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.

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.

Collagen-based biocomposites inspired by bone hierarchical structures for advanced bone regeneration: ongoing research and perspectives.

Bone is a hard-connective tissue composed of matrix, cells and bioactive factors with a hierarchical structure, where the matrix is mainly composed of type I collagen and hydroxyapatite. Collagen fibers assembled by collagen are the template for mineralization and make an important contribution to bone formation and the bone remodeling process. Therefore, collagen has been widely clinically used for bone/cartilage defect regeneration. However, pure collagen implants, such as collagen scaffolds or sponges, have limitations in the bone/cartilage regeneration process due to their poor mechanical properties and osteoinductivity. Different forms of collagen-based composites prepared by incorporating natural/artificial polymers or bioactive inorganic substances are characterized by their interconnected porous structure and promoting cell adhesion, while they improve the mechanical strength, structural stability and osteogenic activities of the collagen matrix. In this review, various forms of collagen-based biocomposites, such as scaffolds, sponges, microspheres/nanoparticles, films and microfibers/nanofibers prepared by natural/synthetic polymers, bioactive ceramics and carbon-based materials compounded with collagen are reviewed. In addition, the application of collagen-based biocomposites as cytokine, cell or drug (genes, proteins, peptides and chemosynthetic) delivery platforms for proangiogenesis and bone/cartilage tissue regeneration is also discussed. Finally, the potential application, research and development direction of collagen-based biocomposites in future bone/cartilage tissue regeneration are discussed.

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.

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.