NAD+ supplementation improves mitochondrial functions and normalizes glaucomatous trabecular meshwork features.

Glaucoma is characterized by pathological elevation of intraocular pressure (IOP) due to dysfunctional trabecular meshwork (TM), which is the primary cause of irreversible vision loss. There are currently no effective treatment strategies for glaucoma. Mitochondrial function plays a crucial role in regulating IOP within the TM. In this study, primary TM cells treated with dexamethasone were used to simulate glaucomatous changes, showing abnormal cellular cytoskeleton, increased expression of extracellular matrix, and disrupted mitochondrial fusion and fission dynamics. Furthermore, glaucomatous TM cell line GTM3 exhibited impaired mitochondrial membrane potential and phagocytic function, accompanied by decreased oxidative respiratory levels as compared to normal TM cells iHTM. Mechanistically, lower NAD + levels in GTM3, possibly associated with increased expression of key enzymes CD38 and PARP1 related to NAD + consumption, were observed. Supplementation of NAD + restored mitochondrial function and cellular viability in GTM3 cells. Therefore, we propose that the aberrant mitochondrial function in glaucomatous TM cells may be attributed to increased NAD + consumption dependent on CD38 and PARP1, and NAD + supplementation could effectively ameliorate mitochondrial function and improve TM function, providing a novel alternative approach for glaucoma treatment.

Hyperglycemia Induces Tear Reduction and Dry Eye in Diabetic Mice through the Norepinephrine-α1 Adrenergic Receptor-Mitochondrial Impairment Axis of Lacrimal Gland.

Dry eye syndrome is a common complication in diabetic patients with a prevalence of up to 54.3%. However, the pathogenic mechanisms underlying hyperglycemia-induced tear reduction and dry eye remain less understood. The present study indicated that both norepinephrine (NE) and tyrosine hydroxylase levels were elevated in the lacrimal gland of diabetic mice, accompanied by increased Fos proto-oncogene (c-FOS)+ cells in the superior cervical ganglion. However, the elimination of NE accumulation by surgical and chemical sympathectomy significantly ameliorated the reduction in tear production, suppressed abnormal inflammation of the lacrimal gland, and improved the severity of dry eye symptoms in diabetic mice. Among various adrenergic receptors (ARs), the α1 subtype played a predominant role in the regulation of tear production, as treatments of α1AR antagonists improved tear secretion in diabetic mice compared with ßAR antagonist propranolol. Moreover, the α1AR antagonist alfuzosin treatment also alleviated functional impairments of the meibomian gland and goblet cells in diabetic mice. Mechanically, the α1AR antagonist rescued the mitochondrial bioenergetic deficit, increased the mitochondrial DNA copy numbers, and elevated the glutathione levels of the diabetic lacrimal gland. Overall, these results deciphered a previously unrecognized involvement of the NE-α1AR-mitochondrial bioenergetics axis in the regulation of tear production in the lacrimal gland, which may provide a potential strategy to counteract diabetic dry eye by interfering with the α1AR activity.

Nicotinamide promotes the differentiation of functional corneal endothelial cells from human embryonic stem cells.

Corneal transplantation represents the primary therapeutic approach for managing corneal endothelial dysfunction, but corneal donors remain scarce. Anterior chamber cell injection emerges as a highly promising alternative strategy for corneal transplantation, with pluripotent stem cells (PSC) demonstrating considerable potential as an optimal cell source. Nevertheless, only a few studies have explored the differentiation of functional corneal endothelial-like cells originating from PSC. In this investigation, a chemical-defined protocol was successfully developed for the differentiation of functional corneal endothelial-like cells derived from human embryonic stem cells (hESC). The application of nicotinamide (NAM) exhibited a remarkable capability in suppressing the fibrotic phenotype, leading to the generation of more homogeneous and well-distinctive differentiated cells. Furthermore, NAM effectively suppressed the expression of genes implicated in endothelial cell migration and extracellular matrix synthesis. Notably, NAM also facilitated the upregulation of surface marker genes specific to functional corneal endothelial cells (CEC), including CD26 (-) CD44 (-∼+-) CD105 (-) CD133 (-) CD166 (+) CD200 (-). Moreover, in vitro functional assays were performed, revealing intact barrier properties and Na+/K+-ATP pump functionality in the differentiated cells treated with NAM. Consequently, our findings provide robust evidence supporting the capacity of NAM to enhance the differentiation of functional CEC originating from hESC, offering potential seed cells for therapeutic interventions of corneal endothelial dysfunction.

Establishment of mouse model of neurotrophic keratopathy through TRPV1 neuronal ablation.

Neurotrophic keratopathy (NK) is a challenging disease with the reduced innervation to the cornea. To establish a genetic and stable mouse model of NK, we utilized the TRPV1-DTR mice with intraperitoneal injection of diphtheria toxin (DT) to selectively eliminate TRPV1 neurons. After DT administration, the mice exhibited robust ablation of TRPV1 neurons in the trigeminal ganglion, accompanied with reduced corneal sensation and nerve density, as well as the decreased calcitonin-gene-related peptide (CGRP) and substance P levels. According to disease progression of TRPV1 neuronal ablation, tear secretion was reduced from day 3, which followed by corneal epithelial punctate lesions from day 7. From day 11 to day 16, the mice exhibited persistent corneal epithelial defects and stromal edema. By day 21, corneal ulceration and stromal melting were observed with the abundant inflammatory cell infiltration, corneal neovascularization, and enhanced cell apoptosis. Moreover, subconjunctival injection of CGRP delayed the NK progression with the characteristics of reduced severe corneal epithelial lesions and corneal inflammation. In addition, the impairments of conjunctival goblet cells, lacrimal gland, and meibomian gland were identified by the diminished expression of MUC5AC, AQP5, and PPARγ, respectively. Therefore, these results suggest that the TRPV1-DTR mice may serve as a reliable animal model for the research of NK pathogenesis.

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.

SRT1720 attenuates UVA-induced corneal endothelial damage via inhibition of oxidative stress and cellular apoptosis.

Corneal endothelium is mostly sensitive to oxidative pressure and mitochondrial dysfunction. However, the oxidative-antioxidant mechanism of corneal endothelial cells (CECs) remains partially defined. Silent information regulator 1 (SIRT1) is a well-studied therapeutic target of oxidative damage. This study aimed to determine the SIRT1 expression in ultraviolet A (UVA)-induced corneal endothelial damage and explore potential drugs to repair corneal endothelial oxidative injury. In this study, we showed that CECs exhibited cellular apoptosis, reactive oxygen species (ROS) accumulation and decreased SIRT1 expression. In addition, UVA induced the imbalance of mitochondrial homeostasis and function, involving in mitochondrial membrane potential, mitochondrial fusion/fission and mitochondrial energy metabolism. SRT1720, the SIRT1 activator, effectively increased SIRT1 expression and attenuated UVA-induced oxidative damage in CECs. The therapeutic effects of SRT1720 for corneal endothelial oxidative damage were also verified in UVA-irradiated mice model. Our findings indicated that SIRT1 maintained the oxidant-antioxidant balance in corneal endothelium, suggesting a new promising therapeutic target for corneal endothelial dysfunction.

Involvement of aberrant acinar cell proliferation in scopolamine-induced dry eye mice.

Dry eye is a multifactorial disease that causes dryness, inflammation and damage of ocular surface. Subcutaneous injection of the muscarinic cholinergic antagonist scopolamine under desiccating stress reduces tear production and induces dry eye symptoms in mice. However, the expression profile and pathogenic changes of the lacrimal gland remain incompletely understood. In the present study, we performed comparative transcriptomic analysis of lacrimal glands from the control and scopolamine-treated mice. Primary analysis identified 677 upregulated genes and 269 downregulated genes in the lacrimal gland of mice with scopolamine treatment. Unexpectedly, KEGG pathway and hierarchical clustering analysis showed the enrichment of "DNA replication" and "cell cycle" categories in the upregulated genes. Subsequently, we confirmed that the acinar cells were the major proliferating cells of lacrimal gland, which exhibited significant increasing of the proliferating cell nuclear antigen (PCNA) expression after scopolamine treatment, accompanied with the upregulation of DNA damage marker γ-H2AX. More importantly, both prophylactic and therapeutic administration of the cyclin-dependent kinase (CDK) inhibitor AT7519 rescued the tear reduction and alleviated dry eye severity in the scopolamine-treated mice, including corneal epithelial barrier function, lacrimal and corneal inflammation, and conjunctival goblet cell density. Therefore, we conclude that aberrant acinar cell proliferation is involved in the scopolamine-induced tear reduction and dry eye onset, which can be improved by AT7519 treatment.

iPSC-Derived Corneal Endothelial Cells.

The corneal endothelium is the innermost monolayer of the cornea that maintains corneal transparency and thickness. However, adult human corneal endothelial cells (CECs) possess limited proliferative capacity, and injuries can only be repaired by migration and enlargement of resident cells. When corneal endothelial cell density is lower than the critical level (400-500 cells/mm2) due to disease or trauma, corneal endothelial dysfunction will occur and lead to corneal edema. Corneal transplantation remains the most effective clinical treatment therapy but is limited by the global shortage of healthy corneal donors. Recently, researchers have developed several alternative strategies for the treatment of corneal endothelial disease, including the transplantation of cultured human CECs and artificial corneal endothelial replacement. Early-stage results show that these strategies can effectively resolve corneal edema and restore corneal clarity and thickness, but the long-term efficacy and safety remain to be further validated. Induced pluripotent stem cells (iPSCs) represent an ideal cell source for the treatment and drug discovery of corneal endothelial diseases, which can avoid the ethical-related and immune-related problems of human embryonic stem cells (hESCs). At present, many approaches have been developed to induce the differentiation of corneal endothelial-like cells from human induced pluripotent stem cells (hiPSCs). Their safety and efficacy for the treatment of corneal endothelial dysfunction have been confirmed in rabbit and nonhuman primate animal models. Therefore, the iPSC-derived corneal endothelial cell model may provide a novel effective platform for basic and clinical research of disease modeling, drug screening, mechanistic investigation, and toxicology testing.

Plasmonically Enhanced Ultrasensitive Epitope-Specific Serologic Assay for COVID-19.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has rapidly spread and resulted in the global pandemic of COVID-19. Although IgM/IgG serology assay has been widely used, with the entire spike or nucleocapsid antigens, they only indicate the presence or absence of antibodies against these proteins but are not specific to the neutralization antibodies, therefore providing only generic information about infection stage and possible future immune protection. Novel technologies enabling easy-to-use and sensitive detection of multiple specific antibodies simultaneously will facilitate precise diagnosis of infection stage, prediction of clinical outcomes, and evaluation of future immune protection upon viral exposure or vaccination. Here, we demonstrate a rapid and ultrasensitive quantification method for epitope-specific antibodies, including different isotypes and subclasses, in a multiplexed manner. Using an ultrabright fluorescent nanolabel, plasmonic-fluor, this novel assay can be completed in 20 min and more importantly, the limit of detection of the plasmon-enhanced immunoassay for SARS-CoV-2 antibodies is as much as 100-fold lower compared to the assays relying on enzymatic amplification of colorimetric signals. Using convalescent patient plasma, we demonstrate that this biodetection method reveals the patient-to-patient variability in immune response as evidenced by the variations in whole protein and epitope-specific antibodies. This cost-effective, rapid, and ultrasensitive plasmonically enhanced multiplexed epitope-specific serological assay has the potential to be broadly employed in the detection of specific antibodies, which may benefit the advanced epidemiology studies and enable improvement of the clinical outcomes and prediction of the future protection against the SARS-CoV-2.

Hyperglycemia induces corneal endothelial dysfunction through attenuating mitophagy.

Hyperglycemia increases the risk of corneal endothelial dysfunction, resulting in damage to corneal endothelial structure and function. However, the effect and mechanism of hyperglycemia-induced corneal endothelial damage remain elusive. In this study, we demonstrated that hyperglycemia reduced the expression of pump-related protein Na+/K+ ATPase and barrier-related protein ZO-1. Moreover, we found hyperglycemia caused abnormal changes of morphological mitochondria and dynamics in vitro. In addition, the decreased levels of mitophagy were further confirmed Western blotting and LC3B-Mitotracker Immunofluorescence. Exogenous application of mitophagy agonist carbonyl cyanide m-chlorophenyl hydrazine (CCCP) increases the expression of Na+/K+ ATPase and ZO-1 in corneal endothelial cells through up-regulated mitophagy in vitro. In addition, CCCP effectively reverses the phenomenon of corneal opacity and increased corneal thickness in diabetic mice. Therefore, our demonstrated the novel function of mitophagy in the pathogenesis of diabetic cornea endothelial dysfunction, and provide potential approach for treating diabetic corneal endothelial dysfunction.

Tannin coordinated nanozyme composite-based hybrid hydrogel eye drops for prophylactic treatment of multidrug-resistant Pseudomonas aeruginosa keratitis.

Pseudomonas aeruginosa infection is a severe acute suppurative ulcer that engulfs virtually the entire tissue in a short period and leads to devastating destruction. Antibiotic therapy is a common approach for the prophylaxis and treatment of P. aeruginosa infection. However, it is often associated with serious side effects, complications, and multidrug resistance. Therefore, it has been a long-standing challenge to explore safe and effective methods for controlling P. aeruginosa infection. Herein, tannin-coordinated nanozyme composite-based hybrid hydrogels (TCNH) are developed and characterized for the prophylactic treatment of P. aeruginosa and multidrug-resistant P. aeruginosa infections using mouse keratitis as the animal model. The TCNH eye drops are constructed by photoinitiated free radical polymerization of acetylated gelatin solution containing self-synthesized tannin-coordinated Co3O4/Ag nanozyme composite. The as-prepared TCNH displays good dispersibility, peroxidase-like activity and in vitro/in vivo biocompatibility. The nanozyme composite in TCNH seems to penetrate the interior of bacteria and exhibited significant broad-spectrum antibacterial activity owing to its intrinsic and nanozymic catalytic properties. Furthermore, TCNH eye drops can be successfully applied to treat P. aeruginosa and multidrug-resistant P. aeruginosa keratitis. The findings of this study reveal the potential of tannin-coordinated nanozyme composite-based hybrid hydrogel eye drops for treating infectious diseases.

Bladder drug mirabegron exacerbates atherosclerosis through activation of brown fat-mediated lipolysis.

Mirabegron (Myrbetriq) is a ß3-adrenoreceptor agonist approved for treating overactive bladder syndrome in human patients. This drug can activate brown adipose tissue (BAT) in adult humans and rodents through the ß3-adrenoreceptor-mediated sympathetic activation. However, the effect of the mirabegron, approved by the US Food and Drug Administration, on atherosclerosis-related cardiovascular disease is unknown. Here, we show that the clinical dose of mirabegron-induced BAT activation and browning of white adipose tissue (WAT) exacerbate atherosclerotic plaque development. In apolipoprotein E-/- (ApoE-/-) and low-density lipoprotein (LDL) receptor-/- (Ldlr-/-) mice, oral administration of clinically relevant doses of mirabegron markedly accelerates atherosclerotic plaque growth and instability by a mechanism of increasing plasma levels of both LDL-cholesterol and very LDL-cholesterol remnants. Stimulation of atherosclerotic plaque development by mirabegron is dependent on thermogenesis-triggered lipolysis. Genetic deletion of the critical thermogenesis-dependent protein, uncoupling protein 1, completely abrogates the mirabegron-induced atherosclerosis. Together, our findings suggest that mirabegron may trigger cardiovascular and cerebrovascular diseases in patients who suffer from atherosclerosis.

Poly(ADP-ribose) polymerase inhibitor PJ34 protects against UVA-induced oxidative damage in corneal endothelium.

Fuchs endothelial corneal dystrophy (FECD) is one of the main causes for corneal endothelial blindness, which is characterized by the progressive decline of corneal endothelial cells. Poly (ADP-ribose) polymerase (PARP) was reported to be involved in cell death and apoptosis of several diseases. However, the role of PARP1 in the progression of FECD remains elusive. In the present study, we reported that UVA irradiation caused the corneal endothelial damage and corneal edema in mice, which was accompanied with the elevated activity of PARP1 and PAR. The PARP1 inhibitor PJ34 resolved the corneal edema and protected corneal endothelium from UVA-induced oxidative damage, mitochondrial dysfunction, and cell apoptosis. Mechanistically, PARP1 inhibition exerted its anti-apoptotic effects through downregulation of the phosphorylation levels of JNK1/2 and p38 MAPK and subsequently the increase of MKP-1. Our results suggest that PARP1 inhibition protects corneal endothelium from UVA-induced oxidative damage, which provides a potential alternative strategy for the therapy of FECD.

Insulin Promotes Corneal Nerve Repair and Wound Healing in Type 1 Diabetic Mice by Enhancing Wnt/ß-Catenin Signaling.

The insulin and Wnt signaling pathways are involved in cell proliferation, tissue homeostasis, and tumorigenesis. However, their interrelationship in the pathophysiological process of diabetic corneal injury remains unclear. In this study, the role of insulin in the diabetic cornea was investigated in vitro, using cultured TKE2 cells and trigeminal ganglion neurons, and in vivo, by assessing corneal wound-healing responses in diabetic mice. A selective Wnt antagonist (XAV-939) and activator (BML-284) were used to regulate the interactions between insulin and the Wnt pathway. The results demonstrated that insulin promoted corneal epithelial wound healing and sensation recovery, whereas the expression of molecules involved in the Wnt/ß-catenin pathway was also up-regulated in the injured corneal epithelium. However, XAV-939 limited the insulin-induced epithelial and corneal nerve repair. By contrast, BML-284 treatment promoted the healing of the corneal epithelium and corneal nerve repair in diabetic mice. These results indicate that insulin, via Wnt signaling, contributes to diabetic corneal epithelial wound healing and nerve injury recovery and is, therefore, a potential protective factor for diabetic corneal epithelial wounds and nerve injury.

Topical calcitriol application promotes diabetic corneal wound healing and reinnervation through inhibiting NLRP3 inflammasome activation.

Vitamin D (VD) deficiency delays corneal wound healing in those with diabetes, which cannot be rescued with supplemental diet. Here, we employed topical calcitriol application to evaluate its efficiency in corneal wound healing and reinnervation in diabetic mice. Type 1 diabetic mice were topically administrated calcitriol, or subconjunctivally injected with NLRP3 antagonist MCC950 or IL-1ß blocking antibody after epithelial debridement. Serum VD levels, corneal epithelial defect, corneal sensation and nerve density, NLRP3 inflammasome activation, neutrophil infiltration, macrophage phenotypes, and gene expressions were examined. Compared with those of normal mice, diabetic mice showed reduced serum VD levels. Topical calcitriol application promoted corneal wound healing and nerve regeneration, as well as sensation recovery in diabetic mice. Moreover, calcitriol ameliorated neutrophil infiltration and promoted the M1-to-M2 macrophage transition, accompanied by suppressed overactivation of the NLRP3 inflammasome. Treatment with NLRP3 antagonist or IL-1ß blockage demonstrated similar improvements as those of topical calcitriol application. Additionally, calcitriol administration upregulated desmosomal and hemidesmosomal gene expression in the diabetic cornea. In conclusion, topical calcitriol application promotes corneal wound healing and reinnervation during diabetes, which may be related to the suppression of the overactivation of NLRP3 inflammasome.

Lipidomic analysis of meibomian glands from type-1 diabetes mouse model and preliminary studies of potential mechanism.

Diabetes is a significant risk factor for meibomian gland dysfunction (MGD), but its mechanism is poorly understood. The main function of the meibomian glands (MGs) is to synthesize, store, and secrete lipids. In this study, we found that the amount of lipids in the meibomian acini in STZ-induced type 1 diabetic mice decreased, and the lipid droplets became larger and irregular. In all, 31 lipid subclasses were identified in the mouse MGs, which contained 1378 lipid species in total through lipidomics analysis based on LC-MS/MS. Diabetes caused a significant increase in the content of ceramides (Cer) in the MGs but a significant decrease in the ration of sphingomyelin to ceramides (SM/Cer). The quantity of meibocytes in diabetic mice was dramatically decreased, and the proliferation activity was alleviated, which may be associated with cell cycle arrest caused by diabetes-induced abnormal Cer metabolism in MGs. We found an increase in macrophage and neutrophils infiltration in the diabetic MGs, which may be related to the significant reduction of AcCa in diabetic MGs. Taken together, the results of the present study demonstrated that diabetes induced disruption of lipid homeostasis in MGs, which may mediate the decreased cell proliferation and increased inflammation caused by diabetes in MGs.

Multiple roles of FGF10 in the regulation of corneal endothelial wound healing.

Corneal endothelial dysfunction usually induces corneal haze and oedema, which seriously affect visual function. The main therapeutic strategy for this condition is corneal transplantation, but the use of this strategy is limited by the shortage of healthy donor corneas. Compared with corneal transplantation, drug intervention is less invasive and more accessible; thus, finding an effective pharmaceutical alternative for cornea transplantation is critical for the treatment of corneal endothelial dysfunction. In this study, we established a rabbit scratch model to investigate the effect of fibroblast growth factor 10 (FGF10) on corneal endothelial wound healing. Results showed that FGF10 injection accelerated the recovery of corneal transparency and increased the protein expression levels of ZO1, Na+/K+-ATPase and AQP-1. Moreover, FGF10 significantly inhibited the expression levels of endothelial-to-mesenchymal transition proteins and reduced the expression levels of the proinflammatory factors IL-1ß and TNF-α in the anterior chamber aqueous humour. FGF10 also enhanced the Na+/K+-ATPase activity by enhancing mitochondrial function as a result of its direct interaction with its conjugate receptor. Thus, FGF10 could be a new pharmaceutical preparation as treatment for corneal endothelial dysfunction.

ß-blocker eye drops affect ocular surface through ß2 adrenoceptor of corneal limbal stem cells.

BACKGROUND: Topical application of ß-blocker eye drops induces damage to the ocular surface in clinical. However, the mechanism involved remains incompletely understood. The purpose of this study was to investigate the influence and mechanism of ß-blocker eye drops on corneal epithelial wound healing. METHODS: Corneal epithelial wound healing models were constructed by epithelial scraping including in the limbal region and unceasingly received eye drops containing 5 mg/mL ß-blocker levobunolol, ß1-adrenoceptor (ß1AR)-specific antagonist atenolol or ß2-adrenoceptor (ß2AR)-specific antagonist ICI 118, 551. For the migration assay, the murine corneal epithelial stem/progenitor cells (TKE2) were wounded and subsequently incubated with levobunolol, atenolol, or ICI 118, 551. The proliferation and colony formation abilities of TKE2 cells treated with levobunolol, atenolol, or ICI 118, 551 were investigated by CCK-8 kit and crystal violet staining. The differentiation marker Cytokeratin 3 (CK3), the stem cell markers-Cytokeratin 14 (CK14) and Cytokeratin 19 (CK19), and corneal epithelium regeneration-related signaling including in Ki67 and the phosphorylated epithelial growth factor receptor (pEGFR) and phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2) were assessed by immunofluorescence staining. RESULTS: Levobunolol and ICI 118, 551 impaired corneal wound healing, decreased the expressions of CK3, CK14, and CK19 after limbal region scraping in vivo and reduced the migration and proliferation of TKE2 in vitro, whereas atenolol had no significant effect. Moreover, levobunolol and ICI 118, 551 inhibited corneal wound healing by mediating the expression of Ki67, and the phosphorylation of EGFR and ERK1/2 in the limbal and regenerated corneal epithelium. CONCLUSION: ß-blocker eye drops impaired corneal wound healing by inhibiting the ß2AR of limbal stem cells, which decreased corneal epithelial regeneration-related signaling. Therefore, a selective ß1AR antagonist might be a good choice for glaucoma treatment to avoid ocular surface damage.

NAD+ precursors protect corneal endothelial cells from UVB-induced apoptosis.

Excessive exposure of the eye to ultraviolet B light (UVB) leads to corneal edema and opacification because of the apoptosis of the corneal endothelium. Our previous study found that nicotinamide (NIC), the precursor of nicotinamide adenine dinucleotide (NAD), could inhibit the endothelial-mesenchymal transition and accelerate healing the wound to the corneal endothelium in the rabbit. Here we hypothesize that NIC may possess the capacity to protect the cornea from UVB-induced endothelial apoptosis. Therefore, a mouse model and a cultured cell model were used to examine the effect of NAD+ precursors, including NIC, nicotinamide mononucleotide (NMN), and NAD, on the UVB-induced apoptosis of corneal endothelial cells (CECs). The results showed that UVB irradiation caused apparent corneal edema and cell apoptosis in mice, accompanied by reduced levels of NAD+ and its key biosynthesis enzyme, nicotinamide phosphoribosyltransferase (NAMPT), in the corneal endothelium. However, the subconjunctival injection of NIC, NMN, or NAD+ effectively prevented UVB-induced tissue damage and endothelial cell apoptosis in the mouse cornea. Moreover, pretreatment using NIC, NMN, and NAD+ increased the survival rate and inhibited the apoptosis of cultured human CECs irradiated by UVB. Mechanistically, pretreatment using nicotinamide (NIC) recovered the AKT activation level and decreased the BAX/BCL-2 ratio. In addition, the capacity of NIC to protect CECs was fully reversed in the presence of the AKT inhibitor LY294002. Therefore, we conclude that NAD+ precursors can effectively prevent the apoptosis of the corneal endothelium through reactivating AKT signaling; this represents a potential therapeutic approach for preventing UVB-induced corneal damage.

DNase I improves corneal epithelial and nerve regeneration in diabetic mice.

DNase I has been reported to improve diabetic wound healing through the clearance of neutrophils extracellular traps (NETs) caused by neutrophil aggregation. However, the function of DNase I on diabetic corneal wound healing remains unclear. Here, we investigated the effect and mechanism of topical DNase I application on diabetic mouse corneal epithelial and nerve regeneration. Corneal epithelial defects, inflammatory response, regeneration-related signalling pathways, oxidative stress, corneal innervation and sensation were examined and compared between the diabetic and normal mice. The results confirmed firstly the increased NETs production during the delayed corneal epithelial wound healing of diabetic mice, which was significantly improved through either DNase I or Cl-amidine administration. Mechanistically, DNase I improved inflammation resolution, reactivated epithelial regeneration-related signalling pathways and attenuated the accumulation of reactive oxygen species (ROS). Moreover, DNase I application also promoted corneal nerve regeneration and restored the impaired corneal sensitivity in diabetic mice. Therefore, these results indicate that topical DNase I application promotes corneal epithelial wound healing and mechanical sensation restoration in diabetic mice, representing the potential therapeutic approach for diabetic keratopathy.