen-induced retinopathy (OIR), providing crucial evidence for lncRNA-MIAT as a potential clinical target. These findings enhance our understanding of the role of exosomal lncRNA-MIAT in retinal angiogenesis, and propose a promising therapeutic strategy against diabetic retinopathy.

Human lens epithelial-secreted exosomes attenuate ocular angiogenesis via inhibiting microglial activation.

The lens is an avascular tissue, where epithelial cells (LECs) are the primary living cells. The role of LECs-derived exosomes (LEC-exos) is largely unknown. In our study, we determined the anti-angiogenic role of LEC-exos, manifested as regressed retinal neovascularization (NV) using the oxygen-induced retinopathy (OIR), and reduced choroidal NV size and pathological vascular leakage using the laser-induced choroidal neovascularization (laser-induced CNV). Furthermore, the activation and accumulation of microglia were also restricted by LEC-exos. Based on Luminex multiplex assays, the expressions of chemokines such as SCYB16/CXCL16, MCP-1/CCL2, I-TAC/CXCL11, and MIP 3beta/CCL19 were decreased after treatment with LEC-exos. Transwell assays showed that LEC-exos restricted the migration of the mouse microglia cell line (BV2 cells). After incubation with LEC-exos-treated BV2 cells, human umbilical vein endothelial cells (hUVECs) were collected for further evaluation using tube formation, Transwell assays, and 5-ethynyl-2'-deoxyuridine (EDU) assays. Using in vitro experiments, the pro-angiogenic effect of microglia was restricted by LEC-exos. Hence, it was investigated that LEC-exos attenuated ocular NV, which might attribute to the inhibition of microglial activation and accumulation.

Lens epithelial cell-derived exosome inhibits angiogenesis in ocular pathological neovascularization through its delivery of miR-146a-5p.

Abnormal ocular neovascularization, a major pathology of eye diseases, leads to severe visual loss. The role of lens epithelial cell (LEC)-derived exosomes (Lec-exo) is largely unknown. Thus, we aimed to investigate whether Lec-exo can inhibit abnormal ocular neovascularization and explore the possible mechanisms. In our study, we proved the first evidence that exosomes derived from LECs attenuated angiogenesis in both oxygen-induced retinopathy and laser-induced choroidal neovascularization mice models. Further in vitro experiments proved that Lec-exo inhibited proliferation, migration, and tube formation capability of human umbilical vein endothelial cells in high glucose condition. Further high-throughput miRNAs sequencing analysis detected that miR-146a-5p was enriched in Lec-exo. Mechanistically, exosomal miR-146a-5p was delivered to endothelial cells and bound to the NRAS coding sequence, which subsequently inactivated AKT/ERK signaling pathway. We successfully elucidated the function of Lec-exo in inhibiting abnormal ocular neovascularization, which may offer a promising strategy for treatment of abnormal ocular neovascularization.

tRNA-derived fragment tRF-30 propels diabetes-induced retinal microvascular complications by regulating STAT3 signaling.

Transfer RNA-derived fragments (tRFs) represent a novel class of non-coding RNA transcripts that possess specific biological functions. However, the involvement of tRFs in retinal microvascular diseases remains poorly understood. In this study, we aimed to reveal whether modulation of tRF-30 expression could attenuate pathological retinal neovascular diseases. Our findings demonstrate a significant upregulation of tRF-30 expression levels in both in vivo models of diabetic retinopathy (DR) and in vitro endothelial sprouting models. Conversely, inhibition of tRF-30 expression suppressed the formation of abnormal neovascularization in the retina in vivo, while reducing the proliferation and migration activity of retinal vascular endothelial cells in vitro. We also found that tRF-30 modulates retinal neovascularization through the tRF-30/TRIB3/signal transducer and activated transcription 3 signaling pathway. Furthermore, we validated a significant upregulation of tRF-30 expression levels in the vitreous humor of DR patients, with high levels of both validity and specificity in diagnostic testing. Collectively, our findings highlight a pro-angiogenic role for tRF-30 in DR. Intervening in the tRF-30 signaling pathway may represent a promising prevention and treatment strategy for retinal angiogenesis.

Epigenetic regulation in the commitment of progenitor cells during retinal development and regeneration.

Retinal development is initiated by multipotent retinal progenitor cells, which undergo several rounds of cell divisions and subsequently terminal differentiation. Retinal regeneration is usually considered as the recapitulation of retinal development, which share common mechanisms underlying the cell cycle re-entry of adult retinal stem cells and the differentiation of retinal neurons. However, how proliferative retinal progenitor cells perform a precise transition to postmitotic retinal cell types during the process of development and regeneration remains elusive. It is proposed that both the intrinsic and extrinsic programming are involved in the transcriptional regulation of the spatio-temporal fate commitment. Epigenetic modifications and the regulatory mechanisms at both DNA and chromatin levels are also postulated to play an important role in the timing of differentiation of specific retinal cells. In the present review, we have summarized recent knowledge of epigenetic regulation that underlies the commitment of retinal progenitor cells in the settings of retinal development and regeneration.

A novel model of subretinal edema induced by DL-alpha aminoadipic acid.

In this study we described a new model of subretinal edema induced by single intraocular injection of DL-alpha-aminoadipic acid (DLAAA) that can be employed to study the mechanism of retinal edema and test the efficacy or potential toxicity of treatments. The progression of subretinal edema was evaluated by fundus photography, fluorescein angiography and optical coherence tomography for up to 4 weeks following DLAAA injection. The VEGF, IL-6, TNF-α, Occludin, ZO-1, AQP4, Kir4.1, GFAP and GS levels were examined in DLAAA models by immunostaining, immumohistochemical staining and Western blot. Additionally, bulk RNA-seq was used to detect the mechanism involved in DLAAA-induced retinal Müller cellular injuries. In vivo and vitro assays were further conducted to confirm the sequencing results. Subretinal edema was successfully induced by DLAAA in New Zealand White rabbits (1.29 mg/eye) and C57BL/6 mice (50 or 100 µg/eye). Our results demonstrated that the disruption of blood-retinal-barrier, including vascular hyperpermeability, inflammation, and Müller cell dysfunction of fluid clearance, was involved in subretinal edema formation in the model. Bulk RNA-seq and in vitro studies indicated the activation of p38 MAPK signaling pathway in DLAAA models. This DLAAA-induced subretinal edema model can be used for mechanistic studies or drug screening.

JP1, a polypeptide specifically targeting integrin αVß3, ameliorates choroidal neovascularization and diabetic retinopathy in mice.

Anti-vascular endothelial growth factor (VEGF) drugs have revolutionized the treatment of neovascular eye diseases, but responses are incomplete in some patients. Recent evidence shows that integrins are involved in the pathogenesis of neovascular age-related macular degeneration and diabetic retinopathy. JP1, derived from an optimized seven-amino-acid fragment of JWA protein, is a polypeptide specifically targeting integrin αVß3. In this study we evaluated the efficacy of JP1 on laser-induced choroidal neovascularization (CNV) and retinal vascular leakage. CNV mice received a single intravitreal (IVT) injection of JP1 (10, 20, 40 µg) or ranibizumab (RBZ, 10 µg). We showed that JP1 injection dose-dependently inhibited laser-induced CNV; the effect of RBZ was comparable to that of 20 µg JP1; a combined IVT injection of JP1 (20 µg) and RBZ (5 µg) exerted a synergistic effect on CNV. In the 3rd month after streptozotocin injection, diabetic mice receiving IVT injection of JP1 (40 µg) or RBZ (10 µg) once a week for 4 weeks showed significantly suppressed retinal vascular leakage. In both in vivo and in vitro experiments, JP1 counteracted oxidative stress and inflammation via inhibiting ROS/NF-κB signaling in microglial cells, and angiogenesis via modulating MEK1/2-SP1-integrin αVß3 and TRIM25-SP1-MMP2 axes in vascular endothelial cells. In addition, intraperitoneal injection of JP1 (1, 5 or 10 mg) once every other day for 3 times also dose-dependently inhibited CNV. After intraperitoneal injection of FITC-labeled JP1 (FITC-JP1) or FITC in laser-induced CNV mice, the fluorescence intensity in the CNV lesion was markedly increased in FITC-JP1 group, compared with that in FITC group, confirming that JP1 could penetrate the blood-retinal barrier to target CNV lesion. We conclude that JP1 can be used to design novel CNV-targeting therapeutic agents that may replace current invasive intraocular injections.

Initial screening for occult congenital ectopia lentis based on ocular biological parameters in preschool children.

BACKGROUND: This study aimed to identify an initial screening tool for congenital ectopia lentis (CEL) by comparing ocular biological parameters in children with myopia. METHODS: A retrospective case-control study was conducted at one tertiary referral centre, from October 2020 to June 2022. Axial length (AL), corneal curvature (CC), refractive astigmatism (RA), corneal astigmatism (CA), internal astigmatism (IA), the difference between the axis of RA and CA [AXIS(RA-CA)], white-to-white corneal diameter (WTW), and axial length-corneal radius ratio (AL/CR) were compared in 28 eyes of CEL patients, and 60 eyes of myopic patients matched for age and refraction. The spherical equivalent of each eye was < -3.00 D. Area under the curve (AUC) of the receiver operating characteristic curves were calculated. RESULTS: The differences in RA, AL, mean keratometry (Kmed), maximum keratometry (Kmax), minimum keratometry (Kmin), CA, IA, AXIS(RA-CA), WTW, and AL/CR between the CEL and myopic groups were statistically significant (p < 0.05; p < 0.001; p < 0.001; p < 0.001; p < 0.001; p < 0.05; p < 0.001; p < 0.001; p < 0.001; p < 0.001, respectively). In logistic regression analysis RA, IA, AXIS(RA-CA), and AL/CR were significantly associated with CEL (p < 0.05). AUCs for RA, IA, AXIS(RA-CA), and AL/CR were 0.694, 0.853, 0.814, and 0.960, respectively. AUCs for AL/CR in SE< -6.00 D subgroup was 0.970, and 0.990 in -6.00 D ≤ SE < -3.00 D group. An AL/CR < 3.024 was the optimal cut-off point differentiating the CEL and control groups (sensitivity, 92.9%; specificity, 88.30%). CONCLUSIONS: A smaller AL/CR could identify CEL in children with myopia. An AL/CR cut-off value of 3.024 may be the most sensitive and specific parameter for the differential diagnosis of CEL in patients with mild to high myopia.

Effectiveness and safety of tafluprost in primary open-angle glaucoma and ocular hypertension: a post-marketing phase IV study in China.

BACKGROUND: Prostaglandin analogs (PGAs) are the first-line treatment for primary open-angle glaucoma (POAG) and ocular hypertension (OH). This study aimed to confirm the effectiveness and safety of Tapros® (0.0015% tafluprost eye drops) in Chinese patients with POAG and OH. METHODS: This phase IV, multicenter, non-comparative, prospective study enrolled patients with POAG and OH in China between 12/27/2017 and 04/15/2020. Patients who were treatment-naïve or untreated within one month (group A) or with unreached intraocular pressure (IOP) target after previous monotherapy of other PGAs (group B) or non-PGA IOP-lowering drugs (group C) were treated with 0.0015% tafluprost for three months. The IOP reduction, response rate, and safety were observed. RESULTS: There were 165, 89, and 31 patients in groups A, B, and C, with baseline IOPs of 22.4 ± 4.7, 21.0 ± 3.5, and 22.5 ± 3.2 mmHg, respectively. The least-square means and percentages of IOP reduction at 3 months for groups A, B, and C were 4.7 (19.8%), 1.6 (6.1%), and 4.6 mmHg (20.3%), respectively. A significant reduction in IOP was observed at each visit compared with baseline (all P < 0.05). At the final visit, 57.0% of the participants in group A achieved an IOP reduction of ≥ 20%, while 40.4% and 77.4% in groups B and C achieved an IOP reduction of ≥ 10%. Fifty-eight treatment-related adverse events occurred in 46 participants (15.7%), of which the most common one was conjunctival hyperemia (34/293, 11.6%). CONCLUSIONS: Tafluprost showed a sustained and significant effect with tolerable adverse events in Chinese patients with POAG and OH who were treatment-naïve or untreated within one month or received prior treatments with unsatisfying outcomes.

Circular Noncoding RNA NR3C1 Acts as a miR-382-5p Sponge to Protect RPE Functions via Regulating PTEN/AKT/mTOR Signaling Pathway.

Age-related macular degeneration (AMD) is a universal leading cause for irreversible blindness in the elderly population. Dedifferentiation of retinal pigment epithelium (RPE) cells initiates early pathological events in atrophic AMD. Herein, we aim to investigate effects of a circular RNA derived from the NR3C1 gene (circNR3C1) on regulating RPE function and AMD pathogenesis. circNR3C1 expression was consistently upregulated along with RPE differentiation and was downregulated in dysfunctional RPE and blood serum of AMD patients. Silencing of circNR3C1 reduced RPE characteristic transcripts and proteins, interrupted phagocytosis, accelerated intracellular reactive oxygen species (ROS) generation, and promoted RPE proliferation in vitro. circN3C1 silencing also decreased expressions of RPE characteristic markers and disturbed the ultrastructure of RPE in vivo, as shown by a thickened RPE with twisted basal infoldings and outer segments. Mechanistically, circNR3C1 acted as an endogenous microRNA-382-5p (miR-382-5p) sponge to sequester its activity, which increased phosphatase and tensin homolog on chromosome 10 (PTEN) expression and inhibited the protein kinase B/mammalian target of rapamycin (AKT/mTOR) pathway. miR-382-5p overexpression and PTEN silencing mimicked effects of circNR3C1 silencing on RPE phenotypes in vivo and in vitro. In conclusion, circNR3C1 prevents AMD progression and protects RPE by directly sponging miR-382-5p to block its interaction with PTEN and subsequently blocks the AKT/mTOR pathway. Pharmacological circNR3C1 supplementations are promising therapeutic options for atrophic AMD.

Vitamin C- and Valproic Acid-Induced Fetal RPE Stem-like Cells Recover Retinal Degeneration via Regulating SOX2.

Retinal pigment epithelial (RPE) cell replacement therapy has provided promising outcomes in the treatment of retinal degenerative diseases (RDDs), but the resulting limited visual improvement has raised questions about graft survival and differentiation. Through combined treatment with vitamin C and valproic acid (together, VV), we activated human fetal RPE (fRPE) cells to become highly proliferative fetal RPE stem-like cells (fRPESCs). In this study, we report that SOX2 (SRY-box 2) activation contributed to mesenchymal-epithelial transition and elevated the retinal progenitor and mesenchymal stromal markers expressions of fRPESCs. These fRPESCs could differentiate into RPE cells, rod photoreceptors, and mesenchymal lineage progenies under defined conditions. Finally, fRPESCs were transplanted into the subretinal space of an RDD mouse model, and a photoreceptor rescue benefit was demonstrated. The RPE and rod photoreceptor differentiation of transplanted fRPESCs may account for the neural retinal recovery. This study establishes fRPESCs as a highly proliferative, multi-lineage differentiation potential (including RPE, rod photoreceptor, and mesenchymal lineage differentiation), mesenchymal-to-epithelial-transitioned retinal stem-like cell source for cell-based therapy of RDDs.

PERFLUOROCARBON LIQUID-ASSISTED INVERTED INTERNAL LIMITING MEMBRANE FLAP TECHNIQUE VERSUS INTERNAL LIMITING MEMBRANE PEELING FOR HIGHLY MYOPIC MACULAR HOLE RETINAL DETACHMENT.

PURPOSE: To compare the efficacy of a modified perfluorocarbon liquid-assisted inverted internal limiting membrane (ILM) flap technique with the standard ILM peeling for the treatment of macular hole retinal detachment in highly myopic eyes. METHODS: This was a retrospective, consecutive, nonrandomized comparative study. Forty-two macular hole retinal detachment eyes of 42 patients were included into either a perfluorocarbon liquid-assisted inverted ILM flap technique group (n = 22, inverted group) or standard ILM removal group (n = 20, peeling group). Outcomes measured were macular hole closure, retinal reattachment, and best-corrected visual acuity at least 6 months after surgery. RESULTS: Macular hole closure was achieved in 20 eyes (90.9%) in the inverted group and in eight eyes (40%) in the peeling group (P < 0.01). Reattachment rates were 100% in the inverted group and 95% in the peeling group (P = 0.476). The mean best-corrected visual acuity improvement from baseline was 27.4 ± 19.9 Early Treatment Diabetic Retinopathy Study letters in the inverted group while the best-corrected visual acuity improvement was 13.6 ± 22.5 Early Treatment Diabetic Retinopathy Study letters in the peeling group (P = 0.044). CONCLUSION: The perfluorocarbon liquid-assisted inverted ILM flap technique was effective in sealing the macular hole, reattaching retina, and improving visual function postoperatively in highly myopic macular hole retinal detachment.

Identification of a novel GPR143 mutation in a large Chinese family with isolated foveal hypoplasia.

BACKGROUND: Pathogenic variants of G-protein coupled receptor 143 (GPR143) gene often leads to ocular albinism type I (OA1) characterized by nystagmus, iris and fundus hypopigmentation, and foveal hypoplasia. In this study, we identified a novel hemizygous nonsense mutation in GPR143 that caused an atypical manifestation of OA1. CASE PRESENTATION: We reported a large Chinese family in which all affected individuals are afflicted with poor visual acuity and foveal hypoplasia without signs of nystagmus. Fundus examination of patients showed an absent foveal reflex and mild hypopigmentation. The fourth grade of foveal hypoplasia and the reduced area of blocked fluorescence at foveal region was detected in OCT. OCTA imaging showed the absence of foveal avascular zone. In addition, the amplitude of multifocal ERG was reduced in the central ring. Gene sequencing results revealed a novel hemizygous mutation (c.939G > A) in GPR143 gene, which triggered p.W313X. However, no iris depigmentation and nystagmus were observed among both patients and carriers. CONCLUSIONS: In this study, we reported a novel nonsense mutation of GPR143 in a large family with poor visual acuity and isolated foveal hypoplasia without nystagmus, which further expanded the genetic mutation spectrum of GPR143.

Optimization of Contact Lenses for Corneal Protection During Vitreoretinal Surgery.

OBJECTIVES: To optimize parameters of contact lenses (CLs) and evaluate their ability to protect the cornea during vitreoretinal surgery. METHODS: We compared the protective effects of balanced saline solution, viscoelastic agent, and CLs on rabbit corneas under conditions simulating vitreoretinal surgery. We evaluated CLs of different thicknesses and compared the protective effects of polymethyl methacrylate (PMMA) and gas-permeable fluorosilicone acrylate (XO) lenses on the corneas of rabbits and patients with severe proliferative diabetic retinopathy (PDR). The corneal fluorescein staining score (FSS) was measured to compare the protective effects of CLs. RESULTS: The FSS was significantly lower in the PMMA group than in the balanced saline solution and viscoelastic agent groups. The thickness of the PMMA lenses had no significant effect on the FSS. The FSS was significantly higher in the PMMA group than in the XO group. In patients with PDR, on day 1 after vitreoretinal surgery, the FSS was significantly higher in the PMMA group than in the XO group, although no significant difference was observed on postoperative day 7. CONCLUSION: The XO lens offers better corneal protection during noncontact wide-angle vitreoretinal surgery and protects the corneal epithelium more efficiently during vitrectomy in patients with PDR, irrespective of its thickness.

Minimal internal limiting membrane peeling with ILM flap technique for idiopathic macular holes: a preliminary study.

BACKGROUND: Internal limiting membrane (ILM) peeling increases the idiopathic macular hole (IMH) closure rate but causes the inner retina dimplings. This study is to introduce a method to minimally peel the ILM, and with the ILM flap to ensure the IMH closure. METHODS: Twelve consecutive IMH eyes were treated with the minimal ILM peeling with ILM flap technique. The ILM around the MH is peeled off in an annular shape with a width of approximately 200 to 300 µm. A tongue-shape ILM flap is created in the superior retina and the inferior margin of ILM is not peeled off. The ILM flap is then inverted to cover the MH, followed by fluid-air exchange and air or silicon tamponade. Spectral domain-optical coherence tomography (SD-OCT) and en face OCT for morphological assessment, best corrected visual acuity (BCVA) and multifocal electroretinogram (ERG) for functional evaluation were performed at baseline and at each postoperative follow-up. RESULTS: All the 12 eyes achieved macular hole closure on SD-OCT after surgery (100%). At baseline, the mean preoperative BCVA was 0.83 ± 0.33 and it improved to 0.39 ± 0.28 postoperatively (p <  0.001). En face OCT showed the inner retinal dimplings were localized only in superior ILM-free retinas (7 eyes). The mERG response density in the central (R1), para-central (R2), R1/R2 ring ratios were remarkably improved at the last follow-up (p = 0.001, p = 0.033, p = 0.018, respectively). CONCLUSIONS: The minimal ILM peeling with ILM flap technique can achieve favorable MH closure with less inner retinal dimplings and has promising visual recovery for IMH eyes.

Protective effects of microRNA-22-3p against retinal pigment epithelial inflammatory damage by targeting NLRP3 inflammasome.

NLRP3, as a crucial inflammasome component, plays important roles in age-related macular degeneration. Though some activators of NLRP3 have been studied, microRNAs (miRNAs) which potentially regulate NLRP3 messenger RNA (mRNA) have not been fully explored in retinal pigment epithelial (RPE) cells and retinopathy. In this study, by miRNA microarray profiling and bioinformatic analysis, we identified that four miRNAs, miR-4286, miR-223-3p, miR-365a, miR-22-3p, may target NLRP3 mRNA in RPE inflammatory damage in vivo. Further, real-time polymerase chain reaction verified that only miR-22-3p was significantly decreased, which was associated with NLRP3 upregulation in blue-light-induced retinopathy. Mechanistically, the dual-fluorescent reporter suggested miR-22-3p directly binds NLRP3 mRNA. Moreover, overexpression of miR-22-3p could significantly reduce whereas inhibition miR-22-3p could increase the mRNA and protein expressions of NLRP3, Caspase-1, and mature IL-1ß. Collectively, our results indicate that miR-22-3p plays a suppressive role in RPE damage by targeting NLRP3, which provides new insights into the future intervention to retinopathy.

Involvement of NYD-SP15 in growth and oxidative-stress responses of ARPE-19.

The aim of this study was to investigate the role of NYD-SP15 in the growth and oxidative-stress responses of ARPE-19 cells. ARPE-19 cell lines overexpressing wild type or RNA interference against NYD-SP15 were established via lentivirus transfection. Cell growth and proliferation, migration, apoptosis, and cell cycle progression were monitored using the Cell Counting Kit-8 assay, the wound scratch assay, and flow cytometry, respectively. Caspase-3/8/9 activity was examined using the caspase-3/8/9 assay kit. An hydrogen peroxide (H 2 O 2 )-induced oxidative-stress damage model was used to study the effect of NYD-SP15 knockdown by examining the activity of reactive oxygen species (ROS). Expressions of Kelch-like ECH-associated protein 1 (Keap-1)/heme oxygenase-1 (HO-1)/nuclear factor erythroid 2-related factor 2 (Nrf2), mitogen-activated protein kinase (MAPK), and Akt were detected by Western blot analysis. The mRNA chip of NYD-SP15 overexpressed ARPE-19 cells as well as controls were performed by one array plus process. Overexpression (OE) of NYD-SP15 inhibited the proliferation and migration of ARPE-19 cells, and led to apoptosis and caspase-3/9 activation. OE of NYD-SP15 inhibited MAPKs and Akt signaling. Downregulation of NYD-SP15 had no effect on the growth of normally cultured ARP19 cells with 10% fetal bovine serum, but promoted the growth of ARP19 cells in the presence of starvation challenge. Gene chip showed that OE of NYD-SP15 led to downregulation of 254 genes and upregulation of 57 genes. Downregulation of NYD-SP15 also exerted a protective effect on H 2 O 2 -induced cell apoptosis and ROS. NYD-SP15 downregulation led to increments in the expression of Nrf2, Keap-1, and HO-1 in response to 200 µM H 2 O 2 . NYD-SP15 might inhibit the growth, proliferation, and migration and promote apoptosis of ARPE-19 cells via MAPK and Akt signaling. Downregulation of NYD-SP15 could protect ARPE-19 cells from H 2 O 2 -induced oxidative damage by active Keap-1/HO-1/Nrf2, Akt, and MAPK signaling.

Gremlin-1: An endogenous BMP antagonist induces epithelial-mesenchymal transition and interferes with redifferentiation in fetal RPE cells with repeated wounds.

Purpose: To investigate the role of Gremlin-1, which is an endogenous antagonist of the bone morphogenetic protein (BMP) signaling pathway, in inducing epithelium-mesenchymal transition (EMT) in fetal RPE cells after repeated wounds. Methods: Subconfluent repetitive passages in fetal RPE cells were regarded as a model of repeated wounds. A phase contrast microscope was used to observe the morphology and pigment formation in cells. The expression of GREM1 (Gene ID: 26585; OMIM 603054) and EMT- or RPE-related genes in cells was evaluated with quantitative PCR (qPCR). Recombinant human protein Gremlin-1 (0.1 µg/ml) was added every day to investigate the molecular effects of Gremlin-1 on fetal RPE cells. The cell migration rate was investigated using a cell wound scratch assay, and western blotting was used to analyze the representative proteins (P-cadherin, ZO-1, vimentin, Smad4, and phosphorylated-Smads). In addition, transfection of siRNA was used to explore the rescue effects on EMT cells through the downregulation of GREM1. Finally, LDN193189, which is a type of pan-inhibitor of BMP receptors, was used to verify whether complete blocking of the BMP pathway interferes with the redifferentiation in low-passage fetal cells, even if the cells were treated with transforming growth factor beta 1 (TGF-ß) inhibitors. Results: In fetal RPE cells, the expression of GREM1 were gradually upregulated with repetitive passages, and at the same time, the function-specific genes in fetal RPE cells (TJP1, PMEL, BEST1, RPE65, and MERTK) were downregulated while the EMT-specific genes were upregulated. In addition, GREM1 had a similar expression pattern as SNAI1, which is a key transcription factor to trigger EMT. Recombinant human Gremlin-1 promoted EMT with the upregulation of SNAI1 and elevated the cell migration rate in a cell scratch assay, as well as decreased the expression of two key transcription factors of RPE embryonic development (MITF and OTX2) and the RPE marker, RPE65. Furthermore, the EMT marker, vimentin, and the TGF-ß pathway downstream transcription factor phosphorylated-Smad2 (p-Smad2) increased, but the epithelial marker, ZO-1, was reduced. Additionally, Smad4, which plays a role as a Snail1 cooperator by binding Smad3, was also increased. In contrast, GREM1 silencing increased the expression of MITF and OTX2, which means there was better redifferentiation in subconfluent fetal RPE cells, but it had little influence on p-Smad2 compared to the negative control group. Finally, by adding LDN193189, the BMP signaling pathway was blocked, and this block led to poor redifferentiation in low-passage cells, although the cells were treated with TGF-ß inhibitors. In addition, as positive feedback to block the BMP pathway, GREM1 was subsequently upregulated. Conclusions: In fetal RPE cells, Gremlin-1 induces EMT and inhibits redifferentiation by promoting the TGF-ß pathway and inhibiting the BMP pathway. GREM1 silencing alleviates EMT and increases the redifferentiation of cells by relieving the blockade of the BMP pathway. However, GREM1 silencing has no effects on the TGF-ß pathway. Thus, Gremlin-1 may serve as a novel target to treat proliferative vitreoretinopathy (PVR) and inhibit subretinal fibrosis, which is a risk factor for influencing the therapeutic effects of anti-vascular endothelial growth factor (anti-VEGF) on neovascular age-related macular degeneration (nAMD).

Targeted next-generation sequencing extends the mutational spectrums for OPA1 mutations in Chinese families with optic atrophy.

Purpose: We aim to reveal the disease-causing mutations in 15 Chinese families with optic atrophy (OA). Methods: In total, 15 families with OA were recruited in the present study. Medical histories were carefully reviewed and comprehensive ophthalmic examinations were received by all recruited patients. Targeted next-generation sequencing (NGS) was selectively performed on all probands for mutation detection. Intrafamilial cosegregation and in-silico analyses were subsequently applied to predict the potential pathogenic effects of identified mutations. Results: All included patients presented bilateral vision loss. Their fundus photographs showed temporal or total pallor of the optic discs. Fourteen mutations in the optic atrophy 1 (OPA1) gene were revealed as disease-causing mutations for the 15 families, including eight novel (c.968A>G, c.193C>G, c.1071dupT, c.987_988del, c.2012+2T>G, c.1036-1G>C, c.2126A>G, and c.1036_1038del) and six recurrent (c.1499G>A, c.1800C>A, c.1034G>A, c.2873_2876del, c.112C>T, and c.804_805del) mutations. Conclusions: In conclusion, our study expands the mutational spectrum for the OPA1 gene and implies targeted NGS as an effective approach for the genetic diagnosis of OA, which might help to improve the clinical diagnosis for patients with OA.

CRB2 mutation causes autosomal recessive retinitis pigmentosa.

Retinitis pigmentosa (RP), the most common form of inherited retinal dystrophies, exhibits significant genetic heterogeneity. The crumbs homolog 2 (CRB2) protein, together with CRB1 and CRB3, belongs to the Crumbs family. Given that CRB1 mutations account for 4% of RP cases, the role of CRB2 mutations in RP etiology has long been hypothesized but never confirmed. Herein, we report the identification of CRB2 as a novel RP causative gene in a Chinese consanguineous family and have analyzed its pathogenic effects. Comprehensive ophthalmic and systemic evaluations confirmed the clinical diagnosis of the two patients in this family as RP. WES revealed a homozygous missense mutation, CRB2 p.R1249G, to segregate the RP phenotype, which was highly conserved among multiple species. In vitro cellular study revealed that this mutation not only interrupted the stability of the transcribed CRB2 mRNA and the encoded CRB2 protein, but also interfered with the wild type CRB2 mRNA/protein and decreased their expression. This mutation was also shown to trigger epithelial-mesenchymal transition (EMT) in retinal pigment epithelium (RPE) cells, thus impairing regular RPE phagocytosis and induce RPE degeneration and apoptosis. Thus, we conclude that CRB2 p.R1249G mutation causes RP via accelerating EMT, dysfunction and loss of RPE cells, and establish CRB2 as a novel Crumbs family member associated with non-syndromic RP. We provide important hints for understanding of CRB2 defects and retinopathy, and for the involvement of EMT of RPE cells in RP pathogenesis.