Phenotypic and transcriptomic changes in the corneal epithelium following exposure to cigarette smoke☆
Graphical abstract
Introduction
Tobacco use is a globally recognized risk factor threatening public health, which causes more than 8 million deaths annually (eport on the Global, 2019). Second-hand smoke refers to exposure to side-stream smoke from the burning cigarette and mainstream smoke exhaled by the smoker (Apelberg et al., 2013); it is a mixture of numerous genotoxic and carcinogenic compounds including nicotine, particulate matter, carbon monoxide, 3-ethenylpyridine, polycyclic aromatic hydrocarbons, nitrosamine, nitrogen oxide, aldehydes, and other organic compounds (Apelberg et al., 2013; Lofroth, 1989). Second-hand smoke is not only strongly associated with chronic respiratory diseases and lung cancer but also initiates or exacerbates cardiovascular diseases and can shorten the average life expectancy (Lois et al., 2008).
The cornea is a highly specialized structure on the anterior surface of the eye that forms the main refractive element and accounts for approximately two-thirds of the total optical power (Casaroli-Marano et al., 2015). Corneal clarity depends on the maintenance of a well-defined intact five to six layered functionally coupled syncytium. It is the most superficial layer of the cornea and is in direct contact with the external environment. This layer is made up of a non-keratinized, stratified squamous epithelial sheet consisting of three differentiating central epithelial cell layers and superficial outermost top layers containing, wing cells as well as proliferating basal cells that are situated in intimate contact with an underlying basement membrane surface (Ehlers et al., 2010; Yazdanpanah et al., 2017). Functionally, the corneal epithelium acts as the frontline ocular defense system to form a critical barrier against various environmental stressors and external pathogen invasion. Loss of the corneal epithelium increases the risk of infection, which can lead to scarring or melting of the corneal stroma and even severe vision loss. Moreover, the corneal epithelium plays an essential role in regulating the passage of solutes and macromolecules into the underlying deeper layers of this tissue. The integrity and homeostasis of the corneal epithelium are essential for preserving corneal transparency, visual acuity, and the integrity of the ocular surface.
Since the corneal epithelium is one of the cell types in direct contact with tobacco smoke, tobacco smoke may have a direct impact on the corneal epithelium. Smoking is one of the primary causes of a range of eye diseases, such as Graves’ ophthalmopathy (Hegedius et al., 2004), dry eye disease (Titiyal et al., 2018), age-related macular degeneration (Woodell and Rohrer, 2014), and cataract (Chowdhury et al., 2018); however, few studies have focused on clarifying underlying mechanism that accounts for how exposure to tobacco smoke damages the cornea. Jetton et al. surveyed patients with corneal abrasions or keratitis and found that the time required for completion of epithelial healing was longer in smokers (Jetton et al., 2014). Thomas et al. demonstrated that smoking causes a series of adverse effects including decreased tear film stability, impaired corneal sensitivity, and enlarged fluorescein staining (Thomas et al., 2012). An investigation of occupational tobacco exposure by Mittal et al. showed that workers in a smoky environment displayed a range of ocular complications such as superficial punctate keratitis, punctate epithelial erosion, eye irritation, and conjunctival hyperplasia (Mittal et al., 2008). While these clinical investigations indicated that tobacco smoke has harmful effects on the ocular surface and leads to many ocular symptoms, the underlying molecular mechanism is still not well defined and requires further exploration. In the present study, we aimed to investigate the phenotypic and transcriptomic impacts of cigarette smoke on the corneal epithelium with a view to clarifying the probable mechanism.
Section snippets
Animals
C57/BL6J mice (aged 6–8 weeks) were obtained from the Experimental Animal Center of Xiamen University. The mice were housed at a temperature of 23 ± 1 °C and a humidity of 60 ± 5% under a 12 h/12 h light/dark cycle. Food and water were provided ad libitum. All experiments were performed based on the Association for Research in Vision and Ophthalmology (ARVO) statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Experimental Animal Ethics Committee of Xiamen
Chemical characteristics of CSE
The generation of mainstream cigarette smoke is a complex and dynamic process with a variety of chemical, physical, and physiological characteristics. Mainstream cigarette smoke contains is a complex aerosol composed of a vapor and a particulate phase containing more than 4000 harmful chemical substances including nicotine, particulate matter, carbon monoxide, polycyclic aromatic hydrocarbons (PAHs), nitrosamine, nitrogen oxide, and other organic compounds. Therefore, it is difficult to analyze
Discussion
The corneal epithelium is the main barrier against exogenous substances, which helps to maintain the homeostasis of the cornea and is one of the first sites of exposure to cigarette smoke. Previous studies have demonstrated that chronic exposure to cigarette smoke has deleterious effects on the ocular surface, typically inducing lipid layer changes, apoptosis, inflammation, ROS-mediated DNA oxidation and autophagy impairment (Miao et al., 2019; Higuchi et al., 2011; Li et al., 2020; Ejaz et
Conclusion
Our results demonstrate that CSE had obvious toxic phenotypic and genetic effects on the corneal epithelium. In order to counter the toxicity of CS, the corneal epithelium developed a series of genetic changes. The antioxidant activity was enhanced and several oxidoreductase enzymes, including GPX, GCL, and GST, were activated to reduce excessive oxidative damage. After sensing a signal, it is likely that the corneal epithelial cells activate Nrf-2, a transcription factor, which binds to
Author statement
Cheng Li and Yuhua Xue are responsible for conceptualization and funding acquisition. Mengyi Jin drafted the manuscript. Cheng Li, Peter S Reinach and Zuguo Liu contributed to original draft, review and editing. Shuiping Wu made contribution to review and editing. Mengyi Jin, Xiaoya An, Honghua Kang, Yixin Wang, Guoliang Wang and Yang Gao performed the experiment. Mengyi Jin and Yanzi Wang made contribution to statistical analysis.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
This study was supported in part by grants from The National Key R&D Program of China (2020YFA0908103, 2018YFA0107301), the National Natural Science Foundation of China (NSFC No. 82070931, 81770891, 81672955, 81900822), and the Huaxia Translational Medicine Fund for Young Scholars (No. 2017-A-001), and the Fundamental Research Funds for the Central Universities (No. 20720200044). The authors thank Jing-Ru Huang from the Biomedical Science Core Facility of Xiamen University for technical
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This paper has been recommended for acceptance by Wen Chen.