Decoding cellular plasticity and niche regulation of limbal stem cells during corneal wound healing.

BACKGROUND: Dysfunction or deficiency of corneal epithelium results in vision impairment or blindness in severe cases. The rapid and effective regeneration of corneal epithelial cells relies on the limbal stem cells (LSCs). However, the molecular and functional responses of LSCs and their niche cells to injury remain elusive. METHODS: Single-cell RNA sequencing was performed on corneal tissues from normal mice and corneal epithelium defect models. Bioinformatics analysis was performed to confirm the distinct characteristics and cell fates of LSCs. Knockdown of Creb5 and OSM treatment experiment were performed to determine their roles of in corneal epithelial wound healing. RESULTS: Our data defined the molecular signatures of LSCs and reconstructed the pseudotime trajectory of corneal epithelial cells. Gene network analyses characterized transcriptional landmarks that potentially regulate LSC dynamics, and identified a transcription factor Creb5, that was expressed in LSCs and significantly upregulated after injury. Loss-of-function experiments revealed that silencing Creb5 delayed the corneal epithelial healing and LSC mobilization. Through cell-cell communication analysis, we identified 609 candidate regeneration-associated ligand-receptor interaction pairs between LSCs and distinct niche cells, and discovered a unique subset of Arg1+ macrophages infiltrated after injury, which were present as the source of Oncostatin M (OSM), an IL-6 family cytokine, that were demonstrated to effectively accelerate the corneal epithelial wound healing. CONCLUSIONS: This research provides a valuable single-cell resource and reference for the discovery of mechanisms and potential clinical interventions aimed at ocular surface reconstruction.

Penetrative Ionic Organic Molecular Cage Nanozyme for the Targeted Treatment of Keratomycosis.

Keratomycosis, caused by pathogenic fungi, is an intractable blinding eye disease. Corneal penetration is an essential requirement for conventional antifungal medications to address keratomycosis. Due to the distinctive anatomical and physiological structure of the cornea, the therapeutic efficacy is hampered by the inadequate penetration capacity. Despite the emergence of diverse antifungal drug delivery systems and advanced antifungal nanomaterials, it has remained challenging to achieve corneal penetration over the past decade. This study fabricates a penetrative ionic organic molecular cage-based nanozyme (OMCzyme) for treating keratomycosis. The synthesis of OMCzyme involved two steps. Initially, the ionic OMC is synthesized by a [2+3] cycloimination reaction of triformylphloroglucinol and 2,3-diaminopropionic acid. Subsequently, OMCzyme is fabricated by coordination of Fe2⁺ with carboxyl anions and phenolic hydroxyls in the organic cage, and further deposition of silver nanoparticles on the surface of OMC-Fe complex. The as-prepared OMCzyme demonstrates excellent water dispersion, peroxidase-like activity, in vitro and in vivo biocompatibility, and corneal penetration. Notably, the nanozyme displays targeted antifungal activity, effectively combating Fusarium solani with negligible cytotoxicity toward human corneal epithelial cells. The hybrid mimic is further demonstrated to be effective in treating keratomycosis in mice, indicating the potential of OMCzyme for curing fungal infectious diseases.

Sleep deprivation induces corneal endothelial dysfunction by downregulating Bmal1.

BACKGROUND: Sleep deprivation (SD) is a common public health problem that contributes to various physiological disorders and increases the risk of ocular diseases. However, whether sleep loss can damage corneal endothelial function remains unclear. This study aimed to determine the effect and possible mechanism of SD on the corneal endothelium. METHODS: Male C57BL/6J mice were subjected to establish SD models. After 10 days, quantitative RT-PCR (qRT-PCR) and western blot or immunostaining for the expression levels of zonula occludens-1 (ZO-1), ATPase Na+/K + transporting subunit alpha 1 (Atp1a1), and core clock genes in the corneal endothelium were evaluated. Reactive oxygen species staining and mitochondrial abundance characterized the mitochondrial function. The regulatory role of Bmal1 was confirmed by specifically knocking down or overexpressing basic helix-loop-helix ARNT like 1 protein (Bmal1) in vivo. In vitro, a mitochondrial stress test was conducted on cultured human corneal endothelial cells upon Bmal1 knockdown. RESULTS: SD damaged the barrier and pump functions of mouse corneal endothelium, accompanied by mitochondrial dysfunction. Interestingly, SD dramatically downregulated the core clock gene Bmal1 expression level. Bmal1 knockdown disrupted corneal endothelial function, while overexpression of Bmal1 ameliorated the dysfunction induced by SD. Mitochondrial bioenergetic deficiency mediated by Bmal1 was an underlying mechanism for SD induced corneal endothelial dysfunction. CONCLUSION: The downregulation of Bmal1 expression caused by SD led to corneal endothelial dysfunction via impairing mitochondrial bioenergetics. Our findings offered insight into how SD impairs the physiological function of the corneal endothelium and expanded the understanding of sleep loss leading to ocular diseases.

Single-Nuclei Characterization of Lacrimal Gland in Scopolamine-Induced Dry Eye Disease.

Purpose: The lacrimal gland (LG) is the main organ responsible for tear secretion and an important pathogenic site for dry eye disease (DED). This study aimed to comprehensively characterize LG cellular heterogeneity under normal and DED conditions using single-nucleus RNA sequencing (snRNA-seq). Methods: Single LG nuclei isolated from mice with or without DED induced by scopolamine (SCOP)/desiccating stress (DS) were subjected to snRNA-seq using the 10x Genomics platform. These cells were clustered and annotated using the t-distributed stochastic neighbor embedding (t-SNE) method and unbiased computational informatic analysis. Cluster identification and functional analysis were performed based on marker gene expression and bioinformatic data mining. Results: The snRNA-seq analysis of 30,351 nuclei identified eight major cell types, with acinar cells (∼72.6%) being the most abundant cell type in the LG. Subclustering analysis revealed that the LG mainly contained two acinar cell subtypes, two ductal cell subclusters, three myoepithelial cell (MECs) subtypes, and four immunocyte subclusters. In the SCOP-induced DED model, three major LG parenchymal cell types were significantly altered, characterized by a reduced proportion of acinar cells with a lowered secretion potential and an augmented proportion of ductal cells and MECs. LG immunocytes in DED scenarios showed an intensified inflammatory response and dysregulated intercellular communication with three major LG parenchymal cells. Conclusions: Overall, this study offers a systemic single-nucleus transcriptomic profile of LGs in both normal and DED conditions and an atlas of the complicated interactions of immunocytes with major LG parenchymal cells. The findings also facilitate understanding the pathogenesis of DED.

NEAT1 Deficiency Promotes Corneal Epithelial Wound Healing by Activating cAMP Signaling Pathway.

Purpose: This study aimed to investigate the role of the long non-coding RNA (lncRNA) NEAT1 in corneal epithelial wound healing in mice. Methods: The central corneal epithelium of wild-type (WT), MALAT1 knockout (M-KO), NEAT1 knockout (N-KO), and NEAT1 knockdown (N-KD) mice was scraped to evaluate corneal epithelial and nerve regeneration rates. RNA sequencing of the corneal epithelium from WT and N-KO mice was performed 24 hours after debridement to determine the role of NEAT1. Quantitative PCR (qPCR) and ELISA were used to confirm the bioinformatic analysis. The effects of the cAMP signaling pathway were evaluated in N-KO and N-KD mice using SQ22536, an adenylate cyclase inhibitor. Results: Central corneal epithelial debridement in N-KO mice significantly promoted epithelial and nerve regeneration rates while suppressing inflammatory cell infiltration. Furthermore, the expression of Atp1a2, Ppp1r1b, Calm4, and Cngb1, which are key components of the cAMP signaling pathway, was upregulated in N-KO mice, indicative of its activation. Furthermore, the cAMP pathway inhibitor SQ22536 reversed the accelerated corneal epithelial wound healing in both N-KO and N-KD mice. Conclusions: NEAT1 deficiency contributes to epithelial repair during corneal wound healing by activating the cAMP signaling pathway, thereby highlighting a potential therapeutic strategy for corneal epithelial diseases.

New dawn for keratoconus treatment: potential strategies for corneal stromal regeneration.

Keratoconus is a progressive, ectatic and blinding disorder of the cornea, characterized by thinning of corneal stroma. As a highly prevalent among adolescents, keratoconus has been a leading indication for corneal transplantation worldwide. However, the severe shortage of donor corneas is a global issue, and the traditional corneal transplantation surgeries may superinduce multiple complications, necessitating efforts to develop more effective strategies for keratoconus treatment. In this review, we summarized several strategies to promote corneal stromal regeneration or improve corneal stromal thickness, including cell-based therapies, biosynthetic alternatives for inducing corneal regeneration, minimally invasive intrastromal implantation and bioengineered tissues for implantation. These strategies provided more accessible but safer alternatives from various perspectives for keratoconus treatment, paving the way for arresting the keratoconus progression in its earlier stage. For the treatments of corneal ectatic diseases beyond keratoconus, these approaches will provide important references and widen the therapy options in a donor tissue-independent manner.

Single-cell RNA sequencing in cornea research: Insights into limbal stem cells and their niche regulation.

The corneal epithelium is composed of stratified squamous epithelial cells on the outer surface of the eye, which acts as a protective barrier and is critical for clear and stable vision. Its continuous renewal or wound healing depends on the proliferation and differentiation of limbal stem cells (LSCs), a cell population that resides at the limbus in a highly regulated niche. Dysfunction of LSCs or their niche can cause limbal stem cell deficiency, a disease that is manifested by failed epithelial wound healing or even blindness. Nevertheless, compared to stem cells in other tissues, little is known about the LSCs and their niche. With the advent of single-cell RNA sequencing, our understanding of LSC characteristics and their microenvironment has grown considerably. In this review, we summarized the current findings from single-cell studies in the field of cornea research and focused on important advancements driven by this technology, including the heterogeneity of the LSC population, novel LSC markers and regulation of the LSC niche, which will provide a reference for clinical issues such as corneal epithelial wound healing, ocular surface reconstruction and interventions for related diseases.

Interference of sympathetic overactivation restores limbal stem/progenitor cells function and accelerates corneal epithelial wound healing in diabetic mice.

Diabetic keratopathy (DK), the diabetic complication in the cornea, is characterized by the delayed epithelial regeneration and sensory nerve degeneration. The involvement of limbal stem/progenitor cells (LSPCs) dysfunction has been reported, however the pathogenic mechanisms remain unclear. Here, we confirmed the dysfunction of LSPCs in diabetic mouse and human corneas. The sympathetic nerve in the cornea was adjacent to LSPCs, and the sympathetic overactivation was found in diabetic mice. Surgical and pharmacological ablation of sympathetic nerves rescued the LSPCs function and promoted corneal epithelial regeneration in diabetic mice. In contrast, both topical norepinephrine (NE) application and chemogenetic sympathetic overactivation directly impaired the stemness and proliferation characteristics of LSPCs, as well as the normal epithelial regeneration. Moreover, we identified that ß2-adrenoceptor (Adrb2) was the predominant adrenergic receptor expressed in LSPCs by corneal limbal single-cell sequencing and real time PCR (RT-PCR) analysis of sorted LSPCs. The Adrb2 knockout mice exhibited the enhancement of epithelial regeneration and LSPCs function, compared with the wild-type mice. Similarly, topical application of the Adrb2 specific antagonist ICI 118, 551 effectively accelerated diabetic corneal epithelial regeneration with the restored LSPCs function. Mechanistically, sonic hedgehog (Shh) activity mediated the downstream effects of NE-Adrb2 signaling pathway in regulating LSPCs and epithelial regeneration. Taken together, our data revealed the involvement of sympathetic overactivation in the impairment of diabetic LSPCs function and corneal epithelial regeneration through the NE-Adrb2-Shh signaling pathway. The interference of sympathetic overactivation may provide novel treatment strategies for diabetic keratopathy.

Targeting Type I IFN/STAT1 signaling inhibited and reversed corneal squamous metaplasia in Aire-deficient mouse.

Corneal transparency and integrity are essential for obtaining good vision; nevertheless, squamous metaplasia (SQM) of ocular epithelium is a kind of serious blinding corneal diseases, without therapeutic medication in clinic. Here, we found that deficiency of the autoimmune regulator (AIRE) in corneas spontaneously developed corneal plaques. Using corneal abrasion model, we revealed that deletion of Aire not only resulted in delayed corneal re-epithelialization, but also promoted a cell-fate transition from transparent corneal epithelium to keratinized epithelium, histopathologically characterized with SQM based on the transcriptomic analysis. Mechanistically, Aire-deficient corneas led to the heightened Type I interferon (IFN-I)/STAT1 signaling after abrasion. Pharmacological blockade of IFN-I/JAK/STAT1 signaling in Aire-knockout (KO) corneas not only accelerated epithelial wound healing, but also alleviated corneal plaques and SQM. Collectively, our findings revealed critical roles of AIRE in governing corneal epithelial homeostasis and pathologic keratinization, and further identified IFN-I/STAT1 signaling as a potential target for treating ocular surface diseases with SQM, and even for treating pathological scenarios related to SQM in other tissues.

Single-cell atlas of keratoconus corneas revealed aberrant transcriptional signatures and implicated mechanical stretch as a trigger for keratoconus pathogenesis.

Keratoconus is a common ectatic corneal disorder in adolescents and young adults that can lead to progressive visual impairment or even legal blindness. Despite the high prevalence, its etiology is not fully understood. In this study, we performed single-cell RNA sequencing (scRNA-Seq) analysis on 39,214 cells from central corneas of patients with keratoconus and healthy individuals, to define the involvement of each cell type during disease progression. We confirmed the central role of corneal stromal cells in this disease, where dysregulation of collagen and extracellular matrix (ECM) occurred. Differential gene expression and histological analyses revealed two potential novel markers for keratoconus stromal cells, namely CTSD and CTSK. Intriguingly, we detected elevated levels of YAP1 and TEAD1, the master regulators of biomechanical homeostasis, in keratoconus stromal cells. Cyclical mechanical experiments implicated the mechanical stretch in prompting protease production in corneal stromal cells during keratoconus progression. In the epithelial cells of keratoconus corneas, we observed reduced basal cells and abnormally differentiated superficial cells, unraveling the corneal epithelial lesions that were usually neglected in clinical diagnosis. In addition, several elevated cytokines in immune cells of keratoconus samples supported the involvement of inflammatory response in the progression of keratoconus. Finally, we revealed the dysregulated cell-cell communications in keratoconus, and found that only few ligand-receptor interactions were gained but a large fraction of interactional pairs was erased in keratoconus, especially for those related to protease inhibition and anti-inflammatory process. Taken together, this study facilitates the understanding of molecular mechanisms underlying keratoconus pathogenesis, providing insights into keratoconus diagnosis and potential interventions.

Pathogenic Role of Endoplasmic Reticulum Stress in Diabetic Corneal Endothelial Dysfunction.

PURPOSE: Progressive corneal edema and endothelial cell loss represent the major corneal complications observed in diabetic patients after intraocular surgery. However, the underlying pathogenesis and potential treatment remain incompletely understood. METHODS: We used streptozotocin-induced type 1 diabetic mice and db/db type 2 diabetic mice as diabetic animal models. These mice were treated with the endoplasmic reticulum (ER) stress agonist thapsigargin; 60-mmHg intraocular pressure (IOP) with the ER stress antagonist 4-phenylbutyric acid (4-PBA); mitochondria-targeted antioxidant SkQ1; or reactive oxygen species scavenger N-acetyl-l-cysteine (NAC). Corneal thickness and endothelial cell density were measured before and after treatment. Human corneal endothelial cells were treated with high glucose with or without 4-PBA. The expression of corneal endothelial- and ER stress-related genes was detected by western blot and immunofluorescence staining. Mitochondrial bioenergetics were measured with an Agilent Seahorse XFp Analyzer. RESULTS: In diabetic mice, the appearance of ER stress preceded morphological changes in the corneal endothelium. The persistent ER stress directly caused corneal edema and endothelial cell loss in normal mice. Pharmacological inhibition of ER stress was sufficient to mitigate corneal edema and endothelial cell loss in both diabetic mice after high IOP treatment. Mechanistically, inhibiting ER stress ameliorated the hyperglycemia-induced mitochondrial bioenergetic deficits and improved the barrier and pump functional recovery of the corneal endothelium. When compared with NAC, 4-PBA and SkQ1 exhibited better improvement of corneal edema and endothelial cell loss in diabetic mice. CONCLUSIONS: Hyperglycemia-induced ER stress contributes to the dysfunction of diabetic corneal endothelium, and inhibiting ER stress may offer therapeutic potential by improving mitochondrial bioenergetics.

Heterogeneity of human corneal endothelium implicates lncRNA NEAT1 in Fuchs endothelial corneal dystrophy.

The corneal endothelium is critical for maintaining corneal clarity by mediating hydration through barrier and pump functions. Progressive loss of corneal endothelial cells during aging has been associated with the development of Fuchs endothelial corneal dystrophy (FECD), one of the main causes of cornea-related vision loss. The mechanisms underlying FECD development remain elusive. Single-cell RNA sequencing of isolated healthy human corneas discovered 4 subpopulations of corneal endothelial cells with distinctive signatures. Unsupervised clustering analysis uncovered nuclear enriched abundant transcript 1 (NEAT1), a long non-coding RNA (lncRNA), as the top expressed gene in the C0-endothelial subpopulation, but markedly downregulated in FECD. Consistent with human corneas, a UVA-induced mouse FECD model validated the loss of NEAT1 expression. Loss of NEAT1 function by an in vivo genetic approach reproduced the exacerbated phenotype of FECD by ablating corneal endothelial cells. Conversely, gain of function by a CRISPR-activated adenoviral delivery system protected corneas from UVA-induced FECD. Our findings provide novel mechanistic insights into the development of FECD, and targeting NEAT1 offers an attractive approach for treating FECD.

Long-term corneal recovery by simultaneous delivery of hPSC-derived corneal endothelial precursors and nicotinamide.

Human pluripotent stem cells (hPSCs) hold great promise for the treatment of various human diseases. However, their therapeutic benefits and mechanisms for treating corneal endothelial dysfunction remain undefined. Here, we developed a therapeutic regimen consisting of the combination of hPSC-derived corneal endothelial precursors (CEPs) with nicotinamide (NAM) for effective treatment of corneal endothelial dysfunction. In rabbit and nonhuman primate models, intracameral injection of CEPs and NAM achieved long-term recovery of corneal clarity and thickness, similar with the therapeutic outcome of cultured human corneal endothelial cells (CECs). The transplanted human CEPs exhibited structural and functional integration with host resident CECs. However, the long-term recovery relied on the stimulation of endogenous endothelial regeneration in rabbits, but predominantly on the replacing function of transplanted cells during the 3-year follow-up in nonhuman primates, which resemble human corneal endothelium with limited regenerative capacity. Mechanistically, NAM ensured in vivo proper maturation of transplanted CEPs into functional CECs by preventing premature senescence and endothelial-mesenchymal transition within the TGF-ß-enriched aqueous humor. Together, we provide compelling experimental evidence and mechanistic insights of simultaneous delivery of CEPs and NAM as a potential approach for treating corneal endothelial dysfunction.

Transcriptional Network Analysis Reveals the Role of miR-223-5p During Diabetic Corneal Epithelial Regeneration.

Diabetes mellitus (DM) is a complex metabolic disorder. Long-term hyperglycemia may induce diabetic keratopathy (DK), which is mainly characterized by delayed corneal epithelial regeneration. MicroRNAs (miRNAs) have been reported to play regulatory roles during tissue regeneration. However, the molecular mechanism by which miRNAs influence epithelial regeneration in DK is largely unknown. In this study, we performed miRNA and mRNA sequencing of regenerative corneal epithelium tissue from streptozotocin-induced type 1 diabetic (T1DM) and wild-type mice to screen for differentially expressed miRNAs and mRNAs. Based on regulatory network analysis, miR-223-5p was selected for subsequent experiments and Hpgds was then identified as a direct target gene. MiR-223-5p downregulation significantly promoted diabetic corneal epithelial wound healing and nerve regeneration. However, the beneficial effects of miR-223-5p inhibition were abolished by an Hpgds inhibitor. Furthermore, mechanistic studies demonstrated that miR-223-5p suppression ameliorated inflammation and enhanced cell proliferation signaling in DK. Taken together, our findings revealed that the regulatory role of miR-223-5p in diabetic corneal epithelial and nerve regeneration by mediating inflammatory processes and cell proliferation signaling. And silencing miR-223-5p may contribute to the development of potential therapeutic strategies for DK.

Revealing the Angiopathy of Lacrimal Gland Lesion in Type 2 Diabetes.

For a better understanding of diabetic angiopathy (DA), the potential biomarkers in lacrimal DA and its potential mechanism, we evaluated the morphological and hemodynamic alterations of lacrimal glands (LGs) in patients with type 2 diabetes and healthy counterparts by color Doppler flow imaging (CDFI). We further established a type 2 diabetic mice model and performed hematoxylin-eosin (HE) staining, immunofluorescence staining of CD31, RNA-sequencing analysis, and connectivity map (CMap) analysis. We found atrophy and ischemia in patients with type 2 diabetes and mice models. Furthermore, we identified 846 differentially expressed genes (DEGs) between type 2 diabetes mellitus (T2DM) and vehicle mice by RNA-seq. The gene ontology (GO) analysis indicated significant enrichment of immune system process, regulation of blood circulation, apoptotic, regulation of secretion, regulation of blood vessel diameter, and so on. The molecular complex detection (MCODE) showed 17 genes were involved in the most significant module, and 6/17 genes were involved in vascular disorders. CytoHubba revealed the top 10 hub genes of DEGs, and four hub genes (App, F5, Fgg, and Gas6) related to vascular regulation were identified repeatedly by MCODE and cytoHubba. GeneMANIA analysis demonstrated functions of the four hub genes above and their associated molecules were primarily related to the regulation of circulation and coagulation. CMap analysis found several small molecular compounds to reverse the altered DEGs, including disulfiram, bumetanide, genistein, and so on. Our outputs could empower the novel potential targets to treat lacrimal angiopathy, diabetes dry eye, and other diabetes-related diseases.

Molecular identity of human limbal heterogeneity involved in corneal homeostasis and privilege.

PURPOSE: The corneal limbus maintains the homeostasis, immune and angiogenic privilege of cornea. This study aimed to depict the landscape of human limbal tissues by single-cell RNA sequencing (scRNA-seq). METHODS: Single cells of human limbus collected from donor corneas were subjected to 10x scRNA-seq, followed by clustering cell types through the t-distributed stochastic neighbor embedding (t-SNE) and unbiased computational informatic analysis. Immunofluorescent staining was performed using human corneas to validate the analysis results. RESULTS: 47,627 cells acquired from six human limbal tissues were collected and subjected to scRNA-seq. 14 distinct clusters were identified and 8 cell types were annotated with representative markers. In-depth dissection revealed three limbal epithelial cell subtypes and refined the X-Y-Z hypothesis of corneal epithelial maintenance. We further unveiled two cell states with higher stemness (TP63+ and CCL20+ cells), and two other differentiated cell states (GPHA2+ and KRT6B + cells) in homeostatic limbal stem/progenitor cells (LSPCs) that differ in transcriptional profiles. Cell-cell communication analysis revealed the central role of LSPCs and their bidirectional regulation with various niche cells. Moreover, comparative analysis between limbus and skin deciphered the pivotal contribution of limbal immune cells, vascular and lymphatic endothelial cells to corneal immune and angiogenic privilege. CONCLUSIONS: The human limbus atlas provided valuable resources and foundations for understanding corneal biology, disease and potential interventions.

A-to-I RNA editing in honeybees shows signals of adaptation and convergent evolution.

Social insects exhibit extensive phenotypic diversities among the genetically similar individuals, suggesting a role for the epigenetic regulations beyond the genome level. The ADAR-mediated adenosine-to-inosine (A-to-I) RNA editing, an evolutionarily conserved mechanism, facilitates adaptive evolution by expanding proteomic diversities. Here, we characterize the A-to-I RNA editome of honeybees (Apis mellifera), identifying 407 high-confidence A-to-I editing sites. Editing is most abundant in the heads and shows signatures for positive selection. Editing behavior differs between foragers and nurses, suggesting a role for editing in caste differentiation. Although only five sites are conserved between bees and flies, an unexpectedly large number of genes exhibit editing in both species, albeit at different locations, including the nonsynonymous auto-editing of Adar. This convergent evolution, where the same target genes independently acquire recoding events in distant diverged clades, together with the signals of adaptation observed in honeybees alone, further supports the notion of recoding being adaptive.