Integrative analysis of gene expression datasets in corneal endothelium samples of Fuchs endothelial corneal dystrophy.

FECD is an age-related progressive ocular disorder characterized by the gradual loss of corneal endothelial cells. Although the exact pathogenesis of FECD remains incompletely understood, differentially expressed genes in the corneal endothelium of FECD patients compared to controls have been reported in several studies. However, a consensus regarding consistently affected genes in FECD has not been established. To address this issue, we conducted a comprehensive meta-analysis incorporating five studies with data that met our predefined inclusion criteria. The combined dataset included 41 FECD patients and 26 controls. We conducted study-level analyses, followed by a meta-analysis, and subsequent functional enrichment analysis targeting the topmost DEGs. Our findings revealed a total of 1537 consistently dysregulated genes in the corneal endothelium of FECD patients. Notably, only 14.6% (224/1537) of these DEGs had been previously identified as statistically significant in individual datasets. Functional enrichment analysis revealed that the upregulated DEGs were significantly enriched in immune-related signaling pathways, with a particularly high enrichment in "The NLRP3 inflammasome" and "Inflammasomes" pathways. In conclusion, we successfully identify a set of consistently dysregulated genes in FECD, which are associated with both established and novel biological pathways. This study highlights the importance of further investigating the role of inflammasomes in FECD pathogenesis and exploring strategies to modulate inflammasome activation for the management of this debilitating condition.

Mitochondria-associated endoplasmic reticulum membranes dysfunction contributes to PARP-1-dependent cell death under oxidative stress in retinal precursor cells.

Persistent poly (ADP-ribose) polymerase 1 (PARP-1) activation has proven detrimental and can lead to PARP-1-dependent cell death. Mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) serve as essential hubs for many biological pathways, such as autophagy and mitochondria fission and fusion. This study aimed to alleviate the effects of hydrogen peroxide (H2 O2 )-induced persistent PARP-1 activation and MAM dysregulation by the usage of a PARP-1 inhibitor. Results showed that receptor-interacting protein kinase (RIPK) 1 inhibitor (necrostatin-1) and PARP-1 inhibitor (olaparib) protected retinal precursor cells from H2 O2 -induced death, while a pan-caspase inhibitor (Z-VAD-FMK) failed to protect R28 cells. Olaparib also alleviated H2 O2 -induced MAM dysregulation, as evidenced by decreased VDAC1/ITPR3 interactions and reduced mitochondrial membrane potential collapse. Additionally, olaparib also inhibited H2 O2 -induced autophagy. Inhibiting autophagic flux increased MAM signaling under both normal and oxidative conditions. Furthermore, H2 O2 treatment caused a reduction in the protein level of mitofusin-2 (MFN2) in a dose- and time-dependent manner. Mfn2 knockdown was found to further magnify MAM dysregulation and mitochondrial dysfunction under normal and oxidative conditions. Mfn2 overexpression surprisingly enhanced H2 O2 -induced MAM signaling and failed to rescue H2 O2 -induced mitochondrial dysfunction. These results indicate that MAMs probably serve as a membrane source for oxidative stress-associated autophagy. MAM dysregulation also contributed to H2 O2 -induced PARP-1-dependent cell death. However, more studies are required to decipher the link between the modulation of Mfn2 expression, changes in MAM integrity, and alterations in mitochondrial performances.

Therapeutic Nanoparticles from Grape Seed for Modulating Oxidative Stress.

The therapeutic potential of nanomaterials toward oxidative damage relevant diseases has attracted great attentions by offering promising advantages compared with conventional antioxidants. Although different kinds of nanoantioxidants have been well developed, the facile fabrication of robust and efficient nanoscavengers is still met with challenges like the use of toxic and high-cost subunits, the involvement of multistep synthetic process, and redundant purification work. Herein, a direct fabrication strategy toward polyphenol nanoparticles with tunable size, excellent biocompatibility, and reactive oxygen species (ROS) scavenging capacities from grape seed via an enzymatic polymerization method is reported. The resulting nanoparticles can efficiently prevent cell damage from ROS and exert promising in vivo antioxidant therapeutic effects on several oxidative stress-related diseases, including accelerating wound healing, inhibiting ulcerative colitis, and regulating the oxidative stress in dry eye disease. This study can stimulate the development of more kinds of low-cost, safe, and efficient biomass-based antioxidative nanomaterials via similar fabrication methodologies.

Blockage of P2X7R suppresses Th1/Th17-mediated immune responses and corneal allograft rejection via inhibiting NLRP3 inflammasome activation.

P2X7R is a vital modifier of various inflammatory and immune-related diseases. However, the immunomodulatory effects of P2X7R on corneal allograft rejection remains unknown. Here we showed that P2X7R expression was significantly upregulated in corneal grafts of allogeneic transplant mice. Pharmacological blockage of P2X7R remarkably prolonged graft survival time, and reduced inflammatory cell infiltration in corneal grafts, in particular Th1/Th17 cells. Meanwhile, the frequencies of Th1/Th17 cells in draining lymph nodes were significantly decreased in P2X7R blocked allogeneic mice. Further results showed that the effect of P2X7R on promoting Th1/Th17 mediated immune responses in corneal allograft rejection relied heavily on its activation on the NLRP3/caspase-1/IL-1ß axis, while P2X7R blockage could mitigate such activation. Nevertheless, the addition of IL-1ß in vivo abrogated the protective effect of P2X7R blockage on promoting corneal graft survival. These findings demonstrate that blockage of P2X7R can substantially alleviate corneal allograft rejection and promote grafts survival, highlighting it as a promising target for preventing or treating corneal allograft rejection.

Calcitriol inhibits apoptosis via activation of autophagy in hyperosmotic stress stimulated corneal epithelial cells in vivo and in vitro.

BACKGROUND: Previously, calcitriol has been demonstrated as a potential therapeutic agent for dry eye, whilst its role on corneal epithelium death remains unclear. This study aims to investigate the relationship between apoptosis and autophagy on dry eye related scenario, as well as the effect of calcitriol and its potential mechanism. METHODS: In vitro, immortalized human corneal epithelial cells (iHCEC) were cultured in hyperosmotic medium with or without various concentrations of calcitriol and other reagents. In vivo, Wistar rats were applied with benzalkonium chloride to induce dry eye. Then rats were topically treated with calcitriol (10-6 M) for 14 days. Autophagy flux (LC3B-II and SQSTM1/P62) was examined by western blotting or immunostaining. To test cell apoptosis, western blotting for cleaved caspase-3, Annexin V/PI double staining and TUNEL assay were used. CCK-8 assay was performed to detect the cell viability. Small interfering RNA was used to knock down the expression of vitamin D receptor in iHCECs. RESULTS: Autophagy activation could protect iHCECs against HS induced apoptosis in vitro, and calcitriol was able to augment autophagy flux via VDR signaling, shown as the remarkably elevated expression of LC3B-II, as well as the declined p62 expression. In vivo results further supported the protective role of calcitriol on corneal epithelium apoptosis through promoting autophagy in dry eye rats. CONCLUSION: The current study indicated that autophagy was an adaptive change of corneal epithelial cells in response to hyperosmotic stress and calcitriol could prevent cells from apoptosis via further activation of autophagy through VDR pathway.

Shared gene signature between pterygium and meibomian gland dysfunction uncovered through gene-expression meta-analysis.

BACKGROUND: Pterygium and meibomian gland dysfunction (MGD) are two clinically correlated ocular diseases. We propose to investigate the shared gene signature between pterygium and MGD. METHODS: Microarray datasets were retrieved from the Gene Expression Omnibus (GEO) database. Initial processing of the data was performed using the R programming package. Gene-expression values were log2 transformed and normalized by quantile normalization. The differentially expressed genes (DEGs) in each individual dataset were analyzed by the limma package. The integration of different pterygium datasets and gene-expression meta-analysis was conducted by the NetworkAnalyst package. A Venn diagram was created to find the overlapped DEGs between MGD and pterygium datasets. Gene ontology enrichment and pathway analysis were performed using the ToppGene Suite. RESULTS: We found 193 DEGs significantly up-regulated in pterygium, with the combined effect sizes ranging from 1.53 to 3.78. A gene signature consisting of 11 DEGs were found to be shared by pterygium and MGD (SPRR3, SERPINB13, NMU, KRT10, IL37, KRT6B, PI3, S100A2, MAL, AURKA, and RGCC), and bioinformatics analyses showed that these overlapped DEGs were significantly enriched in pathways related to keratinization, cell-cycle regulation, and formation of the cornified envelope. CONCLUSION: We identified a shared gene signature between pterygium and MGD through gene-expression meta-analysis. The analysis of this signature underlined that keratinization-related pathways may play important roles in the development of these two clinically correlated pathologies.

MiR-21 promotes pterygium cell proliferation through the PTEN/AKT pathway.

Purpose: To evaluate the effect of the overexpression of miR-21 on the properties of pterygium and examine whether miR-21 promotes the proliferation of pterygium cells through targeting the PTEN/AKT signaling pathway. Methods: Information regarding patient gender, age, and pterygium severity was gathered. Expression of miR-21 was obtained through examination of excised pterygium tissues and superior conjunctiva tissues with real-time PCR. Human pterygium fibroblasts (HPFs) were obtained from pterygium surgery and subjected to primary culture. The HPF cell lines were divided into a negative control group, an miR-21 inhibitor group, and an miR-21 inhibitor + VO-Ohpic trihydrate group, and then the cell viability and apoptosis and the expression of PTEN and AKT were examined. Results: Fifty-eight subjects with unilateral primary pterygium were included. An increase in the miR-21 levels in pterygium tissue was evident compared with that in the paired normal conjunctival tissues (independent-samples t test, p<0.01). As the severity of the pterygium increased, the miR-21 levels increased (p=0.004, rs=0.373, Spearman's rank correlation coefficient). The miR-21 inhibitor suppressed the proliferation and induced apoptosis of HPF cells through increasing the PTEN expression, and further decreasing the expression of p-AKT, which could be reversed by the PTEN inhibitor VO-Ohpic trihydrate. Conclusions: Aberrant miR-21 overexpression in the pterygium could target PTEN, which contributes to abnormal proliferation of the HPF cells through depressing the PTEN/AKT pathway. The results also suggested the potential of miR-21 and the PTEN/AKT pathway as a novel therapeutic strategy for pterygium.

Recombinant ADAMTS 13 Attenuates Brain Injury After Intracerebral Hemorrhage.

BACKGROUND AND PURPOSE: Inflammatory responses and blood-brain barrier (BBB) dysfunction play important roles in brain injury after intracerebral hemorrhage (ICH). The metalloprotease ADAMTS 13 (a disintegrin and metalloprotease with thrombospondin type I motif, member 13) was shown to limit inflammatory responses through its proteolytic effects on von Willebrand factor. In the present study, we addressed the role of ADAMTS 13 after experimental ICH. METHODS: ICH was induced in mice by intracerebral infusion of autologous blood. The peri-hematomal inflammatory responses, levels of matrix metalloproteinase-9 and intercellular adhesion molecule-1, pericyte coverage on brain capillaries, and BBB permeability were quantified at 24 hours. Functional outcomes, cerebral edema, and hemorrhagic lesion volume were quantified at day 3. RESULTS: Treatment with recombinant ADAMTS 13 (rADAMTS 13) reduced the levels of chemokines and cytokines, myeloperoxidase activity, and microglia activation and neutrophil recruitment after ICH. rADAMTS 13 also decreased interleukin-6 expression in brain endothelial cells stimulated by lipopolysaccharide, whereas recombinant von Willebrand factor reversed this effect. The anti-inflammatory effect of rADAMTS 13 was accompanied by reduced expression of intercellular adhesion molecule-1 and less activation of matrix metalloproteinase, enhanced pericyte coverage of brain microvessels, and attenuated BBB disruption. Furthermore, neutrophil depletion protected against BBB damage, and rADAMTS 13 treatment had no further beneficial effect. Finally, treatment of mice with rADAMTS 13 reduced cerebral edema and hemorrhagic lesion volume and improved neurological functions. CONCLUSIONS: Our findings reveal the importance of rADAMTS 13 in regulating pathological inflammation and BBB function and suggest that rADAMTS 13 may provide a new therapeutic strategy for ICH.

Caspase-3 modulates regenerative response after stroke.

Stroke is a leading cause of long-lasting disability in humans. However, currently there are still no effective therapies available for promoting stroke recovery. Recent studies have shown that the adult brain has the capacity to regenerate neurons after stroke. Although this neurogenic response may be functionally important for brain repair after injury, the mechanisms underlying stroke-induced neurogenesis are not known. Caspase-3 is a major executioner and has been identified as a key mediator of neuronal death in the acute stage of stroke. Recently, however, accumulating data indicate that caspase-3 also participates in various biological processes that do not cause cell death. Here, we show that cleaved caspase-3 was increased in newborn neuronal precursor cells (NPCs) in the subventricular zone (SVZ) and the dentate gyrus during the period of stroke recovery, with no evidence of apoptosis. We observed that cleaved caspase-3 was expressed by NPCs and limited its self-renewal without triggering apoptosis in cultured NPCs from the SVZ of ischemic mice. Moreover, we revealed that caspase-3 negatively regulated the proliferation of NPCs through reducing the phosphorylation of Akt. Importantly, we demonstrated that peptide inhibition of caspase-3 activity significantly promoted the proliferation and migration of SVZ NPCs and resulted in a significant increase in subsequent neuronal regeneration and functional recovery after stroke. Together, our data identify a previously unknown caspase-3-dependent mechanism that constrains stroke-induced endogenous neurogenesis and should revitalize interest in targeting caspase-3 for treatment of stroke.

Recombinant ADAMTS13 reduces tissue plasminogen activator-induced hemorrhage after stroke in mice.

OBJECTIVE: Tissue plasminogen activator (tPA) is approved for treatment of acute ischemic stroke, but it increases the risk of cerebral hemorrhage. Accumulating evidence suggests that von Willebrand factor (VWF) plays a pivotal role in thrombus formation and microcirculatory disturbances after ischemic stroke. By cleaving VWF, ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) protects mice from stroke. Therefore, we hypothesized that recombinant ADAMTS13 (rADAMTS13) could increase the safety of tPA thrombolysis in stroke. METHODS: We examined blood-brain barrier (BBB) permeability after intraventricular injection of tPA, VWF, and rADAMTS13 in nonischemic mice. We investigated the role of rADAMTS13 on reducing tPA-induced BBB dysfunction and cerebral hemorrhage in a mouse stroke model. RESULTS: Intraventricular injection of tPA or VWF under nonischemic conditions resulted in a significant increase in BBB permeability. In contrast, rADAMTS13 blocked both tPA- and VWF-induced BBB opening. BBB disruption following stroke was exacerbated by intravenous administration of tPA, but this was attenuated by injection of rADAMTS13. Correspondingly, tPA-associated hemorrhage after stroke was significantly reduced by rADAMTS13. The antihemorrhagic effect of rADAMTS13 was reversed by injection of recombinant VWF. We also showed that rADAMTS13 inhibited tPA-mediated upregulation of vascular endothelial growth factor (VEGF) in vascular endothelium after stroke. The upregulation of VEGF was suppressed by either an Akt inhibitor wortmannin or a Rho kinase inhibitor fasudil. Furthermore, rADAMTS13 downregulated tPA-induced phosphorylation of Akt and activation of RhoA. INTERPRETATION: These findings demonstrate that the VWF-cleaving protease rADAMTS13 reduced tPA-induced hemorrhage by regulating BBB integrity, and suggest that this effect may occur through the Akt/RhoA-mediated VEGF pathways.

A highly selective inhibitor of discoidin domain receptor-1 (DDR1-IN-1) protects corneal epithelial cells from YAP/ACSL4-mediated ferroptosis in dry eye.

BACKGROUND AND PURPOSE: This study investigated the involvement of discoidin domain receptor (DDR) in dry eye and assessed the potential of specific DDR inhibitors as a therapeutic strategy for dry eye by exploring the underlying mechanism. EXPERIMENTAL APPROACH: Dry eye was induced in Wistar rats by applying 0.2% benzalkonium chloride (BAC), after which rats were treated topically for 7 days with DDR1-IN-1, a selective inhibitor of DDR1. Clinical manifestations of dry eye were assessed on Day-7 post-treatment. Histological evaluation of corneal damage was performed using haematoxylin and eosin (H&E) staining. In vitro, immortalized human corneal epithelial cells (HCECs) exposed to hyperosmotic stress (HS) were treated with varying doses of DDR1-IN-1 for 24 h. The levels of lipid peroxidation in dry eye corneas or HS-stimulated HCECs were assessed. Protein levels of DDR1/DDR2 and related pathways were detected by western blotting. The cellular distribution of acyl-CoA synthetase long chain family member 4 (ACSL4) and Yes-associated protein (YAP) was evaluated using immunohistochemistry or immunofluorescent staining. KEY RESULTS: In dry eye corneas, only DDR1 expression was significantly up-regulated compared with normal controls. DDR1-IN-1 treatment significantly alleviated dry eye symptoms in vivo. The treatment remarkably reduced lipid hydroperoxide (LPO) levels and suppressed the expression of ferroptosis markers, particularly ACSL4. Overexpression or reactivation of YAP diminished the protective effects of DDR1-IN-1, indicating the involvement of the Hippo/YAP pathway in DDR1-targeted therapeutic effects. CONCLUSIONS AND IMPLICATIONS: This study confirms the significance of DDR1 in dry eye and highlights the potential of selective DDR1 inhibitor(s) for dry eye treatment.

WNT16b promotes the proliferation and self-renewal of human limbal epithelial stem/progenitor cells via activating the calcium/calcineurin A/NFATC2 pathway.

Our previous finding revealed that WNT16b promoted the proliferation of human limbal epithelial stem cells (hLESCs) through a ß-catenin independent pathway. Here, we aimed to explore its underlying molecular mechanism and evaluate its potential in the treatment of limbal stem cell deficiency (LSCD). Based on the findings of mRNA-sequencing, the expression of key molecules in WNT/calcineurin A/NFATC2 signalling pathway was investigated in WNT16b-co-incubated hLESCs and control hLESCs. An epithelial wound healing model was established on Wnt16b-KO mice to confirm the regulatory effect of WNT16b in vivo. The therapeutic potential of WNT16b-co-incubated hLESCs was also evaluated in mice with LSCD. Our findings showed that WNT16b bound with Frizzled7, promoted the release of Ca2+ and activated calcineurin A and NFATC2. With the translocation of NFATC2 into cell nucleus and the activation of HDAC3, WDR5 and GCN5L2, the expression of H3K4me3, H3K14ac and H3K27ac in the promoter regions of FoxM1 and c-MYC increased, which led to hLESC proliferation. The effect of the WNT16b/calcium/calcineurin A/NFATC2 pathway on LESC homeostasis maintenance and corneal epithelial repair was confirmed in Wnt16b-KO mice. Moreover, WNT16b-coincubated hLESCs could reconstruct a stable ocular surface and inhibit corneal neovascularization in mice with LSCD. In conclusion, WNT16b enhances the proliferation and maintains the stemness of hLESCs by activating the non-canonical calcium/calcineurin A/NFATC2 pathway in vitro and in vivo, and accelerates corneal epithelial wound healing.

WNT16B enhances the proliferation and self-renewal of limbal epithelial cells via CXCR4/MEK/ERK signaling.

Culture of limbal epithelial cells (LECs) provides the principal source of transplanted limbal stem cells (LESCs) for treatment of limbal-stem-cell deficiency. Optimization of the culture conditions for in-vitro-expanded LECs will help to create a graft with an optimized quality and quantity of LESCs. This study aimed to investigate the effects of WNT16B on LECs and corneal wound healing and the underlying mechanism. Treatment with exogenous WNT16B increased the proliferative capacity and self-renewal of LECs in the cultures. We further revealed that C-X-C chemokine receptor type 4 (CXCR4) was vital for the effects of WNT16B, and activation of CXCR4/MEK/ERK signaling was pivotal in mediating the effects of WNT16B on LECs enriched for LESCs. The stimulatory effect of WNT16B on corneal epithelial repair was confirmed in a mouse corneal-wound-healing model. In summary, WNT16B enhances proliferation and self-renewal of LECs via the CXCR4/MEK/ERK signaling cascade and accelerates corneal-epithelial wound healing.

Olaparib, a PARP-1 inhibitor, protects retinal cells from ocular hypertension-associated oxidative damage.

Glaucoma is the most common cause of irreversible blindness worldwide. Elevated intraocular pressure (IOP) and relative hypoxia in the retina stimulate the production of reactive oxygen species (ROS), which, in turn, puts the retina and optic nerve under chronic oxidative stress. Emerging evidence has shown that oxidative stress can trigger PARP-1 overactivation, mitochondrial-associated endoplasmic reticulum membrane (MAM) dysregulation, and NLRP3 activation. Oxidative damage can trigger inflammasome activation, and NLRP3 is the only inflammasome associated with MAM dysregulation. In addition, multiple transcription factors are located on the MAM. This study aimed to investigate the protective effects and underlying mechanisms of a PARP-1 inhibitor (olaparib) against chronic ocular hypertension-associated retinal cell damage. We also mimicked hypoxic stimulation of a retinal precursor cell line by exposing the cells to 0.2% O2 in vitro. We discovered that chronic ocular hypertension (COH) induces oxidative damage and MAM dysregulation in the retinal ganglion cells (RGCs). The protein levels of cleaved-PARP and NLRP3 were upregulated in the retinas of the COH rats. Olaparib, a PARP-1 inhibitor, alleviated COH-induced RGC loss, retinal morphological alterations, and photopic negative response amplitude reduction. Olaparib also relieved hypoxic stimulation-induced loss of cell viability and MAM dysregulation. Additionally, some indicators of mitochondrial performance, such as reactive oxygen species accumulation, mitochondrial Ca2+ influx, and mitochondrial membrane potential collapse, decreased after olaparib treatment. Olaparib attenuated the hypoxia-induced upregulation of NLRP3 protein levels as well as the phosphorylation of ERK1/2 and histone H2A.X. These results suggest that olaparib protects RGCs from chronic intraocular pressure elevation in vivo and alleviates the abnormal MAM dysregulation and mitochondrial dysfunction caused by hypoxia in vitro. This protection may be achieved by inhibiting PARP-1 overactivation, NLRP3 upregulation, and phosphorylation of ERK1/2.

Evaluating the association between single nucleotide polymorphisms in the stonin 2 (STON2) gene and keratoconus in a Han Chinese population.

BACKGROUND: A recent genome-wide association study (GWAS) identified a significant association between the single nucleotide polymorphism (SNP) rs2371597 in the stonin 2 gene (STON2) and keratoconus (KCTN) susceptibility. The current study further explored the association between STON2 and KCTN susceptibility in an independent Han Chinese population. METHODS: Three SNPs (rs2371597, rs8004137, and rs8008602) located in the STON2 gene were examined in 164 Han Chinese patients with KCTN and 239 age- and gender-matched healthy subjects. The TaqMan SNP genotyping assays were performed, and the LDlink, RegulomeDB, and PLINK package were applied for data analyses. The gene expression levels of STON2 were investigated in various murine organ tissues using quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: The SNP rs2371597 was significantly associated with KCTN risk in this Han Chinese population. The frequency of the C allele in KCTN patients was significantly higher than that in healthy subjects [34.8% vs. 26.6%; odds ratio (OR) =1.47; 95% confidence interval (CI): 1.08 to 2.02; P=0.01409]. The genotype distribution of the SNP rs2371597 was also significantly different between KCTN patients and controls. The other two genotyped SNPs allele and genotypic frequencies were not remarkably different between the KCTN group and the control group. However, the haplotype CAT formed by the three SNPs was substantially associated with the risk of KCTN (P=0.04101). Also, gene expression pattern analysis showed a relatively higher expression of STON2 in the cornea in comparison to other tissues. CONCLUSIONS: The current study demonstrated that SNPs in the STON2 gene were associated with an increased risk of developing KCTN in this Han Chinese population, suggesting that the STON2 gene may play an important role in the etiology of KCTN.

Calcitriol Alleviates Hyperosmotic Stress-Induced Corneal Epithelial Cell Damage via Inhibiting the NLRP3-ASC-Caspase-1-GSDMD Pyroptosis Pathway in Dry Eye Disease.

PURPOSE: Inflammasome activation in response to elevated tear osmolarity behaves as an initial signal in dry eye-related corneal inflammation. Pyroptosis is another prominent consequence of inflammasome activation, which is featured by gasdermin D (GSDMD)-driven cell lysis. This study aims to explore the role of pyroptosis in dry eye, and also to verify if calcitriol, a potential therapeutic agent for dry eye, has certain effects against hyperosmotic stress (HS)-induced pyroptosis in human corneal epithelial cells (iHCECs) and the underlying mechanism. METHODS: The expression of pyroptosis executor GSDMD in tears from dry eye patients was examined using western blotting. iHCECs were grown in hyperosmotic medium (450 mOsM) to mimic the feature of elevated tear osmolality of dry eye in vitro. Exogenous calcitriol or pyroptosis inhibitor disulfiram was used. The extent of pyroptosis of iHCECs under various treatments was examined by scanning electron microscopy, caspase-1 and propidium iodide (PI) double staining by flow cytometry, immunofluorescent staining for ASC speck formation, and western blotting. Cell viability was measured by a CCK-8 assay and an LDH release assay. RESULTS: We found that pyroptosis was presented in dry eye patients, shown as the elevation of its effector GSDMD N-terminal domain (N-GSDMD) in patients' tears. Further in vitro results showed that HS promoted pyroptosis in human corneal epithelial cells, while exogeneous supplementation of disulfiram could reduce the number of iHCECs with pyroptotic markers. More importantly, we demonstrated that, in line with the effect of disulfiram, calcitriol could also alleviate HS-induced pyroptosis, through inhibiting the NLRP3-ASC-caspase-1-GSDMD pyroptosis pathway. CONCLUSION: The current study provided direct evidence showing increased pyroptosis in dry eye patients. We demonstrated that calcitriol was able to effectively alleviate HS-induced corneal epithelial cell damage through inhibiting the NLRP3-ASC-caspase-1-GSDMD pyroptosis pathway. This study underlined calcitriol as a promising therapeutic agent for dry eye given its multiple therapeutic targets.

WAY-100635 Alleviates Corneal Lesions Through 5-HT1A Receptor-ROS-Autophagy Axis in Dry Eye.

Purpose: To explore whether 5-HT1A receptors are involved in the dry eye disease (DED) mouse model and reveal its underlying mechanism. Methods: A C57BL/6J mouse DED model was established via the administration of 0.2% benzalkonium chloride twice a day for 14 days. Corneal fluorescein sodium staining score and Schirmer I test were checked before, and on days 7, 14, and 21 after treatment. The experiment was randomly divided into control, DED, 5-HT1A receptor agonist with or without N-acetylcysteine (NAC) and 5-HT1A receptor antagonist with or without NAC groups. The mRNA expression of inflammatory cytokines was measured by reverse transcription-quantitative polymerase chain reaction. Cellular reactive oxygen species (ROS) were detected by 2', 7'-dichlorodihydrofluorescein diacetate assays. Western blot analysis was used to measure the expression levels of autophagic proteins microtubule-associated protein 1 light chain 3 (LC3B-I/II) and autophagy-related gene 5 (ATG5). Results: 5-HT1A receptor agonist (8-OH-DPAT) increased corneal fluorescein sodium staining spots and 5-HT1A receptor antagonist (WAY-100635) decreased them. Treatment with 8-OH-DPAT was associated with the gene expression of more inflammatory cytokines, such as interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), C-C motif chemokine ligand 2 (CCL2) and C-X-C motif chemokine ligand 10 (CXCL10) compared with treatment with WAY-100635. An increased expression of LC3B-I/II and ATG5 was observed in corneal epithelial cells in the mouse model of DED. 8-OH-DPAT significantly enhanced the expression of LC3B-I/II and ATG5 by disrupting ROS levels. WAY-100635 alleviates autophagy by inhibiting ROS production. Conclusion: Excessive ROS release through 8-OH-DPAT induction can lead to impaired autophagy and increased inflammatory response in DED. WAY-100635 reduces corneal epithelial defects and inflammation in DED, as well as alleviates autophagy by inhibiting ROS production. The activation of the 5-HT1A receptor-ROS-autophagy axis is critically involved in DED development.

Whole exome sequencing identified FAM149A as a plausible causative gene for congenital hereditary endothelial dystrophy, affecting Nrf2-Antioxidant signaling upon oxidative stress.

BACKGROUND: Congenital hereditary endothelial dystrophy (CHED) is a rare genetic disease of the corneal endothelium with a very early onset of bilateral corneal edema due to degeneration and dysfunction of the corneal endothelium. Currently SLC4A11 is the only established causative gene for CHED, but not all these reported CHED patients could be explained by SLC4A11 deficiency, indicating that the genetic predisposition of CHED still requires further exploration. METHODS: Trio-based whole-exome sequencing was performed on a CHED patient and his unaffected parents. The GATK2 and an in-house bioinformatics pipeline were applied for variant analyses, following the 2015 American College of Medical Genetics and Genomics (ACMG) guidelines. Potential pathogenic variants were further validated by Sanger sequencing. The expression profiles of FAM149A in cell line, murine tissues or human corneal endothelia were determined by RT-qPCR. Small interfering RNA was used to knock down the expression of FAM149A in vitro. Cell viability was detected by a CCK-8 assay. ROS and 8-OHdG were examined by fluorometric analysis. The nuclear translocation of NRF2 was determined by western blotting. RESULTS: We identified a homozygous mutation (NM_015398.3: c.991A > G; p.R331G) in the FAM149A gene that related to the phenotype of CHED. FAM149A was found to be highly expressed in corneal endothelium, and up-regulated upon oxidative stress. Further functional investigations demonstrated that deficiency in FAM149A impaired Nrf2-antioxidant signaling, rendering cells more vulnerable to oxidative stress. Consistently, the expression of FAM149A was significantly reduced in patients with corneal endothelium dysfunction. CONCLUSION: This study demonstrated, for the first time, FAM149A as a plausible causative gene for CHED etiology, offering new insight for future investigation targeting CHED.

Long Non-coding RNAs Gabarapl2 and Chrnb2 Positively Regulate Inflammatory Signaling in a Mouse Model of Dry Eye.

Purpose: To elucidate the expression profile and the potential role of long non-coding ribonucleic acids (RNAs) (lncRNAs) in a dry eye disease (DED) model. Methods: A DED model was established in C57BL/6J mice with 0.2% benzalkonium chloride (BAC) twice a day for 14 days. The differentially expressed lncRNAs were detected by RNA-seq technology (Gene Expression Omnibus, GEO GSE186450) and the aberrantly expressed lncRNAs were further verified by RT-qPCR. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted to predicate the related candidate genes and potential pathological pathways. Cells from a human corneal epithelial cell line (HCECs) were cultured under hyperosmolarity. The regulation of inflammatory factors by silencing potential targeted lncRNAs was verified in vitro in HCECs. Results: In our study, a significant increase in corneal fluorescence staining and a reduction in tear production were observed in DED mice at all follow-ups compared with the controls, and the differences were increasing over time. In total, 2,649 upregulated and 704 downregulated lncRNAs were identified in DED mice. We selected six aberrantly expressed and most abundant lncRNAs and performed RT-qPCR using the samples for RNA-seq. Chrnb2, Gabarapl2, and Usp31 were thereby confirmed as the most significantly altered lncRNAs. Pathway analysis revealed that the neuroactive ligand-receptor interaction signaling pathway was the most enriched, followed by the calcium signaling pathway and cytokine-cytokine receptor interaction. Following treatment of Gabarapl2 siRNA and Chrnb2 siRNA, tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, and IL-6 were significantly downregulated in the HCECs. Conclusion: Our study suggests that Chrnb2 and Gabarapl2 may be involved in the inflammation response by regulating TNF-α, IL-1ß, and IL-6 in DED. These candidate lncRNAs may be both potential biomarkers and therapeutic targets for DED.

Replication of the Association Between Keratoconus and Polymorphisms in PNPLA2 and MAML2 in a Han Chinese Population.

Keratoconus (KC) is a complex ocular disease that is affected by both genetic and non-genetic triggers. A recent genome-wide association study (GWAS) identified a genome-wide significant locus for KC in the region of PNPLA2 (rs61876744), as well as a suggestive signal in the MAML2 (rs10831500) locus. In order to validate their findings, here we performed a replication study of the Han Chinese population, with 120 sporadic KC cases and 206 gender and age matched control subjects, utilizing the TaqMan SNP genotyping assays. SNP rs10831500, as well as two proxy SNPs for rs61876744, named rs7942159 and rs28633403, were subjected to genotyping. However, we did not find a significant difference (P > 0.05) in all the three genotyped SNPs between KC cases and the controls. A further meta-analysis on four previous cohorts of white patients and this Han Chinese cohort showed a significant genetic heterogeneity within the replicated loci. Thus, the current study suggests that SNP rs61876744 (or its proxy SNPs) and rs10831500 might not be associated with KC susceptibility in this Han Chinese cohort, and a large-scale association analysis focusing on the loci is therefore warranted in further investigations.