Long noncoding RNA ZFAS1: A novel anti-apoptotic target in Fuchs endothelial corneal dystrophy.

Fuchs endothelial corneal dystrophy (FECD) is the leading cause of endothelial keratoplasty without efficacious drug treatment. Recent studies have emphasized the involvement of epigenetic regulation in FECD development. Long non-coding RNAs (lncRNAs) are recognized as crucial epigenetic regulators in diverse cellular processes and ocular diseases. In this study, we revealed the expression patterns of lncRNAs using high-throughput sequencing technology in FECD mouse model, and identified 979 significantly dysregulated lncRNAs. By comparing the data from FECD human cell model, we obtained a series of homologous lncRNAs with similar expression patterns, and revealed that these homologous lncRNAs were enriched in FECD related biological functions, with apoptosis (mmu04210) showing the highest enrichment score. In addition, we investigated the role of lncRNA zinc finger antisense 1 (ZFAS1) in apoptotic process. This study would broaden our understanding of epigenetic regulation in FECD development, and provide potential anti-apoptotic targets for FECD therapy.

Herpes Simplex Keratitis as a Complication of Pterygium Surgery.

BACKGROUND Infectious keratitis after pterygium surgery is a rare but potentially devastating complication. The present study presents 5 cases of herpes simplex keratitis (HSK) after pterygium surgery. CASE REPORT This study was conducted in our clinic in a 5-year period from February 2017 to September 2021. The 5 patients were men, aged between 42 and 73 years, with no prior history of herpes simplex virus (HSV) infections. Symptoms appeared near 1 month (median 30 days, range 10 to 70 days) after primary pterygium surgery. Diagnosis was based on clinical symptoms and laboratory test results, such as tear HSV-sIgA, corneal tissue polymerase chain reaction, and next-generation sequencing of metagenomics. The epithelial (1/5) and stromal (4/5) subtypes of HSK were identified. The patients received topical ganciclovir gel, immunosuppressive eyedrops, and oral acyclovir tablets, along with additional surgical interventions if necessary. Three were healed with conservative therapy, 1 eye required amniotic membrane transplantation due to corneal melt, and 1 was perforated and followed by corneal grafting. Finally, a literature review of previous publications on HSK after ocular surgeries was conducted. CONCLUSIONS HSK is a rare but serious complication that can arise after uneventful pterygium surgery. It is worthy of attention that both epithelial and stromal forms can occur. Timely diagnosis and treatment are crucial to prevent unfavorable outcomes. Consequently, routine corneal fluorescein staining, tear sIgA examination, and corneal scraping for polymerase chain reaction or next-generation sequencing of metagenomics should be performed in any suspected cases.

Single-cell RNA sequencing reveals the complex cellular niche of pterygium.

PURPOSE: Pterygium is a vision-threatening conjunctival fibrovascular degenerated disease with a high global prevalence up to 12 %, while no absolute pharmacotherapy has been applied in clinics. In virtue of single-cell RNA sequencing (scRNA-seq) technique, our study investigated underlying pathogeneses and potential therapeutic targets of pterygium from the cellular transcriptional level. METHODS: A total of 45605 cells from pterygium of patients and conjunctiva of normal controls (NC) were conducted with scRNA-seq, and then analyzed via integrated analysis, pathway enrichment, pseudotime trajectory, and cell-cell communications. Besides, immunofluorescence and western blot were performed in vivo and in vitro to verify our findings. RESULTS: In brief, 9 major cellular types were defined, according to canonical markers. Subsequently, we further determined the subgroups of each major cell lineages. Several newly identified cell sub-clusters could promote pterygium, including immuno-fibroblasts, epithelial mesenchymal transition (EMT)-epithelial cells, and activated vascular endothelial cells (activated-vEndo). Besides, we also probed the enrichment of immune cells in pterygium. Particularly, macrophages, recruited by ACKR1+activated-vEndo, might play an important role in the development of pterygium by promoting angiogenesis, immune suppression, and inflammation. CONCLUSION: An intricate cellular niche was revealed in pterygium via scRNA-seq analysis and the interactions between macrophages and ACKR1+ activated-vEndo might be the key part in the development of pterygia.

Corneal biomechanical properties in vernal keratoconjunctivitis and its subtypes: a preliminary study.

PURPOSE: To explore the corneal biomechanical properties (CBPs) of patients with vernal keratoconjunctivitis (VKC) and the discrepancies among three subtypes of VKC including palpebral, limbal, and mixed forms. METHODS: Forty eyes of 20 VKC patients and twenty eyes of ten non-VKC patients were included in this case-control study. Patients with VKC were further divided into three subtypes (six patients in Palpebral form, five patients in limbal form, and nine patients in mixed form). The CBPs of all patients were obtained from the Corneal Visualization Scheimpflug Technology (Corvis ST). RESULTS: First applanation (A1) length, Ambrosio relational thickness in horizontal (ARTh), and stiffness parameter at first applanation (SP-A1) were significantly lower in the VKC group while A1 velocity was significantly higher in the VKC group (p < 0.05), compared to the non-VKC group. Furthermore, A1 velocity presented a positive correlation with disease course (p < 0.05). In addition, VKC patients of limbal form had lower central corneal thickness (CCT), SP-A1, and higher deformation amplitude ratio (DA ratio), compared to the other two subtypes (p < 0.05). Besides, patients in limbal form had higher A1 velocity, integrated radius, and corneal biomechanical index (CBI) compared with mixed form, and lower A1 length than palpebral form (p < 0.05). CONCLUSIONS: The corneas of VKC patients were softer and more protruded compared with the control group, and the property of steepness was closely related to disease course. VKC patients in limbal form were more inclined to be keratoconus than the other two subtypes due to their CBPs` discrepancies.

Atypical stromal herpes simplex keratitis: clinical features and diagnosis.

PURPOSE: To report atypical clinical features and diagnosis of stromal herpes simplex keratitis (HSK) and to evaluate the diagnostic efficiency of tear HSV-sIgA in atypical HSK. STUDY DESIGN: Prospective observational study. METHODS: Records of keratitis' patients with tear herpes simplex virus (HSV)-sIgA test results acquired between May 2019 and November 2021 were evaluated retrospectively. Positive tear HSV-sIgA test was used to identify herpes simplex virus (HSV) infection. Patients with typical presentations and histories of HSV keratitis (HSK) were excluded. RESULTS: Eleven eyes of 11 patients initially diagnosed as keratitis caused by other etiology were confirmed as having HSV infection by positive results of tear HSV-sIgA. Clinical features of atypical stromal HSK were variable. Antiviral treatment was effective in all patients. CONCLUSION: The appearance of an atypical stromal HSK represents a diagnostic challenge. Tear HSV-sIgA test could help provide a quick diagnosis.

Type 2 Diabetes Mellitus Makes Corneal Endothelial Cells Vulnerable to Ultraviolet A-Induced Oxidative Damage Via Decreased DJ-1/Nrf2/NQO1 Pathway.

Purpose: The purpose of this study was to investigate whether type 2 diabetes mellitus (T2DM) makes corneal endothelial cells (CECs) suffer from more severe ultraviolet A (UVA)-induced oxidative damage and explore its mechanisms via measuring the oxidant level and the antioxidant level in vitro. Methods: Corneas of spontaneous T2DM db/db mice and non-diabetes littermate control mice were irradiated with UVA, leading to oxidative damage of CECs. Anterior segment-optical coherence tomography, corneal image, and CECs immunohistochemistry staining were taken thereafter to measure central corneal thickness, corneal edema degree, and damage extent of CECs. In vitro, human corneal endothelial cells line B4G12 (HCECs) treated with high glucose (HG) and low glucose (LG) were exposed to UVA light separately. Subsequently, cellular proliferation, apoptosis, pro-oxidant factors, such as reactive oxygen species (ROS), antioxidant factors including Parkinson's disease protein 7 (DJ-1), nuclear factor-erythroid 2 related factor 2 (Nrf2), phosphorylated-Nrf2, and NAD(P)H: quinone oxidoreductase 1 (NQO1) were measured. Results: T2DM mice presented greater oxidant damage of CECs and more distinct corneal edema compared with control mice when they were irradiated with the 150 J/cm2 UVA light. In vitro, HCECs in HG condition showed a significant decrease of proliferation, higher apoptosis extent, more ROS generation, lower expressions of DJ-1/Nrf2/NQO1, and distinct reduction of Nrf2 nuclear translocation compared to those in LG condition after exposing to 5 J/cm2 UVA light. Conclusions: Increase of ROS, downregulation of DJ-1/Nrf2/NQO1 expressions, and decrease of Nrf2 nuclear translocation could result in that T2DM makes CECs more vulnerable to oxidative damage.

Construction and verification of a hypoxia-related nine-gene prognostic model in uveal melanoma based on integrated single-cell and bulk RNA sequencing analyses.

Uveal melanoma (UM) is the most common primary intraocular tumor with high metastasis and poor prognosis among adults. Hypoxia participates in the metastasis process in various types of cancers. It is reported that the increased expression of hypoxia inducible factor 1 alpha subunit (HIF1A), a hypoxia-related molecule, is associated with worse prognoses of UM patients. Based on the integrated analysis of single-cell sequencing (scRNA-seq) dataset from Gene Expression Omnibus (GEO) and bulk RNA-seq dataset from the Cancer Genome Atlas (TCGA), we found hypoxia was the key feature in UM progression and identified 47 common hypoxia-related differentially expressed genes (DEGs) for the following research. Univariate cox analysis and LASSO-Cox regression analysis were performed to establish a nine-gene prognostic model. According to this model, UM patients could be divided into high- and low-risk groups, with a significant difference in overall survival and progression free survival between the two groups (P < 0.001). The accuracy of the predictive model was also verified on two other independent datasets. In addition, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses revealed that these hypoxia-related DEGs were enriched in immune and cancer related pathways. The proportion of immune infiltration and the expression of immune biomarkers were different between high- and low-risk UM patients, providing potential targets for UM immunotherapy. Hence, our hypoxia-related nine-gene model could efficiently predict the prognosis and guide personalized therapies for UM patients.

Comprehensive Analysis of circRNA-Associated-ceRNA Networks in Human Corneal Endothelial Dysfunction.

PURPOSE: Circular RNAs (circRNAs) are a novel class of endogenous noncoding RNAs that regulate gene expression through the competitive endogenous RNA (ceRNA) mechanism. CircRNA-associated-ceRNA networks are closely related to oxidative stress-related diseases. Oxidative stress-induced dysfunction of the corneal endothelium (CE) is a major pathological feature in many corneal diseases. This study was aimed to analyze circRNA-associated-ceRNA networks in oxidative stress-induced CE dysfunction. METHODS: A CE dysfunction model was established using human corneal endothelial cells (HCECs) treated with H 2 O 2 at a concentration of 250 µM for 4 hours at 37°C. High-throughput sequencing was conducted to determine the expression profiles of circRNA, miRNA, and mRNA. Bioinformatic analyses, including Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes analysis, were conducted to identify the potential biological modules and pathologic pathways of dysregulated circRNAs. CircRNA-associated-ceRNA networks were established based on the data of sequencing and bioinformatic analyses. RESULTS: We obtained 108 differentially expressed circRNAs, including 77 upregulated and 31 downregulated circRNAs. GO analysis suggested that dysregulated circRNAs were mainly targeted to protein quality control for misfolded or incompletely synthesized proteins (biologic process), nuclear chromatin (cellular component), and ubiquitin protein ligase binding (molecular function). GO terms related to CE functions responding to oxidative stress were also identified. Kyoto Encyclopedia of Genes and Genomes pathway analysis indicated that dysregulated circRNAs were mostly enriched in the adherens junction pathway. Network analysis identified several potential therapeutic targets for CE dysfunction. CONCLUSIONS: CircRNAs are significantly dysregulated in HCECs under oxidative stress. The circRNA-associated-ceRNA networks are closely related to HCEC functions. Targeting these networks might provide novel therapies for CE dysfunction.

Piperlongumine alleviates corneal allograft rejection via suppressing angiogenesis and inflammation.

Background: Neovascularization and inflammatory response are two essential features of corneal allograft rejection. Here, we investigated the impact of Piperlongumine (PL) on alleviating corneal allograft rejection, primarily focusing on pathological angiogenesis and inflammation. Methods: A murine corneal allograft transplantation model was utilized to investigate the role of PL in preventing corneal allograft rejection. PL (10 mg/kg) or vehicle was intraperitoneally injected daily into BALB/c recipients from day -3 to day 14. The clinical signs of the corneal grafts were monitored for 30 days. Corneal neovascularization and inflammatory cell infiltration were detected by immunofluorescence staining and immunohistochemistry. The proportion of CD4+ T cells and macrophages in the draining lymph nodes (DLNs) was examined by flow cytometry. In vitro, HUVECs were cultured under hypoxia or incubated with TNF-α to mimic the hypoxic and inflammatory microenvironment favoring neovascularization in corneal allograft rejection. Multiple angiogenic processes including proliferation, migration, invasion and tube formation of HUVECs in hypoxia with or without PL treatment were routinely evaluated. The influence of PL treatment on TNF-α-induced pro-inflammation in HUVECs was investigated by real-time PCR and ELISA. Results: In vivo, PL treatment effectively attenuated corneal allograft rejection, paralleled by coincident suppression of neovascularization and alleviation of inflammatory response. In vitro, PL distinctively inhibited hypoxia-induced angiogenic processes in HUVECs. Two key players in hypoxia-induced angiogenesis, HIF-1α and VEGF-A were significantly suppressed by PL treatment. Also, TNF-α-induced pro-inflammation in HUVECs was hampered by PL treatment, along with a pronounced reduction in ICAM-1, VCAM-1, CCL2, and CXCL5 expression. Conclusions: The current study demonstrated that PL could exhibit both anti-angiogenic and anti-inflammatory effects in preventing corneal allograft rejection, highlighting the potential therapeutic applications of PL in clinical strategy.

RNA-seq identifies long non-coding RNAs as potential therapeutic targets for human corneal endothelial dysfunction under oxidative stress.

Human corneal endothelial cells (CECs) have limited ability to regenerate in vivo. Oxidative stress has been proposed as one potential reason. Understanding the mechanism of oxidative stress-induced CEC dysfunction might provide novel targets for improving CEC regenerative capacity, and help develop non-surgical therapeutic strategies for CEC dysfunction. Long non-coding RNAs (lncRNAs) are non-coding transcripts with multiple biological functions. The roles of lncRNAs in ocular cells under oxidative stress have been widely studied, such as lens epithelial cells, trabecular meshwork cells, and retinal ganglion cells. In the current study, we established oxidative stress-induced CEC dysfunction model in vitro. By RNA sequencing technology, we identified 824 differentially expressed lncRNAs in CEC dysfunction group, including 667 upregulated lncRNAs and 157 downregulated lncRNAs. We finally demonstrated that CEC functions under oxidative stress, including cellular proliferation, apoptosis, and anti-oxidative stress ability, could be regulated by different lncRNAs, including lncRNA-Z93241.1, lncRNA-XLOC_000818, and lncRNA-AC007952.4. Targeting these lncRNAs might be useful to further elucidate the pathology of CEC dysfunction and develop novel therapeutic strategy.

The evaluation of diagnostic efficiency for stromal herpes simplex keratitis by the combination of tear HSV-sIgA and HSV-DNA.

PURPOSE: To assess the differential diagnostic values for stromal herpes simplex keratitis (HSK) by using tear HSV-sIgA, tear HSV-DNA, and the combination. METHODS: Tear samples for both eyes and the paired serum were collected from 187 stromal HSK and 56 controls. Enzyme-linked immune sorbent assay (ELISA) was used to analyze the tear HSV-sIgA and serum IgG/IgM/IgA. The levels of tear HSV-DNA were measured by polymerase chain reaction (PCR). RESULTS: The positive rates for tear HSV-sIgA and HSV-DNA were 36.90% and 10.96% respectively in stromal HSK patients. Twelve showed positivity for both sIgA and DNA, while 46 cases were positive for sIgA or DNA. The sensitivity, specificity, PPV, and NPV for simultaneous measurement were 39.73%, 98.21%, 98.31%, and 38.46%. The total negative conversion rate of sIgA was 95.71%. CONCLUSIONS: The diagnostic efficiency of HSV-sIgA only is nearly equal to the combination of HSV-sIgA and HSV-DNA, and the positive result is optimum to achieve a reliable diagnosis of stromal HSK even in atypical or unsuspected cases.

Association between poor wound healing and the formation of Salzmann nodules.

We report the case of a 65-year-old woman who presented with a gray-white nodular lesion located at the site of a clear corneal incision in her left eye. Anterior segment optical coherence tomography showed prominent endothelial gaping even 5 years after cataract surgery. Based on the features of clinical and histological examinations, Salzmann nodular degeneration was diagnosed. The formation of the Salzmann nodule was thought to be related to poor healing of the surgical wound. To our knowledge, this is the first case of Salzmann nodular degeneration associated with cataract surgery. FINANCIAL DISCLOSURE: None of the authors has a financial or proprietary interest in any material or method mentioned.