Abstract
Epidemiological studies have indicated that smoking is a pivotal risk factor for the progression of several chronic diseases. Nicotine, the addictive component of cigarettes, has powerful pathophysiological properties in the body. Although the effects of cigarette smoking on corneal re-epithelialization have been studied, the effects of nicotine on corneal wound healing-related neovascularization and fibrosis have not been fully demonstrated. The aim of this study was to evaluate the effects of chronic administration of nicotine on corneal wound healing following acute insult induced by an alkali burn. BALB/C female mice randomly received either vehicle (2% saccharin) or nicotine (100 or 200 μg/ml in 2% saccharin) in drinking water ad libitum. After 1 week, animals were re-randomized and the experimental group was subjected to a corneal alkali burn, and then nicotine was administered until day 14 after the alkali burn. A corneal alkali burn model was generated by placing a piece of 2 mm-diameter filter paper soaked in 1N NaOH on the right eye. Histopathological analysis and the expression level of the pro-angiogenic genes vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP9) revealed that chronic nicotine administration enhanced alkali burn-induced corneal neovascularization. Furthermore, the mRNA expression of the pro-fibrogenic factors α-smooth muscle actin (αSMA), transforming growth factor-β (TGF-β), and collagen α1 (Col1) was enhanced in the high-concentration nicotine-treated group compared with the vehicle group after corneal injury. Immunohistochemical analysis also showed that the αSMA-positive area was increased in chronic nicotine-treated mice after corneal alkali burn. An in vitro assay found that expression of the α3, α7, and β1 nicotinic acetylcholine receptor (nAChR) subunits was significantly increased by chemical injury in human corneal fibroblast cells. Moreover, alkali-induced fibrogenic gene expression and proliferation of fibroblast cells were further increased by treatment with nicotine and cotinine. The proliferation of such cells induced by treatment of nicotine and cotinine was reduced by inhibition of the PI3K and PKC pathways using specific inhibitors. In conclusion, chronic administration of nicotine accelerated the angiogenic and fibrogenic healing processes in alkali-burned corneal tissue.
Highlights
Cigarette smoking, the most common form of tobacco use, is responsible for hundreds of thousands of premature deaths and chronic diseases annually [1]
To further elucidate the effects of nicotine on the formation of new blood vessels, we analyzed the mRNA expression of the angiogenesis-related genes vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP9) in corneal tissue following alkali injury
VEGF and MMP9 expression were significantly increased in mice treated with a high concentration of nicotine compared to control mice on days 4 and 7, respectively (Fig 1B)
Summary
The most common form of tobacco use, is responsible for hundreds of thousands of premature deaths and chronic diseases annually [1]. Nicotine, the addictive component of cigarettes, binds to its receptors, which have several subtypes (α1–10, β1–4, δ, γ, and ε), and thereby exerts a direct action on neuronal and non-neuronal cells [7]. The wound healing response induced by tissue damage is a complex and dynamic process involving various extracellular matrix proteins, growth factors, and cytokines. The main and leading events that happen during the second phase of wound healing, which is known as tissue formation or the proliferative phase, include epithermal restoration, formation of new blood vessels, fibroblast proliferation, and production of extracellular matrix (ECM). Excess inflammation and tissue fibrosis/scarring during the wound healing response disrupt the normal function of tissues and organs [16]
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