Neuromyelitis optica spectrum disorder (NMOSD) is a severe central nervous system disease primarily characterized by optic neuritis and myelitis, which can result in vision loss and limb paralysis. Current treatment options are limited in their ability to prevent relapses and mitigate disease progression, underscoring the urgent need for new drug targets to develop more effective therapies. The objective of this study is to identify potential drug targets associated with a reduced risk of NMOSD attacks or relapses through Mendelian randomization (MR) analysis, thereby addressing the limitations of existing treatment methods and providing better clinical options for patients. To identify therapeutic targets for NMOSD, a MR analysis was conducted. The cis-expression quantitative trait loci (cis-eQTL, exposure) data were sourced from the eQTLGen consortium, which included a sample size of 31,684. NMOSD (outcome) summary data were obtained from two of the largest independent cohorts: one cohort consisted of 86 NMOSD cases and 460 controls derived from whole-genome sequencing data, while the other cohort included 129 NMOSD patients and 784 controls. We performed a two-sample MR analysis to evaluate the association between single nucleotide polymorphisms (SNPs) and copy number variations with NMOSD. The MR analysis utilized the inverse variance weighted (IVW) method, supplemented by MR-Egger, weighted median, simple mode, and weighted mode methods. Sensitivity analyses were conducted to assess the presence of horizontal pleiotropy and heterogeneity. Colocalization analysis was employed to test whether NMOSD risk and gene expression are driven by common SNPs. Additionally, a phenome-wide association study (PheWAS) was performed to detect disease outcomes associated with NEU1. Supplementary analyses included single-nucleus RNA sequencing (snRNA-seq) data analysis, protein-protein interaction (PPI) networks, and drug feasibility assessments to prioritize potential therapeutic targets. Two drug targets, COL4A1 and NEU1, demonstrated significant MR results in two independent datasets. Notably, NEU1 showed substantial evidence of colocalization with NMOSD. Additionally, apart from the association between NEU1 and NMOSD, no other associations were observed between gene-proxied NEU1 inhibition and the increased risk of other NMOSD-related diseases. This study supports the potential of targeting NEU1 for drug inhibition to reduce the risk of NMOSD. Further preclinical research and drug development are warranted to validate the efficacy and safety of NEU1 as a therapeutic target and to explore its potential in NMOSD treatment.

Signals from the lens regulate multiple aspects of eye development, including establishment of eye size, patterning of the presumptive iris and ciliary body in the anterior optic cup and migration and differentiation of neural crest cells. To advance understanding of the molecular mechanism by which the lens regulates eye development, we performed transcriptome profiling of embryonic chicken retinas after lens removal. Genes associated with nervous system development were upregulated in lens-removed eyes, but the presumptive ciliary body and iris region did not adopt a neural retina identity following lens removal. Lens-regulated genes implicated in periocular mesenchyme, cornea and anterior optic cup development were identified, including factors not previously implicated in eye development. Unexpectedly, transcriptomic differences were identified in retinas from male versus female chicken embryos, suggesting sexual dimorphism from early stages. In situ hybridisation of embryonic chicken eyes and analyses of datasets from embryonic mouse and adult human eyes confirmed expression of candidate genes, including multiple WNT genes, in tissues important for anterior eye development and function. Remarkably, pharmacological activation of canonical WNT signalling restored eye development and size in the absence of the lens. These analyses have identified candidate genes and biological pathways involved in eye development, providing avenues for new research in this area.

Use of genome-wide association studies (GWASs) in combination with transcriptomic arrays of different tissues or cell lines can reveal relevant cellular profiles and significantly improve understanding of the mechanisms of diseases. However, due to difficulty of access, few ocular transcriptomics datasets are available. This work aimed to create and make available an expression library platform that can be used with popular and versatile tools such as the linkage disequilibrium score (LDSC) regression techniques to identify specific cell lines enriched in ocular diseases.

We used transcriptome information from six publicly available single-cell and single-nucleus RNA sequence datasets to obtain matrices of normalized gene expression and estimated enrichment of the 10% most expressed transcripts in each cell line. We tested for type 1 error using simulated GWAS datasets and then used LDSC regression analyses to study the enrichment in different eye diseases.

Gene expression databases for over 197 ocular cell lines were generated. Simulations of 1000 random GWASs of varying sample sizes showed no genomic inflation. Cell line-specific analyses of GWAS results found that genes near single nucleotide polymorphisms (SNPs) associated with refractive error were significantly enriched in cones (P = 0.008), rods (P = 0.002) and peripheral retina Müller cells (P = 0.002), juxtacanalicular cribriform cells (P = 0.0017), stromal keratocytes (P = 0.0018), and one beam-cell subpopulation (P = 0.0028) in primary open-angle glaucoma, emphasizing the importance of intraocular pressure in its etiology.

We have built a structured ocular transcriptomics library to estimate cell line enrichment among association signals from genome-wide association studies that may be extended by incorporating other datasets. We identified cells that appear important in the genetics of common eye diseases.

The periocular mesenchyme (POM) gives rise to key structures in the ocular anterior segment, and its malformation leads to anterior segment dysgenesis (ASD) with iridocorneal angle (ICA) abnormalities. However, the transcriptional profile of the POM and the regulatory mechanisms governing cell-fate decision during anterior eye and ICA development remain poorly understood. In this study, we performed a comprehensive time-series analysis by sequencing rat anterior ocular samples collected at five consecutive perinatal stages: embryonic days 16.5 and 18.5, the day of birth, and postnatal days 4 and 8, at the single-cell level and validated a portion of in silico findings with immunostaining. High-quality transcriptomes were obtained from 59,416 cells with diverse embryonic origins. A prominent transcriptional shift was observed in POM cells, coinciding with anatomical alterations around the ICA shortly after birth. We illustrated the molecular signatures of five POM subclusters while tracing their developmental trajectories. Additionally, we identified key driver genes, as well as cell type-specific and stage-wise gene modules underlying lineage specification. Furthermore, the switch of regulon network and cellular crosstalk associated with POM maturation were unveiled. Lastly, we mapped ASD-relevant genes to this single-cell atlas, revealing distinct expression patterns. Collectively, this study provides a transcriptomic blueprint for understanding normal POM and ICA development, as well as a valuable reference for future research into ASD pathogenesis.

Rhabdoid tumours (RT) are an aggressive malignancy affecting <2-year-old infants, characterised by biallelic loss-of-function alterations in SWI/SNF-related BAF chromatin remodelling complex subunit B1 (SMARCB1) in nearly all cases. Germline SMARCB1 alterations are found in ~30% of patients and define the RT Predisposition Syndrome type 1 (RTPS1). Uveal melanoma (UVM), the most common primary intraocular cancer in adults, does not harbour SMARCB1 alterations. We report two cases of a previously undescribed intraocular malignancy that shared some features with UVM and RT, but was also distinct from these entities. Both female patients, aged 23 and 14 years, underwent enucleation, and the tumours were subjected to comprehensive genomic, DNA methylation, and transcriptomic profiling. Pathological examination showed large, amelanotic intraocular tumours with epithelioid features, expressing melanocytic markers [S100P, SOX10, Melan-A, PMEL (HMB45), TYR] as seen using immunohistochemistry (IHC), but with little or no melanin production. Both tumours harboured biallelic loss-of-function SMARCB1 alterations, associated with loss of SMARCB1 (BAF47/INI1) expression on IHC. Their genomic profiles were atypical both for UVM and for RT, and no pathogenic variants were found in other genes tested, including those recurrently altered in UVM. In both patients, a germline SMARCB1 variant was found. However, there was no relevant family history of cancer. Transcriptome and methylome profiling suggested that these tumours were distinct from RT, UVM, and skin melanomas. RNAseq confirmed expression of early and late genes related to melanocytic differentiation. The first patient died of metastatic disease 16 months after diagnosis, the second was disease-free 10 months after completion of treatment. In summary, we report two cases of a previously undescribed, aggressive SMARCB1-deficient intraocular malignancy with melanocytic differentiation, which occurs in young patients, is distinct from UVM and RT, and expands the RTPS1 spectrum. © 2025 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Human lens epithelial cells (hLECs) are critical for lens transparency, and their aberrant metabolic activity and gene expression can lead to cataract. Intracellular delivery to hLECs, especially to sub-cellular organelles (e.g., mitochondrion and nucleus), is a key step in engineering cells for cell- and gene- based therapies. Despite a broad variety of nano- and microparticles can enter cells, their spatial characteristics relevant to cellular uptake and localization remains elusive. To investigate cellular internalization of nanostructures in hLECs, herein, DNA nanotechnology was exploited to precisely fabricate four distinct, mass-controlled DNA-origami nanostructures (DONs) through computer-aided design. Ensembled DONs included the rods, ring, triangle, and octahedron with defined geometric parameters of accessible surface area, effective volume, compactness, aspect ratio, size and vertex number. Atomic force microscopy and agarose gel electrophoresis showed that four DONs self-assembled within 3.5h with up to 59% yield and exhibited structural intactness in cell culture medium for 4 h. Flow cytometry analysis of four Cy5-labelled DONs in hLECs HLE-B3 found time-dependent cellular uptake over 2 h, among which the octahedron and triangle had higher cellular accumulation than the rod and ring. More importantly, the vertex number among other geometric parameters was positively correlated with cellular entry. Confocal images further revealed that four DONs had preferential localization at mitochondria to nucleus at 2 h in HLE-B3 cells, and the degree of their biodistribution varied among DONs as evidenced by Manders' correlation coefficient. This study demonstrates the DONs dependent cellular uptake and intracellular compartment localization in hLECs, heralding the future design of structure-modulating delivery of nanomedicine for ocular therapy.

It is widely recognized that the glycocalyx has significant implications in regulating the self-renewal and differentiation of adult stem cells; however, its composition remains poorly understood. Here, we show that the fucose-binding Aleuria aurantia lectin (AAL) binds differentially to basal cells in the stratified epithelium of the human limbus, hair follicle epithelium, and meibomian gland duct. Using fluorescence-activated cell sorting in combination with single-cell transcriptomics, we find that most epithelial progenitor cells and melanocytes in the limbus display low AAL staining (AALlow) on their cell surface, an attribute that is gradually lost in epithelial cells as they differentiate into mature corneal cells. AALlow epithelial cells were enriched in putative limbal stem cell markers and displayed high clonogenic capacity. Further analyses revealed that AALlow epithelial cells had reduced expression of GDP-mannose-4,6-dehydratase, an enzyme catalyzing the first and limiting step in the de novo biosynthesis of GDP-fucose, and that inhibition of fucosylation using a small-molecule fucose analog stimulated the proliferative potential of limbal epithelial cells ex vivo. These results provide crucial insights into the distinctive composition of the glycocalyx in adult stem cells and underscore the significance of fucose modulation in the therapeutic regeneration of the human limbal stem cell niche.

Marfan syndrome is an inherited connective tissue disorder that affects the cardiovascular, musculoskeletal, and ocular systems. It is caused by pathogenic variants in the fibrillin-1 gene (FBN1). Fibrillin is a primary component of microfibrils, which are found throughout the extracellular matrix (ECM) and provide elasticity and resilience to connective tissue. Microfibrils also play a role in signaling by sequestering growth factors and interacting with cell surface receptors. In many tissues, microfibrils are interwoven with elastin, collagens, and other elements of the ECM. However, uniquely in the ciliary zonule of the eye, microfibrils exist in cell-free bundles largely devoid of other components. This structure offers a rare opportunity to study a pure population of fibrillin microfibrils in a relatively native state. Here, we briefly review the organization of the zonule and describe recent experiments in which we measure zonular biomechanics, providing insights into microfibril dynamics that would be challenging to obtain in other contexts.

To elucidate the genetic basis of primary angle-closure glaucoma (PACG) by identifying pathogenic tissue and critical tissue-specific variants.

The correlations among PACG susceptibility, axial length (AL), and anterior chamber depth (ACD) were evaluated using meta-analyses. Propensity score matching was utilized on 2161 participants from the Handan Eye Study to determine the risk factors independent of ACD and AL for PACG. Subsequently, we employed the assay for transposase-accessible chromatin with sequencing (ATAC-seq) and allele-specific self-transcribing active regulatory region sequencing (STARR-seq) to screen 202 PACG genome-wide association study (GWAS) variants for chromatin accessibility and functional roles.

The meta-analysis found that PACG susceptibility loci are not associated with ACD or AL. However, abnormal iris phenotypes emerged as significant independent risk factors for primary angle-closure disease (PACD), unrelated to ACD and AL. Substantial enrichment of PACG heritability was observed in the open chromatin regions of the human iris. Within the iris-relevant cellular context, 22 out of the 202 PACG GWAS variants could influence enhancer activity. Two variants in the iris open chromatin regions were implicated in the modulation of PLEKHA7 and C10orf53 expression. The downregulation of these two genes affects cytoskeletal organization.

Our findings underscore the importance of the iris in the pathogenesis of PACG and identified iris-specific, enhancer-modulating variants that may influence disease risk. Our approach also provides a generalizable framework for studying ocular diseases from the perspective of enhancer-modulating variants.

Hydrocephalus is a cerebrospinal fluid-related disease that usually manifests as abnormal dilation of the ventricles, with a triad of clinical findings including walking difficulty, reduced attention span, and urinary frequency or incontinence. The onset of congenital hydrocephalus is closely related to mutations in genes that regulate brain development. Currently, our understanding of the mechanisms of congenital hydrocephalus remains limited, and the prognosis of existing treatments is unsatisfactory. Additionally, there are no suitable or dedicated model organisms for congenital hydrocephalus. Therefore, it is significant to determine the mechanism and develop special animal models of congenital hydrocephalus. Recently, zebrafish have emerged as a popular model organism in many fields, including developmental biology, genetics, and toxicology. Its genome shares high similarity with that of humans, and it has fast and low-cost reproduction. These advantages make it suitable for studying the pathogenesis and therapeutic approaches for various diseases, specifically congenital diseases. This study explored the possibility of using zebrafish as a model organism for congenital hydrocephalus. This review describes the characteristics of zebrafish and discusses specific congenital hydrocephalus models. The advantages and limitations of using zebrafish for hydrocephalus research are highlighted, and insights for further model development are provided.

To investigate the associations of tomographic parameters in anterior segment optical coherence tomography (AS-OCT) with sex and age in a cohort study.

A cohort design.

AS-OCT data from 391 Japanese participants aged ≥ 35 years were obtained using swept-source OCT. In the cornea, the keratometric power at the flat (Kf) and steep (Ks) meridians, maximum keratometric power (Kmax), keratometric cylinder, spherical power, regular astigmatism, asymmetry, higher-order irregularity (HOI) from the anterior and posterior surfaces, and the central and thinnest corneal thicknesses were evaluated. Also, anterior chamber depth (ACD), lens thickness, crystalline lens rise (CLR), and nasal and temporal angle opening distances at 500 μm from the scleral spur (AOD500) were assessed. Sex differences and age-related changes were analyzed.

Women exhibited higher anterior Kf, Ks, and Kmax and lower posterior Kf, Ks, and Kmax than men. The ACD and nasal/temporal AOD500 were shorter in women than in men. The CLR was higher in women, whereas the lens thickness did not differ between the sexes, indicating a more anteriorly positioned lens in women. Age-related changes included increased anterior/posterior HOI, increased lens thickness and CLR resulting in decreased ACD and AOD500.

This study reveals sex-related differences in corneal shape, anterior chamber conformation, and lens position, as well as age-related changes in tomographic parameters. ACD, CLR, nasal and temporal AOD500 showed significant sex differences in the 50-70 s, whereas lens thickness showed no difference.

Deficient Angiopoietin-Tie2 signaling is linked to ocular hypertension in glaucoma. Receptor Tie2/TEK expression and signaling at Schlemm's canal (SC) is indispensable for canal integrity and homeostatic regulation of aqueous humor outflow (AHO) and intraocular pressure (IOP), as validated by conditional deletion of Tie2, its ligands (Angpt1, Angpt2 and Angpt3/4) or regulators (Tie1 and PTPRB/VE-PTP). However, these Tie2/TEK knockouts and conditional knockouts are global or endothelial, preventing separation of systemic and ocular vascular defects that impact retinal or renal integrity. To develop a more targeted model of ocular hypertension induced by selective knockdown of Tie2/TEK expressed in SC, we combined the use of viral vectors to target the canal, and two distinct gene-editing strategies to disrupt the Tie2 gene. Adeno-associated virus (AAV2) is known to transduce rodent SC when delivered into the anterior chamber by intracameral injection. First, delivery of Cre recombinase via AAV2.Cre into R26tdTomato/+ reporter mice confirmed preferential and stable transduction in SC endothelium. Next, to disrupt Tie2 expression in SC, we injected AAV2.Cre into homozygous floxed Tie2 (Tie2FL/FL) mice. This led to attenuated Tie2 protein expression along the SC inner wall, decreased SC area and reduced trabecular meshwork (TM) cellularity. Functionally, IOP was significantly and steadily elevated, whereas AHO facility was reduced. In contrast, hemizygous Tie2FL/+ mice responded to AAV2.Cre with inconsistent and low IOP elevation, corroborating the dose-dependency of ocular hypertension on Tie2 expression/activation. In a second model using CRISPR/SaCas9 genome editing, wild-type C57BL/6 J mice injected with AAV2.saCas9-sgTie2 showed similar selective SC transduction and comparable IOP elevation in course and magnitude to that induced by AAV2.Cre in Tie2FL/FL mice. Together, our findings, demonstrate that selective Tie2 knockdown in SC is a targeted strategy that reliably induces chronic ocular hypertension and reproduces glaucomatous damage to the conventional outflow pathway, providing novel models of SC-Tie2 signaling loss valuable for preclinical studies.

Since the trabecular meshwork (TM) is central to intraocular pressure (IOP) regulation and glaucoma, a deeper understanding of its genomic landscape is needed. We present a multimodal, single-cell resolution analysis of mouse limbal cells (includes TM). In total, we sequenced 9,394 wild-type TM cell transcriptomes. We discovered three TM cell subtypes with characteristic signature genes validated by immunofluorescence on tissue sections and whole-mounts. The subtypes are robust, being detected in datasets for two diverse mouse strains and in independent data from two institutions. Results show compartmentalized enrichment of critical pathways in specific TM cell subtypes. Distinctive signatures include increased expression of genes responsible for 1) extracellular matrix structure and metabolism (TM1 subtype), 2) secreted ligand signaling to support Schlemm's canal cells (TM2), and 3) contractile and mitochondrial/metabolic activity (TM3). ATAC-sequencing data identified active transcription factors in TM cells, including LMX1B. Mutations in LMX1B cause high IOP and glaucoma. LMX1B is emerging as a key transcription factor for normal mitochondrial function and its expression is much higher in TM3 cells than other limbal cells. To understand the role of LMX1B in TM function and glaucoma, we single-cell sequenced limbal cells from Lmx1b V265D/+ mutant mice. In V265D/+ mice, TM3 cells were uniquely affected by pronounced mitochondrial pathway changes. This supports a primary role of mitochondrial dysfunction within TM3 cells in initiating the IOP elevation that causes glaucoma in these mice. Importantly, treatment with vitamin B 3 (nicotinamide), to enhance mitochondrial function and metabolic resilience, significantly protected Lmx1b mutant mice from IOP elevation.

Schlemm's canal (SC) is central in intraocular pressure regulation but requires much characterization. It has distinct inner and outer walls, each composed of Schlemm's canal endothelial cells (SECs) with different morphologies and functions. Recent transcriptomic studies of the anterior segment added important knowledge, but were limited in power by SEC numbers or did not focus on SC. To gain a more comprehensive understanding of SC biology, we performed bulk RNA sequencing on C57BL/6 J SC, blood vessel, and lymphatic endothelial cells from limbal tissue (~4,500 SECs). We also analyzed mouse limbal tissues by single-cell and single-nucleus RNA sequencing (C57BL/6 J and 129/Sj strains), successfully sequencing 903 individual SECs. Together, these datasets confirm that SC has molecular characteristics of both blood and lymphatic endothelia with a lymphatic phenotype predominating. SECs are enriched in pathways that regulate cell-cell junction formation pointing to the importance of junctions in determining SC fluid permeability. Importantly, and for the first time, our analyses characterize three molecular classes of SECs, molecularly distinguishing inner wall from outer wall SECs and discovering two inner wall cell states that likely result from local environmental differences. Further, and based on ligand and receptor expression patterns, we document key interactions between SECs and cells of the adjacent trabecular meshwork (TM) drainage tissue. Also, we present cell type expression for a collection of human glaucoma genes. These data provide a new molecular foundation that will enable the functional dissection of key homeostatic processes mediated by SECs as well as the development of new glaucoma therapeutics.

Primary open-angle glaucoma (POAG), a leading cause of irreversible vision loss, is closely linked to increased intraocular pressure (IOP), with the trabecular meshwork (TM) playing a critical role in its regulation. The TM, located at the iridocorneal angle, acts as a sieve, filtering the aqueous humor from the eye into the collecting ducts, thus maintaining proper IOP levels. The transforming growth factor-beta 2 (TGF-β2) signaling pathway has been implicated in the pathophysiology of primary open-angle glaucoma POAG particularly, in the dysfunction of the TM. This study utilizes human TM explants to closely mimic in vivo conditions, thereby minimizing transcriptional changes that could arise from cell culture enabling an exploration of the transcriptomic impacts of TGF-β2. Through bulk RNA sequencing and immunohistological analysis, we identified distinct gene expression patterns and morphological changes induced by TGF-β2 exposure (5 ng/ml for 48 h). Bulk RNA sequencing identified significant upregulation in genes linked to extracellular matrix (ECM) regulation and fibrotic signaling. Immunohistological analysis further elucidated the morphological alterations, including cytoskeletal rearrangements and ECM deposition, providing a visual confirmation of the transcriptomic data. Notably, the enrichment analysis unveils TGF-β2's influence on both bone morphogenic protein (BMP) and Wnt signaling pathways, suggesting a complex interplay of molecular mechanisms contributing to TM dysfunction in glaucoma. This characterization of the transcriptomic modifications on an explant model of TM obtained under the effect of this profibrotic cytokine involved in glaucoma is crucial in order to develop and test new molecules that can block their signaling pathways.

Schlemm's canal (SC) is central in intraocular pressure regulation but requires much characterization. It has distinct inner and outer walls, each composed of Schlemm's canal endothelial cells (SECs) with different morphologies and functions. Recent transcriptomic studies of the anterior segment added important knowledge, but were limited in power by SEC numbers or did not focus on SC. To gain a more comprehensive understanding of SC biology, we performed bulk RNA sequencing on C57BL/6J SC, blood vessel, and lymphatic endothelial cells from limbal tissue (~4500 SECs). We also analyzed mouse limbal tissues by single-cell and single-nucleus RNA sequencing (C57BL/6J and 129/Sj strains), successfully sequencing 903 individual SECs. Together, these datasets confirm that SC has molecular characteristics of both blood and lymphatic endothelia with a lymphatic phenotype predominating. SECs are enriched in pathways that regulate cell-cell junction formation pointing to the importance of junctions in determining SC fluid permeability. Importantly, and for the first time, our analyses characterize 3 molecular classes of SECs, molecularly distinguishing inner wall from outer wall SECs and discovering two inner wall cell states that likely result from local environmental differences. Further, and based on ligand and receptor expression patterns, we document key interactions between SECs and cells of the adjacent trabecular meshwork (TM) drainage tissue. Also, we present cell type expression for a collection of human glaucoma genes. These data provide a new molecular foundation that will enable the functional dissection of key homeostatic processes mediated by SECs as well as the development of new glaucoma therapeutics.

Adding 50% vitreous humor to the media surrounding lens explants induces fiber cell differentiation and a significant immune/inflammatory response. While Fgfr loss blocks differentiation in lens epithelial explants, this blockage is partially reversed by deleting Pten. To investigate the functions of the Fgfrs and Pten during lens fiber cell differentiation, we utilized a lens epithelial explant system and conducted RNA sequencing on vitreous humor-exposed explants lacking Fgfrs, or Pten or both Fgfrs and Pten. We found that Fgfr loss impairs both vitreous-induced differentiation and inflammation while the additional loss of Pten restores these responses. Furthermore, transcriptomic analysis suggested that PDGFR-signaling in FGFR-deficient explants is required to mediate the rescue of vitreous-induced fiber differentiation in explants lacking both Fgfrs and Pten. The blockage of β-crystallin induction in explants lacking both Fgfrs and Pten in the presence of a PDGFR inhibitor supports this hypothesis. Our findings demonstrate that a wide array of genes associated with fiber cell differentiation are downstream of FGFR-signaling and that the vitreous-induced immune responses also depend on FGFR-signaling. Our data also demonstrate that many of the vitreous-induced gene-expression changes in Fgfr-deficient explants are rescued in explants lacking both Fgfrs and Pten.

Uveitis is a heterogenous group of inflammatory eye disease for which current cytokine-targeted immune therapies are effective for only a subset of patients. We hypothesized that despite pathophysiologic nuances that differentiate individual disease states, all forms of eye inflammation might share common mechanisms for immune cell recruitment. Identifying these mechanisms is critical for developing novel, broadly acting therapeutic strategies.

Experimental study.

Biospecimens from patients with active or inactive uveitis and healthy controls.

Protein concentration and single cell gene expression were assessed in aqueous fluid biopsies and plasma samples from deidentified patients with uveitis or healthy controls.

The concentration of 31 inflammatory proteins was measured in all aqueous samples, as well as plasma samples from patients with active uveitis. Chemokine and cytokine ligand and receptor expression were assessed in individual cell types from aqueous biopsies obtained from patients with active uveitis.

We identified 6 chemokines that were both elevated in active uveitis compared with controls and enriched in aqueous compared with plasma during active uveitis (C-C motif chemokine ligand [CCL]2, C-X-C motif chemokine ligand [CXCL]10, CXCL9, CXCL8, CCL3, and CCL14), forming potential gradients for migration of immune cells from the blood to the eye. Of these, CCL2 and CXCL10 were consistently enriched in the aqueous of all patients in our cohort, as well as in a larger cohort of patients from a previously published study. These data suggest that CCL2 and CXCL10 are key mediators in immune cell migration to the eye during uveitis. Next, single cell RNA sequencing suggested that macrophages contribute to aqueous enrichment of CCL2 and CXCL10 during human uveitis. Finally, using chemokine ligand and receptor expression mapping, we identified a broad signaling network for macrophage-derived CCL2 and CXCL10 in human uveitis.

These data suggest that ocular macrophages may play a central role, via CCL2 and CXCL10 production, in recruiting inflammatory cells to the eye in patients with uveitis.

Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Despite the advent of genomic sequencing, molecular diagnosis remains unsolved in approximately half of patients with Mendelian disorders, largely due to unclarified functions of noncoding regions and the difficulty in identifying complex structural variations. In this study, we map a unique form of central iris hypoplasia in a large family to 6q15-q23.3 and 18p11.31-q12.1 using a genome-wide linkage scan. Long-read sequencing reveals a balanced translocation t(6;18)(q22.31;p11.22) with intergenic breakpoints. By performing Hi-C on induced pluripotent stem cells from a patient, we identify two chromatin topologically associating domains spanning across the breakpoints. These alterations lead the ectopic chromatin interactions between APCDD1 on chromosome 18 and enhancers on chromosome 6, resulting in upregulation of APCDD1. Notably, APCDD1 is specifically localized in the iris of human eyes. Our findings demonstrate that noncoding structural variations can lead to Mendelian diseases by disrupting the 3D genome structure and resulting in altered gene expression.

The conjunctiva is a non-keratinized, stratified columnar epithelium with characteristics different from the cornea and eyelid epidermis. From development to adulthood, a distinguishing feature of ocular versus epidermal epithelia is the expression of the master regulator PAX6. A conditionally immortalized conjunctival epithelial cell line (iHCjEC) devoid of stromal or immune cells established in our laboratory spontaneously manifested epidermal metaplasia and upregulated expression of the keratinization-related genes SPRR1A/B and the epidermal cytokeratins KRT1 and KRT10 at the expense of the conjunctival trait. In addition, iHCjEC indicated a significant decrease in PAX6 expression. Dry eye syndrome (DES) and severe ocular surface diseases, such as Sjögren's syndrome and Stevens-Johnson syndrome, cause the keratinization of the entire ocular surface epithelia. We used iHCjECs as a conjunctiva epidermal metaplasia model to test PAX6, serum, and glucocorticoid interventions. Reintroducing PAX6 to iHCjECs resulted in upregulating genes related to cell adhesion and tight junctions, including MIR200CHG and CLDN1. The administration of glucocorticoids or serum resulted in the downregulation of epidermal genes (DSG1, SPRR1A/B, and KRT1) and partially corrected epidermal metaplasia. Our results using an isolated conjunctival epidermal metaplasia model point toward the possibility of rationally "repurposing" clinical interventions, such as glucocorticoid, serum, or PAX6 administration, for treating epidermal metaplasia of the conjunctiva.

To report a 15 year old girl with citrullinemia type 1 and 2 accompanied by neurologic signs and symptoms and a novel ocular complaint in cornea like tyrosinemia type 2.

A 15 year old female was admitted with decreased consciousness and neurologic signs and symptoms. Citrulinemia was discovered through metabolic testing. Later genetic studies revealed mutations in both ASS1 and SLC25A13 genes. Two years after the first presentation, the patient was re-admitted with complaints of bilateral photophobia and tearing. Biomicroscopic examination revealed bilateral corneal haziness with pseudodendritic lesions like tyrosinemia type 2 that were subsided with protein restriction and the use of urea cycle disease (UCD) formula.

Citrullinemia is the inherited autosomal recessive disorder of urea cycle that leads to ammonia and accumulation of other toxic substances in the blood. Two types of Citrullinemia have been defined. Citrullinemia type 1, caused by deficiency or reduction in argininosuccinate synthetase enzyme activity due to damaging mutation in ASS1 gene. Citrullinemia type 2 as another subtype is caused by the absence or dysfunction of the mitochondrial membrane carrier protein (SLC25A13), also called CITRIN. Pseudodendritic keratitis is a rare condition that may be seen with tyrosinemia type 2. The association of this ocular complaint with citrullinemia has not been described previously. Awareness of this phenomenon may improve the diagnosis and management of citrullinemia patients.

Ophthalmic diseases affect many people, causing partial or total loss of vision and a reduced quality of life. The anterior segment of the eye accounts for nearly half of all visual impairment that can lead to blindness. Therefore, there is a growing demand for ocular research and regenerative medicine that specifically targets the anterior segment to improve vision quality. This study aims to generate a microfluidic platform for investigating the formation of the anterior segment of the eye derived from human induced pluripotent stem cells (hiPSC) under various spatial-mechanoresponsive conditions. Microfluidic platforms are developed to examine the effects of dynamic conditions on the generation of hiPSCs-derived ocular organoids. The differentiation protocol is validated, and mechanoresponsive genes are identified through transcriptomic analysis. Several culture strategies is implemented for the anterior segment of eye cells in a microfluidic chip. hiPSC-derived cells showed anterior eye cell characteristics in mRNA and protein expression levels under dynamic culture conditions. The expression levels of yes-associated protein and transcriptional coactivator PDZ binding motif (YAP/TAZ) and PIEZO1, varied depending on the differentiation and growth conditions of the cells, as well as the metabolomic profiles under dynamic culture conditions.

Dry eye disease (DED) affects a substantial worldwide population with increasing frequency. Current single-targeting DED management is severely hindered by the existence of an oxidative stress-inflammation vicious cycle and complicated intercellular crosstalk within the ocular microenvironment. Here, a nanozyme-based eye drop, namely nanoceria loading cyclosporin A (Cs@P/CeO2), is developed, which possesses long-term antioxidative and anti-inflammatory capacities due to its regenerative antioxidative activity and sustained release of cyclosporin A (CsA). In vitro studies showed that the dual-functional Cs@P/CeO2 not only inhibits cellular reactive oxygen species production, sequentially maintaining mitochondrial integrity, but also downregulates inflammatory processes and repolarizes macrophages. Moreover, using flow cytometric and single-cell sequencing data, the in vivo therapeutic effect of Cs@P/CeO2 was systemically demonstrated, which rebalances the immune-epithelial communication in the corneal microenvironment with less inflammatory macrophage polarization, restrained oxidative stress, and enhanced epithelium regeneration. Collectively, our data proved that the antioxidative and anti-inflammatory Cs@P/CeO2 may provide therapeutic insights into DED management.

Inflammatory lymphangiogenesis is intimately linked to immune regulation and tissue homeostasis. However, current evidence has suggested that classic lymphatic vessels are physiologically absent in intraocular structures. Here, we show that neolymphatic vessels were induced in the iris after corneal alkali injury (CAI) in a VEGFR3-dependent manner. Cre-loxP-based lineage tracing revealed that these lymphatic endothelial cells (LECs) originate from existing Prox1+ lymphatic vessels. Notably, the ablation of iridial lymphangiogenesis via conditional deletion of VEGFR3 alleviated the ocular inflammatory response and pathological T cell infiltration. Our findings demonstrate that iridial neolymphatics actively participate in pathological immune responses following injury and suggest intraocular lymphangiogenesis as a valuable therapeutic target for the treatment of ocular inflammation.

Some of the earliest studies of glycans were performed on mammalian corneas and lenses with many of the key concepts we currently recognize as being fundamental to our understanding of basic cell biology arising from these studies. Proteoglycans and their GAG side chains are essential components of the ECM of the lens capsule. They also are present in the anterior corneal epithelial basement membrane and the posterior (Decemet's) basement membrane, and they organize collagen fiber diameters and spacing in the corneal stroma to maintain stromal clarity. Studies using genetically engineered mice and characterization of spontaneously arising mutations in genes controlling proteoglycan synthesis have generated new insight into the roles played by proteoglycans in signal transduction. We now know that proteoglycans and GAGs can regulate cell signaling and the maintenance of avascularity and immune privilege that are hallmarks of these tissues. In addition, proteoglycan-rich matrices provide the pathways for immune cells to populate the surface of the lens as a response to corneal wounding and in a model of Experimental Autoimmune Uveitis. Here we describe what is known about proteoglycans and GAGs in the cornea and lens. This knowledge has begun to provide promising leads into new proteoglycan-based treatments aimed at restoring and maintaining homeostasis in the cornea. Future studies are needed to determine how these new drugs impact the recruitment of immune cells to the lens for functions in restoring/maintaining homeostasis in the eye.

This review highlights the importance of extracellular matrix (ECM) biomaterials in understanding the biology of human trabecular meshwork (TM) and Schlemm's canal (SC) cells under normal and simulated glaucoma-like conditions. We provide an overview of recent progress in the development and application of state-of-the-art 3D ECM biomaterials including cell-derived ECM, ECM scaffolds, Matrigel, and ECM hydrogels for studies of TM and SC cell (patho)biology. Such bioengineered platforms enable accurate and reliable modeling of tissue-like cell-cell and cell-ECM interactions. They bridge the gap between conventional 2D approaches and in vivo/ex vivo models, and have the potential to aid in the identification of the causal mechanism(s) for outflow dysfunction in ocular hypertensive glaucoma. We discuss each model's benefits and limitations, and close with an outlook on future directions.

Vogt-Koyanagi-Harada (VKH) disease and Behcet's uveitis (BU) are the two major vision-threatening uveitis entities. This study performed the first label-free quantitative proteomics on aqueous humor-derived exosomes from 84 patients with VKH or BU to determine their potential roles. Sixty-five differentially expressed proteins (DEPs) and 40 DEPs were detected in the VKH and BU groups, respectively. GO and KEGG analysis showed that DEPs were mainly enriched in the complement-related pathways. The complement C1q subcomponent subunit B (C1QB) was identified as a key exosomal protein, and its expression was significantly increased by western blotting in both diseases. Additionally, the integrated analysis based on the published scRNA-seq data showed that C1QB-containing exosomes were mainly produced by mononuclear macrophages in the anterior segment tissue. Overall, our proteomic profiling highlights that complement-related pathways may be actively involved in the pathogenesis of these two diseases. These pathways may also serve as treatment targets for both diseases.

The origin and evolution of cell types has emerged as a key topic in evolutionary biology. Driven by rapidly accumulating single-cell datasets, recent attempts to infer cell type evolution have largely been limited to pairwise comparisons because we lack approaches to build cell phylogenies using model-based approaches. Here we approach the challenges of applying explicit phylogenetic methods to single-cell data by using principal components as phylogenetic characters. We infer a cell phylogeny from a large, comparative single-cell dataset of eye cells from five distantly related mammals. Robust cell type clades enable us to provide a phylogenetic, rather than phenetic, definition of cell type, allowing us to forgo marker genes and phylogenetically classify cells by topology. We further observe evolutionary relationships between diverse vessel endothelia and identify the myelinating and non-myelinating Schwann cells as sister cell types. Finally, we examine principal component loadings and describe the gene expression dynamics underlying the function and identity of cell type clades that have been conserved across the five species. A cell phylogeny provides a rigorous framework towards investigating the evolutionary history of cells and will be critical to interpret comparative single-cell datasets that aim to ask fundamental evolutionary questions.

Primary open-angle glaucoma (POAG), characterized by retinal ganglion cell death, is a leading cause of irreversible blindness worldwide. However, its molecular and cellular causes are not well understood. Elevated intraocular pressure (IOP) is a major risk factor, but many patients have normal IOP. Colocalization and Mendelian randomization analysis of >240 POAG and IOP genome-wide association study (GWAS) loci and overlapping expression and splicing quantitative trait loci (e/sQTLs) in 49 GTEx tissues and retina prioritizes causal genes for 60% of loci. These genes are enriched in pathways implicated in extracellular matrix organization, cell adhesion, and vascular development. Analysis of single-nucleus RNA-seq of glaucoma-relevant eye tissues reveals that the POAG and IOP colocalizing genes and genome-wide associations are enriched in specific cell types in the aqueous outflow pathways, retina, optic nerve head, peripapillary sclera, and choroid. This study nominates IOP-dependent and independent regulatory mechanisms, genes, and cell types that may contribute to POAG pathogenesis.

Proper differentiation of corneal epithelial cells (CECs) from limbal stem/progenitor cells (LSCs) is required for maintenance of ocular homeostasis and clear vision. Here, using a single-cell transcriptomic atlas, we delineate the comprehensive and refined molecular regulatory dynamics during human CEC development and differentiation. We find that RORA is a CEC-specific molecular switch that initiates and drives LSCs to differentiate into mature CECs by activating PITX1. RORA dictates CEC differentiation by establishing CEC-specific enhancers and chromatin interactions between CEC gene promoters and distal regulatory elements. Conversely, RORA silences LSC-specific promoters and disrupts promoter-anchored chromatin loops to turn off LSC genes. Collectively, our work provides detailed and comprehensive insights into the transcriptional dynamics and RORA-mediated epigenetic remodeling underlying human corneal epithelial differentiation.

Diabetic keratopathy (DK) is a vision-threatening disease that occurs in people with diabetes. Mounting evidence indicates that microRNAs (miRNAs) are indispensable in nerve regeneration within DK. Herein, the role of miRNAs associated with DK, especially focusing on autophagy and apoptosis regulation, was investigated.

To identify differentially expressed miRNAs, we performed miRNA sequencing on trigeminal ganglion (TG) tissues derived from streptozotocin-induced type 1 diabetic mellitus (T1DM) and normal mice. MiR-144-3p was chosen for the subsequent experiments. To explore the regulatory role of miR-144-3p in DK, miRNA antagomir was utilized to inhibit miR-144-3p expression. Bioinformatic tools were used to predict the target genes of miR-144-3p, and a dual-luciferase reporter assay was then applied for validation. Autophagy and apoptosis activities were measured utilizing TUNEL staining, immunofluorescence staining, and Western blotting.

Overall, 56 differentially expressed miRNAs were detected in diabetic versus control mice. In the diabetic mouse TG tissue, miR-144-3p expression was aberrantly enhanced, whereas decreasing its expression contributed to improved diabetic corneal re-epithelialization and nerve regeneration. Fork-head Box O1 (FOXO1) was validated as a target gene of miR-144-3p. Overexpression of FOXO1 could prevent both inadequate autophagy and excessive apoptosis in DK. Consistently, a specific miR-144-3p inhibition enhanced autophagy and prevented apoptosis in DK.

In this study, our research confirmed the target binding relationship between miR-144-3p and FOXO1. Inhibiting miR-144-3p might modulate autophagy and apoptosis, which could generate positive outcomes for corneal nerves via targeting FOXO1 in DK.

Ocular lens development entails epithelial to fiber cell differentiation, defects in which cause congenital cataracts. We report the first single-cell multiomic atlas of lens development, leveraging snRNA-seq, snATAC-seq and CUT&RUN-seq to discover previously unreported mechanisms of cell fate determination and cataract-linked regulatory networks. A comprehensive profile of cis- and trans-regulatory interactions, including for the cataract-linked transcription factor MAF, is established across a temporal trajectory of fiber cell differentiation. Furthermore, we identify an epigenetic paradigm of cellular differentiation, defined by progressive loss of the H3K27 methylation writer Polycomb repressive complex 2 (PRC2). PRC2 localizes to heterochromatin domains across master-regulator transcription factor gene bodies, suggesting it safeguards epithelial cell fate. Moreover, we demonstrate that FGF hyper-stimulation in vivo leads to MAF network activation and the emergence of novel lens cell states. Collectively, these data depict a comprehensive portrait of lens fiber cell differentiation, while defining regulatory effectors of cell identity and cataract formation.

Glaucoma is the leading cause of irreversible blindness worldwide. Currently the only effective treatment for glaucoma is to reduce the intraocular pressure, which can halt the progression of the disease. Highlighting the importance of identifying individuals at risk of developing glaucoma and those with early-stage glaucoma will help patients receive treatment before sight loss. However, some cases of glaucoma do not have raised intraocular pressure. In fact, glaucoma is caused by a variety of different mechanisms and has a wide range of different subtypes. Understanding other risk factors, the underlying mechanisms, and the pathology of glaucoma might lead to novel treatments and treatment of underlying diseases. In this review we present the latest research into glaucoma including the genetics and molecular basis of the disease.

In primary open-angle glaucoma (POAG), lowering intraocular pressure (IOP) is the only proven way of slowing vision loss. Schlemm's canal (SC) is a hybrid vascular and lymphatic vessel that mediates aqueous humour drainage from the anterior ocular chamber. Animal studies support the importance of SC endothelial angiopoietin-TEK signalling, and more recently TIE1 signalling, in maintaining normal IOP. However, human genetic support for a causal role of TIE1 and TEK signalling in lowering IOP is currently lacking.

GWAS summary statistics were obtained for plasma soluble TIE1 (sTIE1) protein levels (N = 35,559), soluble TEK (sTEK) protein levels (N = 35,559), IOP (N = 139,555) and POAG (Ncases = 16,677, Ncontrols = 199,580). Mendelian randomization (MR) was performed to estimate the association of genetically proxied TIE1 and TEK protein levels with IOP and POAG liability. Where significant MR estimates were obtained, genetic colocalization was performed to assess the probability of a shared causal variant (PPshared) versus distinct (PPdistinct) causal variants underlying TIE1/TEK signalling and the outcome. Publicly available single-nucleus RNA-sequencing data were leveraged to investigate differential expression of TIE1 and TEK in the human ocular anterior segment.

Increased genetically proxied TIE1 signalling and TEK signalling associated with a reduction in IOP (- 0.21 mmHg per SD increase in sTIE1, 95% CI = - 0.09 to - 0.33 mmHg, P = 6.57 × 10-4, and - 0.14 mmHg per SD decrease in sTEK, 95% CI = - 0.03 to - 0.25 mmHg, P = 0.011), but not with POAG liability. Colocalization analysis found that the probability of a shared causal variant was greater for TIE1 and IOP than for TEK and IOP (PPshared/(PPdistinct + PPshared) = 0.98 for TIE1 and 0.30 for TEK). In the anterior segment, TIE1 and TEK were preferentially expressed in SC, lymphatic, and vascular endothelium.

This study provides novel human genetic support for a causal role of both TIE1 and TEK signalling in regulating IOP. Here, combined evidence from cis-MR and colocalization analyses provide stronger support for TIE1 than TEK as a potential IOP-lowering therapeutic target.

Single-cell sequencing has revolutionized the scale and resolution of molecular profiling of tissues and organs. Here, we present an integrated multimodal reference atlas of the most accessible portion of the mammalian central nervous system, the retina. We compiled around 2.4 million cells from 55 donors, including 1.4 million unpublished data points, to create a comprehensive human retina cell atlas (HRCA) of transcriptome and chromatin accessibility, unveiling over 110 types. Engaging the retina community, we annotated each cluster, refined the Cell Ontology for the retina, identified distinct marker genes, and characterized cis-regulatory elements and gene regulatory networks (GRNs) for these cell types. Our analysis uncovered intriguing differences in transcriptome, chromatin, and GRNs across cell types. In addition, we modeled changes in gene expression and chromatin openness across gender and age. This integrated atlas also enabled the fine-mapping of GWAS and eQTL variants. Accessible through interactive browsers, this multimodal cross-donor and cross-lab HRCA, can facilitate a better understanding of retinal function and pathology.