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.

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.

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.

Construction of Anterior Hemi-Corneal Equivalents Using Nontransfected Human Corneal Cells and Transplantation in Dog Models.

Tissue-engineered human anterior hemi-cornea (TE-aHC) is a promising equivalent for treating anterior lamellar keratopathy to surmount the severe shortage of donated corneas. This study was intended to construct a functional TE-aHC with nontransfected human corneal stromal (ntHCS) and epithelial (ntHCEP) cells using acellular porcine corneal stromata (aPCS) as a carrier scaffold, and evaluate its biological functions in a dog model. To construct a TE-aHC, ntHCS cells were injected into an aPCS scaffold and cultured for 3 days; then, ntHCEP cells were inoculated onto the Bowman's membrane of the scaffold and cultured for 5 days under air-liquid interface condition. After its morphology and histological structure were characterized, the constructed TE-aHC was transplanted into dog eyes via lamellar keratoplasty. The corneal transparency, thickness, intraocular pressure, epithelial integrity, and corneal regeneration were monitored in vivo, and the histological structure and histochemical property were examined ex vivo 360 days after surgery, respectively. The results showed that the constructed TE-aHC was highly transparent and composed of a corneal epithelium of 7-8 layer ntHCEP cells and a corneal stroma of regularly aligned collagen fibers and well-preserved glycosaminoglycans with sparsely distributed ntHCS cells, mimicking a normal anterior hemi-cornea (aHC). Moreover, both ntHCEP and ntHCS cells maintained positive expression of their marker and functional proteins. After transplantation into dog eyes, the constructed TE-aHC acted naturally in terms of morphology, structure and inherent property, and functioned well in maintaining corneal clarity, thickness, normal histological structure, and composition in dog models by reconstructing a normal aHC, which could be used as a promising aHC equivalent in corneal regenerative medicine and aHC disorder therapy.

Construction of a human corneal stromal equivalent with non-transfected human corneal stromal cells and acellular porcine corneal stromata.

A tissue-engineered human corneal stroma (TE-HCS) has been developed as a promising equivalent to the native corneal stroma for replacement therapy. However, there is still a crucial need to improve the current approaches to render the TE-HCS equivalent more favorable for clinical applications. At the present study, we constructed a TE-HCS by incubating non-transfected human corneal stromal (HCS) cells in an acellular porcine corneal stromata (aPCS) scaffold in 20% fetal bovine serum supplemented DMEM/F12 (1:1) medium at 37 °C with 5% CO2in vitro. After 3 days of incubation, the constructed TE-HCS had a suitable tensile strength for transplantation, and a transparency that is comparable to native cornea. The TE-HCS had a normal histological structure which contained regularly aligned collagen fibers and differentiated HCS cells with positive expression of marker and functional proteins, mimicking a native HCS. After transplantation into rabbit models, the TE-HCS reconstructed normal corneal stroma in vivo and function well in maintaining corneal clarity and thickness, indicating that the completely biological TE-HCS could be used as a HCS equivalent. The constructed TE-HCS has promising potentials in regenerative medicine and treatment of diseases caused by corneal stromal disorders.

Role of hemoglobin from blood clam Scapharca kagoshimensis beyond oxygen transport.

The evolutionary race between hosts and pathogens has led to a variety of adaptations. Little is known about the immunological role of hemoglobin (Hb) in antimicrobial immune responses. Results showed that a 31.2 kDa monodimer Hb (skHbI) and a 57.8 kDa heterotetramer Hb (skHbII) from the blood clam, Scapharca kagoshimensis, had phenoloxidase (PO)-like activities and antimicrobial activities. Both were found capable of oxidizing l-DOPA, catechol and hydroquinone. Their PO-like activities were visibly greatly inhibited by oxidase inhibitors, EDTA, and divalent metal ions, and greatly enhanced by isopropanol and Fe(2+), indicating that they have the properties of a metalloenzyme and a catecholase-type PO as well. They also showed obvious anti-bacterial activities against gram-positive bacteria but not against either gram-negative bacteria nor fungi. The anti-bacterial activities levels were a result of the generation of reactive oxygen species (ROS) of superoxide anions. These results indicate that skHbI and skHbII, not only function as iron-containing oxygen carriers, but also exert anti-bacterial activities and catecholase-type oxidizing activities. The fact that skHbII exerts high level of PO-like activity indicates different roles in the innate immunodefense system. These results may improve understanding of the multiple functions of invertebrate Hbs beyond serving as oxygen carriers and may provide insight into how the fundamental and universal mode of the innate immune system has persisted in respiratory proteins throughout the course of evolution.

Dose dependent cytotoxicity of pranoprofen in cultured human corneal endothelial cells by inducing apoptosis.

Pranoprofen (PPF), a non-steroidal anti-inflammatory drugs (NSAIDs), is often used in keratitis treatment in clinic. Several studies have assessed in vitro the cytotoxicity of topical NSAIDs to corneal epithelial cells due to its importance for predicting human corneal toxicity. Damage by cytotoxic drugs can result in excessive loss of human corneal endothelial (HCE) cells which lead to decompensation of the endothelium and eventual loss of visual acuity. However, the endothelial cytotoxicity of PPF has not yet been reported using an in vitro model of HCE cells. This study assessed the cytotoxicity of PPF to HCE cells and its underlying mechanism. Cellular viability was determined using inverted phase contrast light microscopy, and plasma membrane permeability, genomic DNA fragmentation, and ultrastructure were detected by acridine orange/ethidium bromide staining, DNA agarose gel electrophoresis, and transmission electron microscopy (TEM), respectively. The results on cellular viability showed that PPF at concentrations ranging from 0.0625 to 1.0 g/l had poignant cytotoxicity to HCE cells, and the extent of its cytotoxicity was dose- and time-dependent. Further characterization indicated that PPF induced plasma membrane permeability elevation, DNA fragmentation, and apoptotic body formation, proving its apoptosis inducing effect on HCE cells. In conclusion, PPF above 0.0625 g/l has poignant cytotoxicity on HCE cells in vitro by inducing cell apoptosis, and should be carefully employed in eye clinic.

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.

Transplantation of tissue-engineered human corneal endothelium in cat models.

PURPOSE: To evaluate the performance of reconstructed tissue-engineered human corneal endothelium (TE-HCE) by corneal transplantation in cat models. METHODS: TE-HCE reconstruction was performed by culturing 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)-labeled monoclonal HCE cells on denuded amniotic membranes (dAMs) in 20% fetal bovine serum-containing Dulbecco's Modified Eagle's Medium/Ham's Nutrient Mixture F12 (1:1) medium and 5% CO(2) at 37 ° C on a 24-well culture plate. The reconstructed TE-HCE was transplanted into cat corneas via lamellar keratoplasty with all of the endothelium and part of Descemet's membrane stripped. Postsurgical corneas were monitored daily with their histological properties examined during a period of 104 days after transplantation. RESULTS: The reconstructed TE-HCE at a density of 3,413.33 ± 111.23 cells/mm(2) in average established intense cell-cell and cell-dAM junctions. After lamellar keratoplasty surgery, no obvious edema was found in TE-HCE-transplanted cat corneas, which were transparent throughout the monitoring period. In contrast, intense corneal edema developed in dAM-transplanted cat corneas, which were turbid. The corneal thickness gradually decreased to 751.33 ± 11.37 µm on day 104 after TE-HCE transplantation, while that of dAM eye was over 1,000 µm in thickness during the monitoring period. A monolayer of endothelium consisting of TE-HCE-originated cells at a density of 2,573.33 ± 0.59 cells/mm(2) attached tightly to the surface of remnant Descemet's membrane over 104 days; this was similar to the normal eye control in cell density. CONCLUSIONS: The reconstructed TE-HCE was able to function as a corneal endothelium equivalent and restore corneal function in cat models.

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.

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.

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.

Establishment of a continuous untransfected human corneal endothelial cell line and its biocompatibility to denuded amniotic membrane.

PURPOSE: To establish an untransfected human corneal endothelial (HCE) cell line and characterize its biocompatibility to denuded amniotic membrane (dAM). METHODS: Primary culture was initiated with a pure population of HCE cells in DMEM/F12 media (pH 7.2) containing 20% fetal bovine serum and various supplements. The established cell line was characterized by growth property, chromosome analysis, morphology recovery, tumorigenicity assay, and expression of marker proteins, cell-junction proteins, and membrane transport proteins. The biocompatibility of HCE cells to dAM was evaluated by light microscopy, alizarin red staining, immunofluorescence assay, and electron microscopy. RESULTS: HCE cells proliferated to confluence 6 weeks later in primary culture and have been subcultured to passage 224 so far. A continuous untransfected HCE cell line with a population doubling time of 26.20 h at passage 101 has been established. Results of chromosome analysis, morphology, combined with the results of expression of marker protein, cell-junction protein and membrane transport protein, suggested that the cells retained HCE cell properties and potencies to form cell junctions and perform membrane transport. Furthermore, HCE cells, without any tumorigenicity, could form confluent cell sheets on dAMs. The single layer sheets that attached tightly to dAMs had similar morphology and structure to those of HCE in situ and had an average cell density of 3,413±111 cells/mm². CONCLUSIONS: An untransfected and non-tumorigenic HCE cell line has been established, and the cells maintained positive expression of marker proteins, cell-junction proteins and membrane transport proteins. The cell line, with excellent biocompatibility to dAM, might be used for in vitro reconstruction of HCE and provides a promising method for the treatment of diseases caused by corneal endothelial disorders.

A prophenoloxidase from Artemia sinica: cDNA cloning, expression and activity analysis during early development.

To understand the defense mechanisms of Crustacean animals, brine shrimp Artemia sinica prophenoloxidase (AsproPO) cDNA was cloned and its expression at early developmental stages was examined by reverse-transcription PCR (RT-PCR) and semi-quantitative RT-PCR, and activity of phenoloxidase (PO) at different developmental stages was further detected by using l-3,4-dihydroxyphenylalanine (l-DOPA) as a specific substrate in this study. It was found that the full-length of AsproPO cDNA is 2125 bp and it contains an open reading frame of 2100 bp encoding a protein of 699 amino acids. The deduced amino acid sequence of AsproPO has two putative copper binding sites highly conserved in Arthropods. Semi-quantitative RT-PCR analyses showed that the gene of AsproPO expressed at Emergence, Instar I and Instar II stages but did not at 0 h and 6 h stages. Activity measurement showed that PO activity could only be detected at Instar II stage but the other measured stages. All these implied that Artemia proPO immune system was complexly modulated during early development.

Purification and characterization of phenoloxidase from brine shrimp Artemia sinica.

Phenoloxidase from Artemia sinica (AsPO) was purified by Superdex 200 gel-filtration and Q Sepharose fast flow ion-exchange chromatography, and its properties were characterized biochemically and enzymatically by using L-dihydroxyphenylalanine (L-DOPA) as the specific substrate. Results showed that AsPO was isolated as a monomeric protein of 125.5 kDa in molecular mass. The optimal pH value and temperature are 7.0 and 50°C, respectively, for its PO activity. The AsPO had an apparent K(m) value of 4.2 mM on L-DOPA, and 10.9 mM on catechol, respectively. Oxidase inhibitor on PO activity showed that the AsPO was extremely sensitive to ascorbic acid, sodium sulfite, and citric acid; and was very sensitive to cysteine, benzoic acid, and 1-phenyl-2-thiourea. Combined with its specific enzyme activity on L-DOPA and catechol, it can be concluded that AsPO is most probably a typical catechol-type O-diphenoloxidase. Its PO activity was also sensitive to metal ions and chelators, and 20 mM DETC-inhibited PO activity was obviously recovered by 15 mM Cu(2+), indicating that AsPO is most probably a copper-containing metalloenzyme. All these data about specific substrate, sensitivity to oxidase inhibitor metal ions and chelators indicate that the AsPO has the properties of a catechol-type copper-containing O-diphenoloxidase that functions as a vital humoral factor in host defense via melaninization as in other Crustaceans.

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.