**1. Introduction**

According to the World Health Organization (WHO) World Health Statistics 2016, the world's highest cigarette smoking rates were 76.2% for men in Indonesia, and 52.0% for women in Nauru. Ranked second place was Jordan for men (70.2%), and Kiribati for women (40.9%),

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

third place was Kiribati for men (63.9%) and Serbia for women (39.7%). As of 2015, the gender smoking ratio was estimated as 33.7% men and 10.6% women in Japan [1]. Globally, an estimated 93.3 million people smoke, the majority of whom reside in developing countries, where smoking rates are estimated to be as high as 50% for men. It has been shown that men tend to use all tobacco products at a higher rate than women [2]. Atherosclerosis is more common in men than women [3]. It may be derived from this that men have more arteriosclerotic diseases.

thickens as the result of invasion, accumulation of white blood cells and fatty materials such as foam cells and cholesterol [22, 23] (**Figure 1**). It has also been known that accumulation of vascular smooth muscle cells (VSMCs) can be observed in atherosclerotic lesions (**Figure 1**). The proliferation of VSMCs with the subsequent formation of intimal thickening is a major event in the development of atherosclerotic lesions [24, 25] (**Figure 1**). Normally, the differentiated VSMCs constitute the tunica media of the artery and are responsible for the vasoconstriction function. However, why the VSMCs accumulate during arteriosclerotic plaque

Effects of Nicotine Contained in Tobacco Mainstream Smoke on Vascular Smooth Muscle Cells

http://dx.doi.org/10.5772/intechopen.77010

49

In this chapter, we describe that nicotine in tobacco mainstream smoke causes dedifferentiation of VSMCs to migration-proliferation types via nicotinic acetylcholine receptors (nAChRs)

nAChRs are transmembrane ligand-gated ion channels expressed in the cell membrane of all mammalian cells, and their endogenous ligand is acetylcholine [26]. We were the first to report that nAChRs were expressed on VSMCs [27]. Also, we found that nicotine promotes cell migration of VSMCs GbaSM-4 cells isolated from basilar arteries of guinea pigs, and this cell migration is inhibited by methyllycaconitine, an antagonist of nAChRs [27]. That was the first report on the effect of nAChRs on VSMCs [27]. In subsequent studies of other groups, it was reported that nicotine promoted the chemotaxis and migration of VSMCs isolated from rats and humans [28, 29]. Thereafter, various types of nAChRs have been discovered and reported by real-time qPCR, Western blots, etc. in several tis-

We exposed cell line AC01 cells derived from mouse aortic smooth muscle to 0.1 μM nicotine [32], and performed exhaustive gene expression analysis using DNA microarray for gene expression after 48 h. As a result of whole gene expression analysis, α1, α2, α6, α7, α9, β1, β2, β4, δ, ε, and γ subunits of nAChRs were detected in AC01 cells (**Figure 2A**). After AC01 cells were exposed to nicotine for 48 h, a change was observed in the ratio of the fluorescence intensity of cy3 / cy5 indicating the amount of transcription of mRNA for each subunit. As a result, the α1, α6, α7, β2, and δ subunits increased by 2.6, 2.3, 2.4, 2.0, and 3.1 times, respectively,

Furthermore, we measured the expression levels of nAChRs in human vascular smooth muscle cells (HVSMCs) using real-time qPCR. As a result, α2, α6, α7, and β1 subunits of the nAChRs were detected. The expression level of the α2 subunit was relatively low, and it disappeared within 72 h of nicotine exposure. The expression level of the α6 subunit increased with time, to about 20-fold after 72 h as compared with the control (0 h). The α7 subunit was the most frequently expressed in the HVSMCs. The expression level of the β1 subunit was in trace amounts, and from this result, there was no clear influence of exposure to nicotine. Thus, it was discovered that nAChRs were expressed in response to

expressed in the VSMCs, which is a cause of arteriosclerotic plaque formation.

**2. The nicotinic acetylcholine receptors (nAChRs) on VSMCs**

formation is not well understood (**Figure 1**).

sues [30, 31].

compared to the control (**Figure 2**).

nicotine in HVSMCs [33].

Smoking, as well as second-hand smoke, induces circulatory diseases, heart attacks, strokes, cancers, and respiratory diseases [4–7]. Several studies have suggested that cigarette smoke has 7357 chemical compounds from different classes [8]. Nicotine is the most predominant alkaloid (approximately 90–95%), found in the tobacco plant, *Nicotiana tabacum* [9–10]. Plasma nicotine levels have been reported as 4–30 ng/ml after smoking a cigarette, 8–10 ng/ml after chewing nicotine gum, and 22 ng/ml after smoking a pipe [11, 12]. Nicotine is a toxic compound that should be handled with care, as it has been reported that more than 0.5 g of oral nicotine is required to kill an adult [13, 14].

Epidemiological studies show that cigarette smoking has long been known as a major risk factor for atherosclerosis [15–18]. In particular, nicotine in the cigarette smoke promotes atherogenesis [17–20]. However, little is known about the mechanism by which nicotine induces arteriosclerosis. Atherosclerosis is a specific form of arteriosclerosis in which an artery wall

**Figure 1.** Atherosclerotic plaque is due to the cooperation of three types of cells. The first one is macrophages. Their invasion into tunica intima preludes the formation of plaque. The second is endothelial cells. It secretes some growth factors which affects the VSMC. The third is VSMCs. Under the stimulation, their phenotype changes before migrating to tunica intima and proliferating there. This phenotypic change is referred to as contractile-to-synthetic-type transition and it contributes to the development of plaque. This phenotypic change could be induced by different kinds of stimuli. Nicotine is one of them. Nicotine is a main pharmacological compound in cigarette smoke. It is reported to promote cell migration of rat and human VSMCs. However, little is known about whether nicotine promotes the phenotypic change of human VSMC.

thickens as the result of invasion, accumulation of white blood cells and fatty materials such as foam cells and cholesterol [22, 23] (**Figure 1**). It has also been known that accumulation of vascular smooth muscle cells (VSMCs) can be observed in atherosclerotic lesions (**Figure 1**). The proliferation of VSMCs with the subsequent formation of intimal thickening is a major event in the development of atherosclerotic lesions [24, 25] (**Figure 1**). Normally, the differentiated VSMCs constitute the tunica media of the artery and are responsible for the vasoconstriction function. However, why the VSMCs accumulate during arteriosclerotic plaque formation is not well understood (**Figure 1**).

In this chapter, we describe that nicotine in tobacco mainstream smoke causes dedifferentiation of VSMCs to migration-proliferation types via nicotinic acetylcholine receptors (nAChRs) expressed in the VSMCs, which is a cause of arteriosclerotic plaque formation.
