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.

CGRP Released by Corneal Sensory Nerve Maintains Tear Secretion of the Lacrimal Gland.

Purpose: This study aims to elucidate the calcitonin gene-related peptide (CGRP) mediation and primary mechanism of corneal sensory nerves on tear production of the lacrimal gland. Methods: Mouse corneal denervation models were constructed through surgical axotomy, pharmacologic treatment with capsaicin or resiniferatoxin, and Trpv1-Cre/DTR mice with diphtheria toxin injection. The capsaicin-treated mice received subconjunctival injection of CGRP or substance P, while the normal C57BL/6J mice were administered with CGRP receptor antagonist BIBN-4096. Furthermore, double immunostaining of c-FOS+ and choline acetyltransferase was used to evaluate the activation of the superior salivatory nucleus (SSN). Mouse lacrimal glands were collected for transcriptomic sequencing and subsequent RNA and protein expression analysis. Results: The corneal denervated mice exhibited a significant reduction in corneal sensitivity and tear secretion. In capsaicin-treated mice, tear secretion decreased to 2.5 ± 0.5 mm compared to 6.3 ± 0.9 mm in control mice (P < 0.0001). However, exogenous administration of CGRP in capsaicin-treated mice increased tear secretion from 2.6 ± 0.5 mm to 4.5 ± 0.5 mm (P = 0.0009), while BIBN-4096 treatment reduced tear secretion to 3.4 ± 0.5 mm when compared to 7.3 ± 0.7 mm in control mice (P = 0.0022). Furthermore, c-FOS+ cell number in the SSN increased by twofold (P = 0.0168) after CGRP administration compared with capsaicin-treated mice. In addition, the expressions of CCNA2, Ki67, PCNA, and CDK1 in acinar cells of the lacrimal gland were impaired by corneal denervation and alleviated by CGRP administration. Conclusions: CGRP released by corneal sensory nerves mediates tear secretion of the lacrimal gland, providing a new strategy for improving tear secretion in patients with neurotrophic keratitis.

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.

The role of corneal endothelium in macular corneal dystrophy development and recurrence.

Macular corneal dystrophy (MCD) is a progressive, bilateral stromal dystrophic disease that arises from mutations in carbohydrate sulfotransferase 6 (CHST6). Corneal transplantation is the ultimate therapeutic solution for MCD patients. Unfortunately, postoperative recurrence remains a significant challenge. We conducted a retrospective review of a clinical cohort comprising 102 MCD patients with 124 eyes that underwent either penetrating keratoplasty (PKP) or deep anterior lamellar keratoplasty (DALK). Our results revealed that the recurrence rate was nearly three times higher in the DALK group (39.13%, 9/23 eyes) compared with the PKP group (10.89%, 11/101 eyes), suggesting that surgical replacement of the corneal endothelium for treating MCD is advisable to prevent postoperative recurrence. Our experimental data confirmed the robust mRNA and protein expression of CHST6 in human corneal endothelium and the rodent homolog CHST5 in mouse endothelium. Selective knockdown of wild-type Chst5 in mouse corneal endothelium (ACsiChst5), but not in the corneal stroma, induced experimental MCD with similar extracellular matrix synthesis impairments and corneal thinning as observed in MCD patients. Mice carrying Chst5 point mutation also recapitulated clinical phenotypes of MCD, along with corneal endothelial abnormalities. Intracameral injection of wild-type Chst5 rescued the corneal impairments in ACsiChst5 mice and retarded the disease progression in Chst5 mutant mice. Overall, our study provides new mechanistic insights and therapeutic approaches for MCD treatment by high-lighting the role of corneal endothelium in MCD development.

A Versatile Hydrogel with Antibacterial and Sequential Drug-Releasing Capability for the Programmable Healing of Infectious Keratitis.

Hydrogels have attracted tremendous attention as favorable corneal substitutes for treating severe infectious keratitis (IK). However, current hydrogel-based corneal substitutes were majorly designed to promote the single stage of corneal regeneration, which falls short in meeting the clinical management needs of severe IK including the multiple phases of corneal wound healing. Herein, we introduce a versatile hybrid hydrogel (SQPV) composed of silk fibroin and chitosan, which exhibits spatiotemporal properties for drug release. The SQPV is fabricated by incorporating verteporfin-loaded poly(lactic-co-glycolic)-polyethylene glycol-o-nitrobenzene micelles into a hydrogel network, which is formed from methacrylate silk fibroin and glycidyl methacrylate functionalized quaternized chitosan containing polydeoxyribonucleotide. This double network approach results in a material with exceptional anti-inflammatory, antibacterial, and proliferative stimulation and tissue remodeling regulation capabilities. Furthermore, SQPV showcases mechanical strength and transparency akin to those of native cornea. Extensive in vitro and in vivo studies validate SQPV's ability to effectively eliminate residual bacteria, mitigate inflammation, foster regeneration of corneal epithelium and stroma, prevent corneal scarring, and ultimately expedite wound healing. In summary, the SF/CS-based hybrid hydrogel may represent a promising substitute for comprehensive corneal repair and regeneration in severe IK.

Loss of TRPM8 Exacerbate Herpes Simplex Keratitis Infection in Mice by Promoting the Infiltration of CD11b+ Ly6G+ Cells and Increasing the Viral Load in the Cornea.

Purpose: To reveal the role of transient receptor potential cation subfamily M member 8 (TRPM8) channels in herpes simplex keratitis (HSK). Methods: HSK models were established using TRPM8 knockout (TRPM8-/-) mice and their wild-type (WT) littermates. The infected corneas were graded and harvested to evaluate the mRNA levels of inflammatory factors through quantitative real-time polymerase chain reaction (RT-PCR), as well as the infiltration of inflammatory cells through immunofluorescence staining and flow cytometry. Viral titers were determined by plaque assay and absolute quantitative method. RNA-sequencing was conducted to elucidate the transcriptome of corneal epithelium in response to TRPM8 knockout after infection. The anti-inflammatory effect of TRPM8 agonist menthol was documented via subconjunctival administration. Results: Compared to their wild-type counterparts, TRPM8-deficient mice exhibited exacerbated infection symptoms and thicker corneas in HSK models. Infection in TRPM8-deficient mice resulted in significant lymphocyte infiltration, primarily consisting of Ly6G+ CD11b+ cells. Additionally, TRPM8-deficient mice displayed increased levels of corneal viral titers after infection, along with decreased expression of interferon-stimulated genes (ISGs). Subconjunctival administration of menthol effectively alleviated infection-induced symptoms and Ly6G+ CD11b+ cell infiltration in herpes simplex virus type 1 (HSV-1)-treated mice. Conclusions: TRPM8 promoted host resistance to HSV-1 infection by suppressing the accumulation of Ly6G+ CD11b+ cells and virus replication. These findings suggest that targeting TRPM8 could be valuable for therapeutic interventions against HSV-1 infections.

Development of Thermoplastic Polyurethane Films for the Replacement of Corneal Endothelial Function of Transparency Maintenance.

Endothelial keratoplasty is the main surgical procedure for treating corneal endothelial dysfunction (CED), which is limited by the global shortage of donor corneas. Herein, we developed and evaluated the modified thermoplastic polyurethane (M-TPU) films with gelatin-glycidyl methacrylate to replace the corneal endothelial function and maintain corneal transparency. The films displayed comparable light transmission characteristics with normal corneas and clinically favorable mechanical properties for surgical manipulation. After surface modification, the hydrophilicity and biocompatibility of M-TPU films were significantly improved. In the rabbit CED model, the M-TPU implants exhibited firm adhesion to the exposed stromal surface. The rabbit corneal transparency and thickness could be restored completely within 1 week of M-TPU film implantation. There was no significant inflammatory reaction and immune rejection during the follow-up of 1 month. Proteomic analysis suggested that the complement inhibition, the increase of mineral absorption, and the decrease of P53 apoptosis signaling pathway and lysine degradation might be beneficial in maintaining the corneal transparency. Overall, our study demonstrated the potential of M-TPU films as artificial implants for the replacement of corneal endothelial function to restore corneal thickness and transparency.

Ex vivo cultivated retinal pigment epithelial cell transplantation for the treatment of rabbit corneal endothelial dysfunction.

OBJECTIVE: Stem cell therapy is a promising strategy for the treatment of corneal endothelial dysfunction, and the need to find functional alternative seed cells of corneal endothelial cells (CECs) is urgent. Here, we determined the feasibility of using the retinal pigment epithelium (RPE) as an equivalent substitute for the treatment of corneal endothelial dysfunction. METHODS: RPE cells and CECs in situ were obtained from healthy New Zealand male rabbits, and the similarities and differences between them were analyzed by electron microscopy, immunofluorescent staining, and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). Rabbit primary RPE cells and CECs were isolated and cultivated ex vivo, and Na+/K+-ATPase activity and cellular permeability were detected at passage 2. The injection of cultivated rabbit primary RPE cells, CECs and human embryonic stem cell (hESC)-derived RPE cells was performed on rabbits with corneal endothelial dysfunction. Then, the therapeutic effects were evaluated by corneal transparency, central corneal thickness, enzyme linked immunosorbent assay (ELISA), qRT-PCR and immunofluorescent staining. RESULTS: The rabbit RPE cells were similar in form to CECs in situ and ex vivo, showing a larger regular hexagonal shape and a lower cell density, with numerous tightly formed cell junctions and hemidesmosomes. Moreover, RPE cells presented a stronger barrier and ionic pumping capacity than CECs. When intracamerally injected into the rabbits, the transplanted primary RPE cells could dissolve corneal edema and decrease corneal thickness, with effects similar to those of CECs. In addition, the transplantation of hESC-derived RPE cells exhibited a similar therapeutic effect and restored corneal transparency and thickness within seven days. qRT-PCR results showed that the expressions of CEC markers, like CD200 and S100A4, increased, and the RPE markers OTX2, BEST1 and MITF significantly decreased in the transplanted RPE cells. Furthermore, we have demonstrated that rabbits transplanted with hESC-derived RPE cells maintained normal corneal thickness and exhibited slight pigmentation in the central cornea one month after surgery. Immunostaining results showed that the HuNu-positive transplanted cells survived and expressed ZO1, ATP1A1 and MITF. CONCLUSION: RPE cells and CECs showed high structural and functional similarities in barrier and pump characteristics. Intracameral injection of primary RPE cells and hESC-derived RPE cells can effectively restore rabbit corneal clarity and thickness and maintain normal corneal function. This study is the first to report the effectiveness of RPE cells for corneal endothelial dysfunction, suggesting the feasibility of hESC-derived RPE cells as an equivalent substitute for CECs.

A Novel Inorganic Aluminum Phosphate-Based Flame Retardant and Thermal Insulation Coating and Performance Analysis.

Currently, most thin-layer expandable coatings are polymer-based, with very few inorganic expandable coatings. Due to the high environmental friendliness of inorganic coatings, studying new types of inorganic coatings is of great significance. A novel amorphous aluminum phosphate-based flame-retardant coating was prepared by modifying it with nano-silica, hollow silica beads, hollow glass microspheres, and boron carbide. A comprehensive study was conducted on the flame retardancy and thermal insulation performance, composition and structural evolution under flame and physical and chemical properties, and the mechanisms of flame retardancy and thermal insulation were elucidated. Large-plate combustion testing, bonding strength testing, XRD, IR, TG-DSC, and SEM testing were all applied in this work. The synergistic effect of the four fillers was very obvious, and a series of AP22XY (nano-silica/silica beads/hollow glass microspheres/boron carbide = 2:2:0:4, 2:2:1:3, 2:2:2:2, 2:2:3:1, 2:2:4:0) coatings were prepared. The change in the ratio of glass microspheres to boron carbide had a significant impact on the composition and structural evolution of the coating, thus reflecting its effectiveness as a flame retardant and thermal insulation. Although decreasing the ratio would promote the formation of borosilicate glass and Al18B4O33 and improve the thermal stability of coatings, the structure inside of the coating, especially the skeleton, would be dense, which is not conducive to thermal insulation. When the ratio of glass microspheres to boron carbide is 3:1, AP2231 shows the best fire resistance. Under the combustion of butane flame at about 1200-1300 °C, the backside temperature reaches a maximum of 226 °C at 10 min, and then the temperature gradually decreases to 175 °C at 60 min. This excellent performance is mainly attributed to three aspects: (1) the foaming and expandability of coatings when exposed to fire, (2) the multiple endothermic reactions the coating undergoes, and (3) the improvement effect of boron carbide. Additionally, AP2231 shows the best bonding performance with a strength of close to 4.5 MPa after combustion, because of the appropriate content matching between borosilicate glass, Al18B4O33, and hollow glass microspheres. The coating has potential application prospects in the construction and transportation fields, such as the protection of structural steel, fire prevention in subways and tunnels, and the prevention of lithium battery fires.

Decellularized Porcine Conjunctiva in Treating Severe Symblepharon.

This prospective study aimed to evaluate the effectiveness of decellularized porcine conjunctiva (DPC) in the management of severe symblepharon. Sixteen patients with severe symblepharon were enrolled in this study. After symblepharon lysis and Mitomycin C (MMC) application, tarsus defects were covered with residual autologous conjunctiva (AC), autologous oral mucosa (AOM), or DPC throughout the fornix, and DPC was used for all the exposed sclera. The outcomes were classified as complete success, partial success, or failure. Six symblepharon patients had chemical burns and ten had thermal burns. Tarsus defects were covered with DPC, AC, and AOM in two, three, and eleven cases, respectively. After an average follow-up of 20.0 ± 6 months, the anatomical outcomes observed were complete successes in twelve (three with AC+DPC, four with AC+AOM+DPC, and five with AOM+DPC) (75%) cases, partial successes in three (one with AOM+DPC and two with DPC+DPC) (18.75%) cases, and failure in one (with AOM+DPC) (6.25%) case. Before surgery, the depth of the narrowest part of the conjunctival sac was 0.59 ± 0.76 mm (range, 0-2 mm), tear fluid quantity (Schirmer II tests) was 12.5 ± 2.26 mm (range, 10-16 mm), and the distance of the eye rotation toward the opposite direction of the symblepharon was 3.75 ± 1.39 mm (range, 2-7 mm). The fornix depths increased to 7.53 ± 1.64 mm (range, 3-9 mm), eye movement was significantly improved, and the distance of eye movement reaching 6.56 ± 1.24 mm (range, 4-8 mm) 1 month after the operation; the postoperative Schirmer II test (12.06 ± 2.90 mm, range, 6-17 mm) was similar to that before surgery. Goblet cells were finally found in fifteen patients by conjunctival impression cytology in the transplantation area of DPC, except for one patient who failed. DPC could be considered an alternative for ocular surface reconstruction of severe symblepharon. Covering tarsal defects with autologous mucosa is necessary for extensive reconstruction of the ocular surface.

Uncovering Dislocation- and Precipitate-Induced Viscoplastic Damage in Al-Zn-Mg Alloy.

The existing phenomenological theories of plastic forming of sheet metal lack the predictability of the influences of dislocations and precipitates on viscoplastic damage in Al-Zn-Mg alloys. This study examines the evolution of grain size that occurs when the Al-Zn-Mg alloy undergoes a hot deformation process, specifically concentrating on dynamic recrystallization (DRX). The uniaxial tensile tests are conducted at deformation temperatures ranging from 350 to 450 °C and strain rates of 0.01-1 s-1. The intragranular and intergranular dislocation configurations and their interactions with dynamic precipitates are revealed by transmission electron microscopy (TEM). In addition, the MgZn2 phase induces microvoid formation. Subsequently, an improved multiscale viscoplastic constitutive model is established that emphasizes the effect of precipitates and dislocations on the evolution of microvoid-based damage. Using a calibrated and validated micromechanical model, the simulation of hot-formed U-shaped parts is conducted through finite element (FE) analysis. During the hot U-forming process, the formation of defects is expected to have an impact on both the distribution of thickness and the level of damage. In particular, the damage accumulation rate is influenced by temperature and strain rate, and local thinning is caused by the damage evolution of U-shaped parts.

Norepinephrine as the Intrinsic Contributor to Contact Lens-Induced Pseudomonas aeruginosa Keratitis.

Purpose: Contact lens wear (CLW) is one of the leading risk factors for Pseudomonas aeruginosa keratitis (PAK). However, the intrinsic factors that contribute to the high susceptibility to keratitis during CLW remain to be elucidated. CLW over an extended period can elevate corneal norepinephrine (NE) concentration. In this study, we investigated the role of NE in promoting PAK. Methods: We constructed an injury-induced PAK model and a CLW-induced PAK model to confirm the impact of NE during corneal infection. Pharmacological blockage of NE and gene knockdown mouse were used to investigate the downstream effector of NE. RNA sequencing was performed to explore the cellular alterations during NE treatment. Non-parametric Mann-Whitney U test or Kruskal-Wallis test were used to ascertain the significance (P < 0.05). Results: Supplementation of NE led to PAK even without artificial corneal injury during CLW. The effect was mediated by the ß2-adrenergic receptor (ß2-AR) in the corneal epithelium. The ß2-AR blockage by the NE antagonist ICI118,551 (ICI) or by deleting of its encoding gene Adrb2 significantly alleviated infection during CLW. Conversely, ß2-AR activation compromised the integrity of the epithelium and significantly increased the cortical plaque marker ezrin. Transcriptome analysis identified that the protective effect of ICI on the keratitis was mediated by dual-specificity phosphatases. Suramin, a Dusp5 antagonist, abrogated the protective effect of ICI. Conclusions: These data reveal a new mechanism by which NE acts as an intrinsic factor that promotes CLW-induced PAK and provide novel therapeutic targets for treating keratitis by targeting NE-ß2-AR.

ECM-Like Adhesive Hydrogel for the Regeneration of Large Corneal Stromal Defects.

The repair of large-diameter corneal stroma defects is a major clinical problem. Although some studies have attempted to use hydrogels to repair corneal damage, most of these hydrogels can only be used for focal stromal defects that are ≤3.5 mm in diameter due to poor hydrogel adhesion. Here, a photocurable adhesive hydrogel that mimics the extracellular matrix (ECM) with regard to composition for repairing 6 mm-diameter corneal stromal defects in rabbits is investigated. This ECM-like adhesive can be rapidly cured after light exposure, with high light transmittance and good mechanical properties. More importantly, this hydrogel maintains the viability and adhesion of cornea-derived cells and promotes their migration in vitro in 2D and 3D culture environments. Proteomics analysis confirms that the hydrogel promotes cell proliferation and ECM synthesis. Furthermore, in rabbit corneal stromal defect repair experiments, it is proven by histological and proteomic analysis that this hydrogel can effectively promote corneal stroma repair, reduce scar formation, and increase corneal stromal-neural regeneration at the six months follow-up. This work demonstrates the great application of ECM-like adhesive hydrogels for the regeneration of large-diameter corneal defects.

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.

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.

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.

Overactivation of Norepinephrine-ß2-Adrenergic Receptor Axis Promotes Corneal Neovascularization.

Purpose: To investigate the role of the sympathetic nervous system in corneal neovascularization (CNV) and to identify the downstream pathway involved in this regulation. Methods: Three types of CNV models were constructed with C57BL/6J mice, including the alkali burn model, suture model, and basic fibroblast growth factor (bFGF) corneal micropocket model. Subconjunctival injection of the sympathetic neurotransmitter norepinephrine (NE) was administered in these three models. Control mice received injections of water of the same volume. The corneal CNV was detected using slit-lamp microscopy and immunostaining with CD31, and the results were quantified by ImageJ. The expression of ß2-adrenergic receptor (ß2-AR) was stained with mouse corneas and human umbilical vein endothelial cells (HUVECs). Furthermore, the anti-CNV effects of ß2-AR antagonist ICI-118,551 (ICI) were examined with HUVEC tube formation assay and with a bFGF micropocket model. Additionally, partial ß2-AR knockdown mice (Adrb2+/-) were used to establish the bFGF micropocket model, and the corneal CNV size was quantified based on the slit-lamp images and vessel staining. Results: Sympathetic nerves invaded the cornea in the suture CNV model. The NE receptor ß2-AR was highly expressed in corneal epithelium and blood vessels. The addition of NE significantly promoted corneal angiogenesis, whereas ICI effectively inhibited CNV invasion and HUVEC tube formation. Adrb2 knockdown significantly reduced the cornea area occupied by CNV. Conclusions: Our study found that sympathetic nerves grow into the cornea in conjunction with newly formed vessels. The addition of the sympathetic neurotransmitter NE and activation of its downstream receptor ß2-AR promoted CNV. Targeting ß2-AR could potentially be used as an anti-CNV strategy.

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.

Norepinephrine as an Enhancer Promoting Corneal Penetration of Riboflavin for Transepithelial Corneal Crosslinking.

Purpose: Previously, we found norepinephrine (NE) could affect the corneal epithelial integrity, herein we investigated the feasibility and safety of NE serving as a chemical enhancer to promote corneal penetration of riboflavin during transepithelial corneal crosslinking (CXL). Methods: The dosage of NE that could promote riboflavin diffusion through the healthy epithelial barrier without inducing epithelial damage in C57BL/6 mice was determined. The safety of NE treatment was confirmed by morphological and histological examinations of the whole cornea. The efficacy of NE in promoting riboflavin penetration was verified by slit lamp and scanning electron microscope (SEM), and corneal biomechanical measurement after CXL. To better fit the clinical scenario, increased NE dosage and shortened riboflavin infiltration time were further evaluated. Results: The lowest dosage of NE (1 mg/mL) that facilitated transepithelial riboflavin permeation was 2 µL. No visible corneal structure alteration was observed after NE treatment. SEM indicated dissociation of intercellular junctions among corneal epithelial cells. Homogenous distribution of riboflavin throughout corneal stroma was observed. NE-treated corneas reached comparable biomechanical properties after CXL, including stress-relaxation curve and elastic modulus, with corneas treated with the commercially available transepithelial drug Peschke TE. To better fit the clinical scenario, increasing NE up to 5.5 µL helped riboflavin infiltrate the corneal stroma within 30 minutes. After CXL with 9 mW/cm2 ultraviolet-A (UVA) for 2.5 minutes, the cornea showed significantly enhanced corneal biomechanical properties with undisturbed corneal endothelium. Conclusions: NE serves as an effective enhancer in increasing riboflavin diffusion with limited impairment on corneal epithelium and has great potential for clinical application. Translation Relevance: NE serves as an effective enhancer for riboflavin penetration and clinical transepithelial CXL.