Pathologic microvasculature plays a crucial role in innumerable diseases causing death and major organ impairment. A major clinical challenge is the development of selective therapies to remove these diseased microvessels without damaging surrounding tissue. This report describes our development of novel photo-mediated ultrasound therapy (PUT) technology for precisely removing choroidal blood vessels in the eye. PUT selectively removes microvessels by concurrently applying nanosecond laser pulses with ultrasound bursts. In PUT experiments on rabbit eyes in vivo, we applied 55-75 mJ/cm2 of light fluence at the retinochoroidal surface at 532-nm and 0.5 MPa of ultrasound pressure at 0.5 MHz. PUT resulted in significantly reduced blood perfusion in the choroidal layer which persisted to four weeks without causing collateral tissue damage, demonstrating that PUT is capable of removing choroidal microvasculature safely and effectively. With its unique advantages, PUT holds potential for the clinical management of eye diseases associated with microvessels and neovascularization.

Diabetic retinopathy (DR) is a well-known microvascular complication related to inflammation. Mcc950 is a potent and specific inhibitor of the NLRP3 inflammasome but its influence on DR has not been studied. Thus, we evaluated the anti-inflammatory effects of Mcc950 on high-glucose-induced human retinal endothelial cells (HRECs) and the potential underlying mechanism. In surgical excised proliferative membranes from DR patients, high expression of NLRP3, caspase 1 and IL-1β was observed and co-localization of NLRP3 and IL-1β occurred in CD31+ labeled HRECs. Moreover, in high-glucose-stimulated HRECs, increased production of the NLRP3 inflammasome activation and severe apoptosis were rescued with Mcc950 treatment. Additionally, the inhibitory effect of Mcc950 was mimicked through downregulation of NEK7 by siRNA in high-glucose-induced HRECs and Mcc950 treatment remarkably inhibited Nek7 and NLRP3 interactions by co-immunoprecipitation, suggesting that Mcc950 may be a potentially protective agent against inflammation, likely via downregulation of the Nek7-NLRP3 pathway. In conclusion, Mcc950 inhibited HREC dysfunction under high-glucose conditions and this research may offer insight for future pharmaceutical approaches for treating DR.

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.

Increasing evidence has indicated that long non-coding RNAs (lncRNAs) play significant roles in various diseases; however, their roles in age-related macular degeneration (AMD) remain unclear. Dedifferentiation and dysfunction of retinal pigment epithelium (RPE) cells have been shown to contribute to AMD etiology in several studies. Herein, we found that lncRNA LINC00167 was downregulated in RPE-choroid samples of AMD patients and dysfunctional RPE cells, and it was consistently upregulated along with RPE differentiation. In vitro study indicated that reduced endogenous LINC00167 expression resulted in RPE dedifferentiation, which was typified by attenuated expression of RPE markers, reduced vascular endothelial growth factor A secretion, accumulation of mitochondrial reactive oxygen species, and interrupted phagocytic ability. Mechanistically, LINC00167 functioned as a sponge for microRNA miR-203a-3p to restore the expression of the suppressor of cytokine signaling 3 (SOCS3), which further inhibited the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway. Taken together, our study demonstrated that LINC00167 showed a protective role in AMD by maintaining RPE differentiation through the LINC00167/miR-203a-3p/SOCS3 axis and might be a potential therapeutic target for AMD.

Retinoblastoma (RB) is the most common childhood malignant intraocular tumor. The clinical efficacy of vincristine (VCR) in the treatment of RB is severely limited by drug resistance. Here, we found that CD24, a GPI-anchored protein, was overexpressed in human RB tissues and RB cell lines, and was associated with the sensitivity of RB cells in response to VCR therapy. We demonstrated that CD24 plays a critical role in impairing RB sensitivity to VCR via regulating autophagy. Mechanistically, CD24 recruits PTEN to the lipid raft domain and regulates the PTEN/AKT/mTORC1 pathway to activate autophagy. Lipid raft localization was essential for CD24 recruitment function. Collectively, our findings revealed a novel role of CD24 in regulating RB sensitivity to VCR and showed that CD24 is a potential target for improving chemotherapeutic sensitivity and RB patient outcomes.

Idiopathic epiretinal membrane (iERM) or idiopathic macular hole (iMH) is frequently used as a "healthy" control in comparison of vitreous cytokines with other vitreoretinal diseases. This study aimed to investigate if there is a difference in vitreal cytokines expression between patients with iERM and iMH.

We aim to reveal the disease-causing mutations in 15 Chinese families with optic atrophy (OA).

Age-related macular degeneration (AMD) causes irreversible blindness in people aged over 50 worldwide. The dysfunction of the retinal pigment epithelium is the primary cause of atrophic AMD. In the current study, we used the ComBat and Training Distribution Matching method to integrate data obtained from the Gene Expression Omnibus database. We analyzed the integrated sequencing data by the Gene Set Enrichment Analysis. Peroxisome and tumor necrosis factor-α (TNF-α) signaling and nuclear factor kappa B (NF-κB) were among the top 10 pathways, and thus we selected them to construct AMD cell models to identify differentially expressed circular RNAs (circRNAs). We then constructed a competing endogenous RNA network, which is related to differentially expressed circRNAs. This network included seven circRNAs, 15 microRNAs, and 82 mRNAs. The Kyoto Encyclopedia of Genes and Genomes analysis of mRNAs in this network showed that the hypoxia-inducible factor-1 (HIF-1) signaling pathway was a common downstream event. The results of the current study may provide insights into the pathological processes of atrophic AMD.

This cross-sectional study aims to investigate the prevalence and causes of visual impairment (VI) and blindness in Jiangsu Province, China in 2022 during the COVID-19 pandemic. Participants (n = 13,208, aged 18-93) underwent comprehensive ocular examinations. The prevalence and causes of binocular VI (presenting visual acuity [VA] ≥ 20/400 and < 20/63 in the better eye) and blindness (presenting VA < 20/400 in the better eye) were assessed according to the World Health Organization (WHO) criteria. The estimation of refractive error prevalence was conducted using the following classification: myopia ≤  - 0.50 diopters (D), high myopia ≤  - 6.00 D, hyperopia ≥ 0.50 D, and anisometropia ≥ 1.00 D. The overall prevalence of binocular VI and blindness was 21.04% (95% confidence interval [CI] 20.35-21.74%) and 0.47% (95% CI 0.37-0.60%). The highest prevalence of binocular VI was in the population aged 18-24 years old (46.29%, [95% CI 44.30-48.28%]), those with education at university and above (43.47%, [95% CI 41.93-45.02%]), students (54.96%, [95% CI 52.73-57.17%]). Uncorrected refractive error (URE) was the leading cause of presenting binocular VI (93.40%) and blindness (50.79%). The prevalence of myopia was 54.75% (95% CI 53.90-55.60%). Actions are needed to control URE and myopia within the adult Chinese population, with a particular emphasis on the younger, well-educated demographic.

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.

It is known that oxidative stress plays a pivotal role in age-related macular degeneration (AMD) pathogenesis. Alpha-mangostin is the main xanthone purified from mangosteen known as anti-oxidative properties. The aim of the study was to test the protective effect of alpha-mangostin against oxidative stress both in retina of light-damaged mice model and in hydrogen peroxide (H2O2)-stressed RPE cells. We observed that alpha-mangostin significantly inhibited light-induced degeneration of photoreceptors and 200 μM H2O2-induced apoptosis of RPE cells. 200 μM H2O2-induced generation of reactive oxygen species (ROS) and light-induced generation of malondialdehyde (MDA) were suppressed by alpha-mangostin. Alpha-mangostin stimulation resulted in an increase of superoxide dismutase (SOD) activity, glutathione peroxidase (GPX) activity and glutathione (GSH) content both in vivo and vitro. Furthermore, the mechanism of retinal protection against oxidative stress by alpha-mangostin involves accumulation and the nuclear translocation of the NF-E2-related factor (Nrf2) along with up-regulation the expression of heme oxygenas-1 (HO-1). Meanwhile, alpha-mangostin can activate the expression of PKC-δ and down-regulate the expression of mitogen-activated protein kinases (MAPKs), including ERK1/2, JNK, P38. The results suggest that alpha-mangostin could be a new approach to suspend the onset and development of AMD.

The development of the mammalian retina is a complicated process involving the generation of distinct types of neurons from retinal progenitor cells (RPCs) in a spatiotemporal-specific manner. The progression of RPCs during retinogenesis includes RPC proliferation, cell-fate commitment, and specific neuronal differentiation. In this study, by performing single-cell RNA sequencing of cells isolated from human embryonic stem cell (hESC)-derived 3D retinal organoids, we successfully deconstructed the temporal progression of RPCs during early human retinogenesis. We identified two distinctive subtypes of RPCs with unique molecular profiles, namely multipotent RPCs and neurogenic RPCs. We found that genes related to the Notch and Wnt signaling pathways, as well as chromatin remodeling, were dynamically regulated during RPC commitment. Interestingly, our analysis identified that CCND1, a G1-phase cell-cycle regulator, was coexpressed with ASCL1 in a cell-cycle-independent manner. Temporally controlled overexpression of CCND1 in retinal organoids demonstrated a role for CCND1 in promoting early retinal neurogenesis. Together, our results revealed critical pathways and novel genes in early retinogenesis of humans.

We verified whether fetal RPE (fRPE) cells and mesenchymal stem cells (MSCs) cotransplantation can combine the features of these two cell types and alleviate retinal degeneration in a retinal degenerative disease mouse model.

Diabetic retinopathy is a heterogeneous retinal degenerative disease with the microvascular dysfunction being recognized as a hallmark of the advanced stage. In this study, we demonstrated that exosomes collected from the vitreous humor of proliferative diabetic retinopathy patients promoted proliferation, migration and tube formation ability of primary human retinal endothelial cells via its elevated miR-9-3p expression level. Müller glia cells were further recognized as the sole source of the aberrantly expressed miR-9-3p, and both in vitro and in vivo experiments validated that Müller glia-derived exosomes aggravate vascular dysfunction under high glucose. Mechanistically, exosomal miRNA-9-3p was transferred to retinal endothelial cells and bound to the sphingosine-1-phosphate receptor S1P1 coding sequence, which subsequently activated VEGFR2 phosphorylation and internalization in the presence or absence of exogenous VEGF-A. We successfully orchestrated the dynamic crosstalk between retinal Müller glia cells and endothelial cells in pathological condition, which may provide a novel biomarker or promising therapeutic agents for the treatment of diabetic retinopathy.

Age-related macular degeneration (AMD), the leading cause of visual loss after the age of 60 years, is a degenerative retinal disease involving a variety of environmental and hereditary factors. Although it has been implicated that immune system is involved in the disease progression, the exact role that microglia has is still unclear. Here we demonstrated that knockout of Ccr2 gene could alleviate photoreceptor cell death in mice retinas exposed to chronic blue light. In Ccr2-/- mice, a damaged microglia recruitment was shown in retina and this could protect the visual function in electroretinogram and alleviate the photoreceptor apoptosis, which thus helped attenuate the blue light-induced retinopathy. We further found an increased co-location of NLRP3, Iba-1, and IL-1β in fluorescence and a concomitant increased protein expression of NLRP3, caspase-1, and IL-1β in western blotting in chronic blue light-induced retinopathy. Moreover, the activation of microglia and their cellular NLRP3 inflammasomes occurred as an earlier step before the structural and functional damage of the mice retinas, which collectively supported that microglial NLRP3 inflammasome might be the key to the chronic blue light-induced retinopathy.

Diabetic retinopathy (DR) is a common diabetic eye disease which is well-known as the result of microvascular retinal changes. Although the potential biological functions of astragaloside IV (AS IV) have long been described in traditional system of medicine, its protective effect on DR remains unclear. This study aims to investigate the function and mechanism of AS IV on type 2 diabetic db/db mice.

Abnormally expressed long noncoding RNA (lncRNA) high expression in hepatocellular carcinoma (HEIH) has been implicated in many types of human cancer, and plays crucial roles in tumor development and progression. However, little is known about its function in retinoblastoma.

The use of Cre recombinase for conditional targeting permits the controlled removal or activation of genes in specific tissues and at specific times of development. The Rho-Cre mice provide an improved tool for studying gene ablation in rod photoreceptor cells. To establish a robust expression of Rho-Cre transgenic mice that would be useful for the study of various protein functions in photoreceptor cells, a total 11,987 kb fragment (pNCHS4 Rho-NLS-cre) containing human rhodopsin promoter was cloned. The Rho-Cre plasmid was digested with EcoR1 and I Ceu-1, and the 9.316 kb fragment containing the hRho promoter and Cre recombinase gel was purified. To generate transgenic mice, the purified DNA fragment was injected into fertilized oocytes according to standard protocols. ROSA26R reported the steady expression of Rho-Cre especially in photoreceptor cells, allowing further excising proteins in rod photoreceptors across the retina. This Rho-Cre transgenic line should thus prove useful as a general deletor line for genetic analysis of diverse aspects of retinopathy.

Dedifferentiation of retinal pigment epithelium (RPE) cells and choroidal neovascularization (CNV) contributes to the pathogenesis of age-related macular degeneration (AMD). MicroRNAs (miRNAs) have crucial roles in AMD onset and progression. We thus aim to investigate the effects of miRNAs on RPE dedifferentiation and endothelium cell (EC) behavior, and analyze its downstream pathways. We have previously identified miR-302d-3p as the most downregulated miRNA signature along with RPE differentiation. Herein, in vitro study supported that miR-302d-3p induces RPE dedifferentiation typified by reduction of RPE characteristic markers, interrupts its phagocytosis, and promotes its migration, proliferation, and cell-cycle progression. c-Jun was identified as a potential upstream transcript factor for MIR302D, which might modulate RPE function by regulating miR-302d-3p expression. P21Waf1/Cip1, a cyclin-dependent kinase inhibitor encoded by the CDKN1A gene, was identified as a downstream target of miR-302d-3p. Our data suggested that p21Waf1/Cip1 could promote RPE differentiation, and inhibit its proliferation, migration, and cell-cycle progression. We also demonstrated that miR-302d-3p suppresses RPE differentiation through directly targeting p21Waf1/Cip1. In addition, the miR-302d-3p/CDKN1A axis was also involved in regulating tube formation of ECs, indicating its potential involvement in CNV formation. Taken together, our study implies that miR-302d-3p, regulated by c-Jun, contributes to the pathogenesis of both atrophic and exudative AMD. MiR-302d-3p promotes RPE dedifferentiation, migration, proliferation and cell-cycle progression, inhibits RPE phagocytosis, and induces abnormal EC behavior by targeting p21Waf1/Cip1. Pharmacological miR-302d-3p inhibitors are prospective therapeutic options for prevention and treatment of AMD.

Using the paradigm of in vitro differentiation of hESCs/iPSCs into retinal pigment epithelial (RPE) cells, we have recently profiled mRNA and miRNA transcriptomes to define a set of RPE mRNA and miRNA signature genes implicated in directed RPE differentiation. In this study, in order to understand the role of DNA methylation in RPE differentiation, we profiled genome-scale DNA methylation patterns using the method of reduced representation bisulfite sequencing (RRBS). We found dynamic waves of de novo methylation and demethylation in four stages of RPE differentiation. Integrated analysis of DNA methylation and RPE transcriptomes revealed a reverse-correlation between levels of DNA methylation and expression of a subset of miRNA and mRNA genes that are important for RPE differentiation and function. Gene Ontology (GO) analysis suggested that genes undergoing dynamic methylation changes were related to RPE differentiation and maturation. We further compared methylation patterns among human ESC- and iPSC-derived RPE as well as primary fetal RPE (fRPE) cells, and discovered that specific DNA methylation pattern is useful to classify each of the three types of RPE cells. Our results demonstrate that DNA methylation may serve as biomarkers to characterize the cell differentiation process during the conversion of human pluripotent stem cells into functional RPE cells.