Different concentrations of betaxolol switch cell fate between necroptosis, apoptosis, and senescence in human corneal stromal cells.

Betaxolol is commonly used to manage glaucoma in clinical practice. However, its long-term use may damage the cornea. Thus, the cytotoxicity and mechanisms of betaxolol in human corneal stromal cells (HCSCs) warrant further study. In this study, we used in vitro HCSCs and in vivo rabbit corneal models to investigate betaxolol cytotoxic effects and mechanism of action. At near-clinical concentrations (0.28% and 0.14%), betaxolol inhibited caspase-8 activity, activated receptor-interacting protein kinase (RIPK)1, RIPK3, and mixed-spectrum kinase-like domain (MLKL), and phosphorylated MLKL to induce necroptosis in HCSCs. Similarly, moderate concentrations of betaxolol (0.07%-0.0175%) activated caspase-8 to trigger the exogenous apoptotic pathway. Through the intrinsic apoptotic pathway, betaxolol upregulated the expression of Bcl-2 family apoptotic proteins Bax and Bad and downregulated that of anti-apoptotic proteins Bcl-2 and Bcl-xL. This subsequently disrupted the mitochondrial membrane potential and cytoplasmic transfer of cytochrome c and apoptosis-inducing factor, activated caspase-9, and induced apoptosis in HCSCs. Furthermore, continuous treatment with low betaxolol concentrations (0.00875%) for three generations of HCSCs prevented apoptosis by promoting the expression of Bcl-xL and suppressing that of Bax. However, its toxic effects initiated cellular senescence by increasing reactive oxygen species, leading to the disruption of energy metabolism and DNA damage. Finally, clinical concentrations of betaxolol had a pro-apoptotic effect on rabbit corneal stromal cells in vivo. These results suggest that betaxolol induces cytotoxicity in a concentration-dependent manner in HCSCs, and that caspase-8 and Bcl-2 family proteins may be critical switches in the conversion of different HCSC death mechanisms.

Carteolol triggers senescence via activation of ß-arrestin-ERK-NOX4-ROS pathway in human corneal endothelial cells in vitro.

Carteolol is a commonly-used topical medication for primary open-angle glaucoma. However, long-term and frequent ocular application of carteolol entails its residuals at low concentration in the aqueous humor for a long duration and may exert latent toxicity in the human corneal endothelial cells (HCEnCs). Here, we treated the HCEnCs in vitro with 0.0117% carteolol for 10 days. Thereafter, we removed the cartelolol and normally cultured the cells for 25 days to investigate the chronical toxicity of carteolol and the underlying mechanism. The results exhibited that 0.0117% carteolol induces senescent features in the HCEnCs, such as increased senescence-associated ß-galactosidase positive rates, enlarged relative cell area and upregulated p16INK4A and senescence-associated secretory phenotypes, including IL-1α, TGF-ß1, IL-10, TNF-α, CCL-27, IL-6 and IL-8, as well as decreased Lamin B1 expression and cell viability and proliferation. Thereby, further exploration demonstrated that the carteolol activates ß-arrestin-ERK-NOX4 pathway to increase reactive oxygen species (ROS) production that imposes oxidative stress on energetic metabolism causing a vicious cycle between declining ATP and increasing ROS production and downregulation of NAD+ resulting in metabolic disturbance-mediated senescence of the HCEnCs. The excess ROS also impair DNA to activate the DNA damage response (DDR) pathway of ATM-p53-p21WAF1/CIP1 with diminished poly(ADP-Ribose) polymerase (PARP) 1, a NAD+-dependent enzyme for DNA damage repair, resulting in cell cycle arrest and subsequent DDR-mediated senescence. Taken together, carteolol induces excess ROS to trigger HCEnC senescence via metabolic disturbance and DDR pathway.

NAD+-Consuming Enzymes in Stem Cell Homeostasis.

Nicotinamide adenine dinucleotide (NAD+) is a coenzyme used in redox reactions, energy metabolism, and mitochondrial biogenesis. NAD+ is also required as a cofactor by nonredox NAD+-dependent enzymes. Hundreds of enzymes that consume NAD+ have been identified. The NAD+-consuming enzymes are involved in a variety of cellular processes such as signal transduction, DNA repair, cellular senescence, and stem cell (SC) homeostasis. In this review, we discussed how different types of NAD+-consuming enzymes regulate SC functions and summarized current research on the roles of the NAD+ consumers in SC homeostasis. We hope to provide a more global and integrative insight to the mechanism and intervention of SC homeostasis via the regulation of the NAD+-consuming enzymes.

Ultraviolet B irradiation induces senescence of human corneal endothelial cells in vitro by DNA damage response and oxidative stress.

The human corneal endothelial cells (HCEnCs) play a vital role in the maintenance of corneal transparency and visual acuity. In our daily life, HCEnCs are inevitably exposed to ultraviolet B (UVB) radiation leading to decreases of visual acuity and corneal transparency resulting in visual loss eventually. Therefore, understanding the UVB-induced cytotoxicity in HCEnCs is of importance for making efficient strategies to protect our vision from UVB-damage. However, in-depth knowledge about UVB-induced cytotoxicity in HCEnCs is missing. Herein, we pulse-irradiated the HCEnCs in vitro with 150 mJ/cm2 UVB (the environmental dose) at each subculture for 4 passages to explore the insights into UVB-induced phototoxicity. The results showed that the UVB-treated HCEnCs exhibit typical senescent characteristics, including significantly enlarged relative cell area, increased senescence-associated ß-galactosidase positive staining, and upregulated p16INK4A and senescence associated secretory phenotypes (SASPs) such as CCL-27, IL-1α/6/8/10, TGF-ß1 and TNF-α, as well as decreased cell proliferation and Lamin B1 expression, and translocation of Lamin B1. Furthermore, we explored the causative mechanisms of senescence and found that 150 mJ/cm2 UVB pulse-irradiation impairs DNA to activate DNA damage response (DDR) pathway of ATM-p53-p21WAF1/CIP1 with downregulated DNA repair enzyme PARP1, leading to cell cycle arrest resulting in DDR-mediated senescence. Meanwhile, UVB pulse-irradiation also elicits a consistent increase of ROS production to aggravate DNA damage and impose oxidative stress on energy metabolism leading to metabolic disturbance resulting in metabolic disturbance-mediated senescence. Altogether, the repeated pulse-irradiation of 150 mJ/cm2 UVB induces HCEnC senescence via both DDR pathway and energy metabolism disturbance.

Tissue-Engineered Corneal Endothelial Sheets Using Ultrathin Acellular Porcine Corneal Stroma Substrates for Endothelial Keratoplasty.

Tissue-engineered cornea endothelial sheets (TECES), created using a biocompatible thin and transparent carrier with corneal endothelial cells, could alleviate the shortage of donor corneas and provide abundant functional endothelial cells. In our previous clinical trials, the effectiveness and safety of the acellular porcine corneal stroma (APCS) applied in lamellar keratoplasty have been confirmed. In this study, we optimized the method to cut APCS into multiple 20 µm ultrathin lamellae by a cryostat microtome and investigated the feasibility of TECES by seeding rabbit corneal endothelial cells (RCECs) on ultrathin APCS. Cell adhesion, proliferation, and functional gene expression of RCECs on tissue-culture plastic and APCS of different thicknesses were compared. The results indicated that ultrathin lamellae were superior in increasing cell viability and maintaining cell functions. Analyzing with histology, electron microscopy, and immunofluorescence, we found that RCECs cultured on 20 µm ultrathin APCS for 5 days grew into a confluent monolayer with a density of 3726 ± 223 cells/mm2 and expressed functional biomarkers Na+/K+-ATPase and zonula occludens. After 14 days, RCECs formed an early stage of Descemet's membrane-like structure by synthesizing collagen IV and laminin. Human corneal endothelial cells were also used to further validate the supportive effect of ultrathin APCS on cells. The resulting constructs were flexible and tough enough to implant into rabbits' anterior chambers through small incisions. TECES adhered to the posterior corneal stroma, and the thickness of cornea gradually reduced to normal after grafting. These results indicate that the ultrathin APCS can serve as a tissue engineering carrier and might be a suitable alternative for endothelial cells expansion in endothelial keratoplasty.

Commentary: Novel Cell Culture Paradigm Prolongs Mouse Corneal Epithelial Cell Proliferative Activity In Vitro and In Vivo.

[This corrects the article DOI: 10.3389/fcell.2021.675998.].

Construction of tissue-engineered human corneal endothelium for corneal endothelial regeneration using a crosslinked amniotic membrane scaffold.

Descemet's membrane endothelial keratoplasty (DMEK) may provide fast visual rehabilitation in the therapy of corneal endothelial disorders. However, due to shortage of donated corneas, how to construct a corneal endothelial substitute with powerful functions that can be used for DMEK is still unsolved. Herein, we introduced the method of corneal crosslinking (CXL) and conjugated the components of native Descemet's membrane (DM) to improve the mechanical properties and the biocompatibility of denuded amniotic membrane (dAM), further assessed their effects on cell adhesion, proliferation, YAP translocation, and metabolic activity in human corneal endothelial (HCE) cells. Using modified crosslinked dAM (mcdAM) and non-transfected HCE cells, we constructed a tissue-engineered HCE (TE-HCE) and evaluated its functions in cat and monkey models as well. Our results showed that the mechanical properties of mcdAM were improved effectively by CXL, and the adhesion, proliferation, and YAP translocation of HCE cells were dose-dependently improved after ECM modification. The combination of 0.01 mg/mL laminin with 0.1 mg/mL fibronectin showed the highest efficacy. Then, the TE-HCE was constructed in vitro, with a high density of 3612 ± 243 cells/mm2. Results of DMEK in animal models showed that corneal transparency was maintained, accompanied with normal morphology and histological structure of the regenerated corneal endothelium. Therefore, CXL combined with DM-mimic-coating methods could effectively improve the mechanical properties of dAM and enhance the biocompatibility with HCE cells. The constructed TE-HCE had normal histological structure and functioned well in animal models via DMEK, which could be used as a promising powerful equivalent of HCE. STATEMENT OF SIGNIFICANCE: Using high-quality corneal endothelium and an appropriate endothelial keratoplasty is the most effective way for the treatment of corneal endotheliopathy. Descemet's membrane endothelial keratoplasty (DMEK) which can provide better visual acuity, lower immunological rejection rates, and improved graft survival is an ideal surgery at present. However, due to the shortage of donated corneas, it is urgent to find an equivalent substitute of corneal endothelial donor which is suitable for the DMEK surgery to solve the problem of corneal endothelial regeneration. Herein, we introduced the clinical cornea-crosslinking and Descemet's membrane-mimic-coating methods to build the modified crosslinked denuded amniotic membrane scaffold and further constructed a high-quality corneal endothelial functional substitute that can be used in DMEK surgery.

Timolol induces necroptosis, apoptosis and senescence concentration-dependently in rabbit Limbal stem cells in vitro.

Limbal stem cells (LSCs) are crucial for corneal transparency and vision. Any damages to LSCs might lead to limbal stem cell deficiency resulting in corneal opacification and even blindness. Here, we investigated the cytotoxicity of timolol and its underlying mechanisms in rabbit LSCs (rLSCs) in vitro. High concentrations of 0.5% and 0.25% timolol induced necroptosis in rLSCs to upregulate receptor interacting protein kinase (RIPK)1, RIPK3, mixed lineage kinase domain-like (MLKL) and phosphorylated MLKL along with downregulation of caspase-8 and caspase-2 within 4 h. While, median concentrations of 0.125% to 0.0625% timolol induced apoptosis in the rLSCs within 28 h. The apoptotic mechanism in the median-concentration timolol-treated rLSCs is probably via extrinsic apoptosis pathway by activating caspase-2, caspase-8 and caspase-3 and intrinsic apoptosis pathway triggered by excessive generation of ROS and subsequent DNA damage to upregulate Bax and Bad, downregulate Bcl-2 and Bcl-xL, subsequently disrupt mitochondrial membrane potential, cytosolically translocate cytochrome c and apoptosis-inducing factor, and activate caspase-9. In addition, low concentration of 0.03125% timolol induced senescence in the rLSCs by elevating ROS level and increasing number of senescence associated ß-galactosidase positive cells at 28 h. Our findings reveal that timolol induces necroptosis, apoptosis and senescence concentration-dependently in rLSCs in vitro.

Diclofenac Sodium Triggers p53-Dependent Apoptosis in Human Corneal Epithelial Cells via ROS-Mediated Crosstalk.

Diclofenac sodium (DFS), a nonsteroidal anti-inflammatory drug, is frequently used in ophthalmology, but it causes negative effects on corneas. The mechanisms underlying the toxicities to corneas remains unclear. The present study was designed to assess the cytotoxicity of DFS to human corneal epithelial (HCEP) cells in vitro and further investigate its related mechanisms. The HCEP cells were treated with DFS at different concentrations ranging from 0.003 125% to 0.1%. DFS showed a dose- and time-dependent cytotoxicity to HCEP cells including abnormal morphology and declined viability. The 0.05% DFS-treated HCEP cells presented cell cycle arrest at S phase, reactive oxygen species (ROS) overproduction, and positive staining of phosphorylated H2AX, suggesting that DFS caused ROS-mediated DNA damage. The upregulation of p53 expression, formation of apoptotic body, phosphatidylserine externalization, and DNA ladder demonstrated that the p53-dependent apoptosis pathway was involved in the cytotoxicity of DFS. Furthermore, DFS activated caspase-8, caspase-9, and caspase-3 altered the expression levels of Bcl-2 family proteins including tBid, Bax, and Bcl-2, as well as increased poly(ADP-ribose) polymerase (PARP) cleavage. DFS also induced ΔΨm disruption, resulting in the release of cytochrome c and apoptosis-inducing factor into the cytoplasm. Additionally, the DFS-induced apoptosis was alleviated by p53 inhibitor. Taken together, DFS triggered p53-dependent apoptosis in HCEP cells via ROS-mediated crosstalk between the extrinsic and intrinsic pathways.

Norfloxacin induces apoptosis and necroptosis in human corneal epithelial cells.

Norfloxacin (NOR) is applied clinically to treat keratitis. However, NOR has brought severe side-effects for human corneal epithelium (HCEP) due to overdose and potential toxicity. In this study, two in vitro experimental models including monolayer HCEP cells and tissue-engineered human corneal epithelium (TE-HCEP) were used to explore the cytotoxicity and its related mechanisms. The HCEP cells treated with NOR at concentrations from 0.1875 to 3.0 mg/mL displayed abnormal morphology, declined viability, and increased plasma membrane permeability. Moreover, 0.75 mg/mL NOR induced chromatin condensation, S phase arrest, phosphatidylserine externalization, and formation of apoptotic body through activation of caspase-2/-8/-9/-3, downregulation of Bcl-2 and Bcl-xL, upregulation of Bad and Bax, mitochondrial transmembrane potential disruption and release of cytochrome c and apoptosis inducing factor into cytosol, whereas 1.5 mg/mL and 3.0 mg/mL NOR upregulated the expressions of receptor-interacting protein kinase 1 (RIPK1), RIPK3 and mixed lineage kinase domain-like (MLKL) together with inactivation of caspase-2/-8. Furthermore, 0.1875-3.0 mg/mL NOR destroyed the multilayer structure of TE-HCEP model due to a dose-dependent cytotoxicity, which validated the above results. Overall, low-dose (0.1875-0.75 mg/mL) NOR induced apoptosis through mitochondrion-dependent and death receptor-mediated pathways, and high-dose (1.5-3.0 mg/mL) NOR triggered necroptosis via RIPK1-RIPK3-MLKL cascade in HCEP cells.

Dose- and Time-Dependent Cytotoxicity of Carteolol in Corneal Endothelial Cells and the Underlying Mechanisms.

Carteolol is a non-selective ß-adrenoceptor antagonist used for the treatment of glaucoma, and its abuse might be cytotoxic to the cornea. However, its cytotoxicity and underlying mechanisms need to be elucidated. Herein, we used an in vivo model of feline corneas and an in vitro model of human corneal endothelial cells (HCECs), respectively. In vivo results displayed that 2% carteolol (clinical dosage) could induce monolayer density decline and breaking away of feline corneal endothelial (FCE) cells. An in vitro model of HCECs that were treated dose-dependently (0.015625-2%) with carteolol for 2-28 h, resulted in morphological abnormalities, declining in cell viability and elevating plasma membrane (PM) permeability in a dose- and time- dependent manner. High-dose (0.5-2%) carteolol treatment induced necrotic characteristics with uneven distribution of chromatin, marginalization and dispersed DNA degradation, inactivated caspase-2/-8, and increased RIPK1, RIPK3, MLKL, and pMLKL expression. The results suggested that high-dose carteolol could induce necroptosis via the RIPK/MLKL pathway. While low-dose (0.015625-0.25%) carteolol induced apoptotic characteristics with chromatin condensation, typical intranucleosomal DNA laddering patterns, G1 cell-cycle arrest, phosphatidylserine (PS) externalization, and apoptotic body formation in HCECs. Meanwhile, 0.25% carteolol treatment resulted in activated caspase-2, -3, -8, and -9, downregulation of Bcl-2 and Bcl-xL, upregulation of Bax and Bad, ΔΨm disruption, and release of cytoplasmic cytochrome c (Cyt.c) and AIF into the cytoplasm. These observations suggested that low-dose carteolol could induce apoptosis via a caspase activated and mitochondrial-dependent pathway. These results suggested that carteolol should be used carefully, as low as 0.015625% cartelol caused apoptotic cell death in HCECs in vitro.

Establish an In Vitro Cell Model to Explore the Impacts of UVA on Human Corneal Endothelial Wound Healing.

PURPOSE: To provide scientific data for clinical practice in making strategies for accelerating corneal endothelial wound healing, we investigated the impact of UVA on the corneal endothelial wound healing process and the underlying mechanism using an in vitro cell model. MATERIALS AND METHODS: An in vitro cell model for corneal endothelial wound healing was established by scratching the in vitro cultured human corneal endothelial cell (HCEnC) confluent layer. Then, we investigated the impacts of UVA irradiation and Ascorbic acid-2-phosphate (Asc-2p) on the wound healing process of the in vitro HCEnC model by examining wound-healing index, F-actin+ rate, Ki-67+ rate, and ROS production. RESULTS: After scratching, the Ki-67+ and F-actin+ HCEnCs occupied the scratching gap. Furthermore, the F-actin+ rates were significantly higher than Ki-67+ rates in the wound closure area. After irradiated with UVA, the wound-healing indexes, Ki-67+ rates and F-actin+ rates of the wound-healing model significantly reduced, whereas the ROS production significantly increased in a dose-dependent manner. Pretreatment with Asc-2p significantly reduced the ROS production as well as increased the wound-healing indexes, Ki-67+rates and F-actin+ rates of the UVA irradiated wound-healing model. CONCLUSION: The migration of HCEnC plays a major role in the wound healing process of the established cell model, which is like the wound healing process in vivo. UVA decreases the wound closure of the in vitro HCEnC model dose-dependently, while antioxidant Asc-2p can attenuate the damage to UVA to HCEnCs probably via reducing ROS to improve their migration.

Apoptotic effects of norfloxacin on corneal endothelial cells.

Norfloxacin, a frequently used ocular antibiotic, might have cytotoxic effect on human corneal endothelial cells (HCECs), subsequently damage the cornea and finally impair human vision. However, the possible mechanisms of cytotoxicity of norfloxacin to HCEC line are unclear. Herein, we investigated the cytotoxicity of norfloxacin and its underlying cellular and molecular mechanisms using in vitro cultured non-transfected HCECs and verified the cytotoxicity with cat corneal endothelium in vivo. In the present study, the cytotoxicity of norfloxacin in the in vitro cultured HCECs was recognized by causing abnormal morphology such as cell shrinkage and detachment from plate bottom, and decline of viability of in vitro cultured HCECs. Then, its cytotoxicity was verified by inducing reduction of cell density and morphological abnormality of in vivo cat corneal endothelial cells. Furthermore, the cytotoxicity of norfloxacin in HCECs was corroborated as apoptosis by elevation of plasma membrane permeability, S phase arrest, phosphatidylserine externalization, DNA fragmentation, and apoptotic body formation in in vitro cultured HCECs and apoptosis-like swollen cells in the in vivo model. Moreover, norfloxacin induced extrinsic death receptor-mediated apoptosis pathway by activating caspase-2/-8/-3 and intrinsic mitochondrion-dependent apoptosis pathway by downregulating anti-apoptotic Bcl-2 and upregulating of pro-apoptotic Bad, which disrupted mitochondrial transmembrane potential, subsequently upregulated cytoplasmic cytochrome c and apoptosis-inducing factor and finally activated caspase-9/-3. Generally, norfloxacin induces HCE cell apoptosis via a death receptor-mediated and mitochondrion-dependent signaling pathway.

Insights into mechanisms of pranoprofen-induced apoptosis and necroptosis in human corneal stromal cells.

Pranoprofen (PPF) is a wildly used anti-inflammatory ophthalmic drug. It was reported that PPF could decrease early epithelialization of scrape wounds in rabbit cornea and could reduce cell activities of cultured human corneal endothelial cells. However, effects of PPF on corneal stromal cells playing important roles in corneal wound healing remain unknown. In this study,in vitro model of cultured human corneal stomal (HCS) cells and in vivo model of rabbit corneas were used to investigate the effects and underlying mechanisms of PPF. Our findings showed that high concentrations of PPF treatment (0.1 % to 0.0125 %) caused limited chromatin condensation and quickly decreased cell viability that was proved to initiate necroptosis in HCS cells through activating receptor interacting protein kinase (RIPK) and mixed lineage kinase domain-like (MLKL). While low concentrations of PPF treatment (0.00625 %) induced DNA fragmentation, apoptotic body formation, ROS generation, activation of caspases and increase in cytoplasmic content of Bad, Bax and cytoplasmic cytochrome c that suggested apoptosis happened through ROS-mediated caspase-dependent and caspase-independent pathways. Studies of rabbit corneas treated with 0.1 % PPF (the clinical concentration) showed that PPF could induce apoptosis of rabbit corneal stromal cells. This work would be helpful for better understanding cytotoxic effects PPF on human corneal cells.

Gatifloxacin inducing apoptosis of stromal fibroblasts through cross-talk between caspase-dependent extrinsic and intrinsic pathways.

AIM: To reveal the cytotoxicity and related mechanisms of gatifloxacin (GFX) to stromal fibroblasts (SFs) in vitro. METHODS: SFs were treated with GFX at different concentrations (0.009375%-0.3%), and their viability was detected by MTT method. The cell morphology was observed using light/transmission electron microscope. The plasma membrane permeability was measured by AO/EB double-staining. Then cell cycle, phosphatidylserine (PS) externalization, and mitochondrial transmembrane potential (MTP) were analyzed by flow cytometry. DNA damage was analyzed by electrophoresis and immunostaining. ELISA was used to evaluate the caspase-3/-8/-9 activation. Finally, Western blotting was applied for detecting the expressions of apoptosis-related proteins. RESULTS: Morphological changes and reduced viability of GFX-treated SFs demonstrated that GFX above 0.009375% had cytotoxicity to SFs with dependence of concentration and time. GFX-treating cells also showed G1 phase arrest, increased membrane permeability, PS externalization and DNA damage, which indicated that GFX induced apoptosis of SFs. Additionally, GFX could activate the caspase-8, caspase-9, and caspase-3, induce MTP disruption, downregulate B-cell leukemia-2 (Bcl-2) and B-cell leukemia-XL (Bcl-XL), and upregulate Bcl-2 assaciated X protein (Bax), Bcl-2-associated death promoter (Bad), Bcl-2 interacting domain (Bid) and cytoplasmic cytochrome C in SFs, suggesting that caspase-dependent extrinsic and intrinsic pathways were related to GFX-contributed apoptosis of SFs. CONCLUSION: The cytotoxicity of GFX induces apoptosis of SFs through triggering the caspase-dependent extrinsic and intrinsic pathways.

Phenylephrine induces necroptosis and apoptosis in corneal epithelial cells dose- and time-dependently.

In the present study, the toxicity of phenylephrine, a selective α1-adrenergic receptor agonist, in corneal epithelial cells and its underlying mechanisms were investigated using an in vitro model of human corneal epithelial cells (HCEPCs) and an in vivo model of New Zealand white rabbit corneas. The HCEPCs treated with phenylephrine at concentrations from 10% to 0.078125% displayed abnormal morphology, decline of cell viability and elevation of plasma membrane permeability time- and dose-dependently. Moreover, 10%-1.25% phenylephrine induce necrosis characteristics of marginalization and uneven distribution of chromatin through up-regulation of RIPK1, RIPK3 and MLKL along with inactivation of caspase-8 and caspase-2, whereas 0.625% phenylephrine induced condensed chromatin, S phase arrest, phosphatidylserine externalization, DNA fragmentation and apoptotic body formation in the HCECs through activation of caspase-2, -8, -9 and -3 as well as down-regulation of Bcl-2, up-regulation of Bad, ΔΨm disruption and release of cytochrome c and AIF into cytosol. At last, 10% phenylephrine induced destruction of the corneal epithelia and apoptosis of corneal epithelial cells in rabbit corneas. In conclusion, 10% to 1.25% phenylephrine cause necroptosis via RIPK1-RIPK3-MLKL axis and 0.625% phenylephrine induce apoptosis via a mitochondrion-dependent and death receptor-mediated signal pathway in HCEPCs.

Fibronectin regulates the self-renewal of rabbit limbal epithelial stem cells by stimulating the Wnt11/Fzd7/ROCK non-canonical Wnt pathway.

Microenvironmental factors regulate stem cell fate. Fibronectin (FN), a key extracellular matrix component of the microenvironment, has been linked to various stem cell behaviors. However, how FN controls self-renewal, proliferation, and homeostasis of limbal stem cells remains unclear. Our study investigated the roles of FN in the self-renewal of rabbit limbal epithelial stem cells (rLESCs) by assessing rLESC proliferation and stemness in the presence and absence of FN. We further examined the effect of FN on non-canonical Wnt signaling during rLESC proliferation by evaluating the expression of cell cycle regulators. We found that rLESC proliferation increased after FN treatment and that 12.5 µg/cm2 FN maintained rLESC stemness. FN facilitated rLESC self-renewal by promoting Wnt11 and Fzd7 interaction. Furthermore, FN modulated cell cycle regulators to enhance rLESC proliferation via the upregulation of ROCK1 and ROCK2. Our study provides new insights into the mechanism through which FN regulates the self-renewal of rLESCs; specifically, this occurs via stimulation of the Wnt11/Fzd7/ROCK non-canonical Wnt pathway. The roles of FN in the self-renewal of limbal epithelial stem cells should be further investigated for the potential treatment of limbal deficiency.

Construction of a high cell density human corneal endothelial equivalent and its transplantation in primate models.

BACKGROUND: Recently, many patients with corneal blindness caused by endothelial dysfunction have no opportunity to receive keratoplasty therapy because of the extremely limited number of donor corneas. Corneal tissue engineering opens a new path for in vitro reconstruction of tissue-engineered HCE which will cure the corneal endotheliopathy by clinical corneal transplantation. In this study, we construct a human corneal endothelium (HCE) equivalent with non-transfected monoclonal HCE (mcHCE) cells and modified denuded amniotic membrane (mdAM), and evaluate its functions in monkey models. METHODS: Tissue-engineered HCE (TE-HCE) was constructed by culturing DiI-labeled mcHCE cells on mdAMs in 20% fetal bovine serum-containing DMEM/Ham's Nutrient Mixture F12 (1:1) medium and 5% CO2 at 37°C on a 24-well culture plate. The constructed TE-HCE was transplanted into monkey corneas via penetrating keratoplasty with Descemet's membrane and endothelium stripped. The corneal transparency, thickness, and intraocular pressure were monitored in vivo, and the corneal morphology and histological structure were examined ex vivo 181 days after surgery. RESULTS: The constructed TE-HCE, with an average density of 3602.22 ± 45.22 cells/mm2 , mimicked its natural counterpart both in morphology and histological structure. In vivo, corneal transparency was maintained, and the corneal thickness gradually decreased to 567.33 ± 72.77 µm at day 181 after TE-HCE transplanted into monkey eyes, while intense corneal edema and turbid were found in mdAM-transplanted eyes with their corneal thicknesses maintained over 1000 µm during the monitoring period. Ex vivo, a monolayer of corneal endothelium, consisting of mcHCE cells at a density of 2795.65 ± 156.83 cells/mm2 , was reconstructed in transplanted monkey eyes. The cells in the transplanted area had the hexagonal or polygonal morphology and normal ultrastructure, and established plenty of cell-cell and cell-stromal matrix junctions. Besides, huge membrane-bounded flat stacks with electric dense inclusions were found in mcHCE cells beneath the plasma membrane at the stromal side. CONCLUSIONS: The constructed TE-HCE has normal histological property and functions well in monkey models. The TE-HCE could be used as a promising HCE equivalent in therapy of corneal endothelium dysfunction and corneal regenerative medicine.

Sodium Ferulate Attenuates Lidocaine-Induced Corneal Endothelial Impairment.

The introduction of intracameral anaesthesia by injection of lidocaine has become popular in cataract surgery for its inherent potency, rapid onset, tissue penetration, and efficiency. However, intracameral lidocaine causes corneal thickening, opacification, and corneal endothelial cell loss. Herein, we investigated the effects of lidocaine combined with sodium ferulate, an antioxidant with antiapoptotic and anti-inflammatory properties, on lidocaine-induced damage of corneal endothelia with in vitro experiment of morphological changes and cell viability of cultured human corneal endothelial cells and in vivo investigation of corneal endothelial cell density and central corneal thickness of cat eyes. Our finding indicates that sodium ferulate from 25 to 200 mg/L significantly reduced 2 g/L lidocaine-induced toxicity to human corneal endothelial cells, and 50 mg/L sodium ferulate recovered the damaged human corneal endothelial cells to normal growth status. Furthermore, 100 mg/L sodium ferulate significantly inhibited lidocaine-induced corneal endothelial cell loss and corneal thickening in cat eyes. In conclusion, sodium ferulate protects human corneal endothelial cells from lidocaine-induced cytotoxicity and attenuates corneal endothelial cell loss and central corneal thickening of cat eyes after intracameral injection with lidocaine. It is likely that the antioxidant effect of sodium ferulate reduces the cytotoxic and inflammatory corneal reaction during intracameral anaesthesia.

Tetracaine induces apoptosis through a mitochondrion-dependent pathway in human corneal stromal cells in vitro.

PURPOSE: Tetracaine is a local anesthetic widely used in ocular diagnosis and ophthalmic surgery and may lead to some adverse effects and complications at a clinical dose. To assess the cytotoxicity and molecular toxicity mechanisms of tetracaine, we used human corneal stromal (HCS) cells as an in vitro model to study the effects of tetracaine on HCS cells. MATERIALS AND METHODS: The cytotoxicity of tetracaine on HCS cells was investigated by examining the changes of cell growth, morphology, viability and cell cycle progressing when HCS cells were treated with tetracaine at concentrations from 10 g/L to 0.078125 g/L. To prove the hypothesis that the cytotoxicity of tetracaine on HCS cells was related with apoptosis induction, we further detected multiple changes in HCS cells, including plasma membrane (PM) permeability, phosphatidylserine (PS) orientation, genomic DNA integrality, and cell ultrastrcuture after treated with tetracaine. Furthermore, the pro-apoptotic signalling pathway induced by tetracaine was explored through detecting the activation of various caspases, the changes of mitochondrial transmembrane potential (MTP), the expression level of Bcl-2 family proteins and the amount of mitochondria-released apoptosis regulating proteins in cytoplasm. RESULTS: Tetracaine at concentrations above 0.15625 g/L had a dose- and time-dependent cytotoxicity to HCS cells, which resulted cell growth inhibition, proliferation retardation, morphological abnormalities and decreased viability. Meanwhile, we found that the HCS cells treated with tetracaine had typical features associated with apoptosis, including an increase in PM permeability, PS externalization, DNA fragmentation and apoptotic body formation. Tetracaine not only resulted in caspase-3, caspase-8 and caspase-9 activation and disruption of MTP but also downregulated Bcl-2 and Bcl-xL and upregulated Bad and Bax, along with the upregulation of cytoplasmic cytochrome c (Cyt. c) and apoptosis-inducing factor (AIF). CONCLUSIONS: These results suggested that tetracaine-induced apoptosis might be triggered through Fas death receptors and mediated by Bcl-2 family proteins in the mitochondria-dependent pathway. Our findings identified the cytotoxicity and molecular mechanisms of tetracaine, which could provide a reference value for the safety of this medication and prospective therapeutic interventions in eye clinic.