Disruption of retinal pigment epithelial cell properties under the exposure of cotinine

Xiao-Yu Zhang,Kwok Ping Chan,Chi Pui Pang,Jasmine Sum Yee Yung,Tsz Kin Ng,Di Wu,Shaodan Zhang,Di Cao,Yumeng Wang,Mårten Erik Brelén,Sun On Chan

doi:10.1038/s41598-017-03283-x

Abstract

Cigarette smoking is a major risk factor for age-related macular degeneration (AMD), in which progressive retinal pigment epithelial (RPE) cell degeneration is a major pathological change. Nicotine is a major biologically active component in cigarette smoke. It is continuously catabolized into cotinine, which has longer half-life and higher concentration in tissue cells and fluids. Here we hypothesized that continuous exposure of cotinine has more potent effects on human RPE cell properties than nicotine. Human RPE cell line (ARPE-19) was treated continuously with 1–2 µM of nicotine and/or cotinine for 7 days. RPE cells treated with 2 μM cotinine and nicotine-cotinine mixture has lower MTT signals without significant changes in cell apoptosis or integrity. Moreover, RPE cell migration was retarded under cotinine treatments, but not nicotine. Both nicotine and cotinine treatments attenuated the phagocytotic activity of RPE cells. In addition, cotinine and nicotine-cotinine mixture suppressed VEGF and IL-8 expression and upregulated TIMP-2 expression. Expressions of autophagy genes were upregulated by the cotinine treatment, whereas expressions of epithelial-to-mesenchymal transition markers were downregulated. In conclusion, our study, for the first time, demonstrated that cotinine, rather than nicotine, affects the properties of RPE cells in vitro, which could explain the smoking-induced RPE pathology.

Highlights

Age-related macular degeneration (AMD) is a progressive retinal degenerative disease affecting more than 150 million people in the world[1]
To understand the capability of ARPE-19 cells to respond to nicotine and cotinine, we examined the expression of nicotinic acetylcholine receptor gene by polymerase chain reaction (PCR)
Our results showed that (1) cotinine reduces retinal pigment epithelial (RPE) cell proliferation; (2) nicotine and cotinine did not cause RPE cell apoptosis; (3) nicotine and cotinine did not affect RPE cell integrity; (4) cotinine retards RPE cell migration and downregulates epithelial-to-mesenchymal transition (EMT) marker expression; (5) cotinine reduces pro-angiogenic factor and enhances anti-angiogenic factor expression; (6) nicotine and cotinine attenuate RPE phagocytotic activity; (7) cotinine increases autophagy pathway gene expression in RPE cells

Summary

Introduction

Age-related macular degeneration (AMD) is a progressive retinal degenerative disease affecting more than 150 million people in the world[1]. Nicotine is a key component and the determinant for addiction to smoking[9, 10]. It is the major component in different cigarette replacements, including the recently popular electronic cigarette and nicotine patches. In human primary fetal RPE culture, exposure of 1 μM nicotine for 3 days changes RPE morphology without affecting cell proliferation, and reduces interleukin-8 (IL8), metalloproteinase-2 (MMP2) and MMP9, but not vascular endothelial growth factor (VEGF) expression[11]. Exposure of 10 nM nicotine for 3 days did not induce ARPE-19 cell death or proliferation, but upregulated VEGF and downregulated PEDF expression[12]. The expression of CHRNB1 was downregulated in the 1 μM nicotine, 2 μM cotinine as well as 2 μM nicotine-cotinine mixture groups. ‘*’p < 0.05; ‘**’p < 0.01; ‘***’p < 0.001

Methods

Results

Conclusion