**4. New challenges on HVSMC exposure to nicotine**

a time-dependent manner to approximately 3-fold after 48 h [33]. During Western blot experiments using specific antibodies against each of the marker proteins, the protein expression of myosin II isoform 10 increased after 48 h exposure to nicotine. The amount of myosin II isoform 11 decreased by approximately 0.6-fold after the 48-h nicotine exposure. The myosin II isoform 10 level was increased to about 1.2-fold after exposure to nicotine [33]. These results indicated that the isoforms of myosin II had changed to the non-muscle (proliferative) type

Subsequently, α-actin and β-actin were used as contractile-type and synthetic-like type marker genes, respectively [43]. After exposure of HVSMCs to nicotine, the α-actin mRNA level decreased by approximately 0.4-fold, whereas, the β-actin mRNA level increased to approximately 1.7-fold after 48 h, respectively [33]. Using Western blot experiments, the protein expression of α-actin levels did not significantly change. In contrast, β-actin levels significantly increased to approximately 1.6-fold after the nicotine exposure [33]. These results indicated that the actin isoform also changed to the synthetic-like type from the contractile-

SM22 and high-molecular-weight caldesmon (H-caldesmon) are major smooth muscle differentiation markers [44, 45]. The SM22 mRNA level decreased by approximately 0.9-fold after the 48-h exposure of HVSMCs to nicotine. The mRNA level of the H-caldesmon, a smooth muscle contractile-type marker protein, was about 0.7-fold after 48 h [33]. Using Western blot experiments, H-caldesmon and SM22 levels, significantly decreased by approximately 0.4- and 0.7-fold, respectively after nicotine exposure [33]. The decreased H-caldesmon and SM22 expression levels also indicated the transformation to the synthetic-like type from the

Notch receptors are intimately involved in HVSMC differentiation. Activation of Notch receptors by cell-cell adhesion induces the expression differentiation marker proteins of contractile-type on smooth muscles [46]. However, when HVSMCs at 100% confluence were exposed to nicotine in our study, the expression of Notch receptors did not increase [33]. This indicated

Mitogen-activated protein kinases (MAPKs) play an important role in cell proliferation and migration [46, 47]. MAPKs are also intimately involved in VSMC growth and migration [48, 49]. It has been reported that nicotine induces the production of growth factors such as vascular endothelial growth factor (VEGF), Platelet-derived growth factor (PDGF-BB), and Fibroblast growth factor (FGF-2) from VSMCs, and that PDGF-BB and FGF-2 promoted the proliferation of VSMCs [29, 50–52]. Nicotine-induced VEGF production was mediated by nAChRs via activation of the VEGF and its receptor as well as the extracellular signalregulated kinase (ERK)1/2 pathway [27]. PDGF-BB caused cytoskeletal protein remodeling, enhanced the proliferation, and migration of VSMCs [51]. In our study, the phosphorylation levels of the p38 MAPK, ERK1/2, and c-jun N-terminal kinase increased after 48 h of nicotine exposure [33]. Activation of MAPKs signaling indicated that the characteristics of VSMCs

Our results suggest that nicotine can decrease the expression of differentiation marker proteins in HVSMCs, and change these cells from the contractile-type to synthetic-like type,

that nicotine had suppressed the expression and function of the Notch receptors.

from the smooth muscle contractile type because of nicotine exposure (**Figure 4**).

type after nicotine exposure (**Figure 4**).

52 Atherosclerosis - Yesterday, Today and Tomorrow

contractile-type after nicotine exposure (**Figure 4**).

changed to migration-type cells after nicotine exposure.

Regarding the influence of nicotine on HVSMCs, a new problem was found during our research. It was about how nicotine works as a signal in HVSMCs. It has been shown that nicotine binds to nAChRs, and opens the ion channels in these receptors to significantly increased intracellular Ca2+ levels [54, 55]. We measured the changes in intracellular Ca2+ level in HVSMCs upon nicotine stimulation. Our results indicated that nicotine stimulation significantly increased intracellular Ca2+ levels in HVSMCs. In addition, mecamylamine, a non-selective nAChR blocker, effectively blocked the nicotine effect in the nicotine-treated HVSMCs. However, mecamylamine did not exhibit complete inhibition of the nicotine stimulation. This suggests that nicotine is involved in intracellular signal transduction through receptors other than nAChRs. From the results of our comprehensive gene analysis, several receptors whose gene expression were increased by nicotine exposure have been discovered. In the future, it would be expedient to clarify the functions of these novel nicotine receptors (**Figure 5**).

Furthermore, the transformation of VSMCs by nicotine shown in our study suggested that nicotine itself promoted arteriosclerosis. In addition to cigarettes, nicotine is also contained in

**Figure 5.** A schematic diagram showing the relationship between nicotine exposure and the phenotypic change in HVSMCs. The solid line arrows indicate an effect based on our results. The break line arrows indicate an effect based on our speculation.

therapeutic nicotine patches and gums used for smoking cessation. Thus, there is a possibility that these nicotine patches or gums promote atheromatous plaque formation. Moreover, smokeless tobacco contains large amounts of sodium, which enhance nicotine absorption [56]. These problems should also be considered sufficiently because nicotine used even during smoking cessation treatment and avoidance of tobacco sidestream smoke induces arteriosclerosis.

gho/publications/world\_health\_statistics/en/. http://apps.who.int/iris/bitstream/10665/

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

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

55

[2] al'Absi M, Nakajima M, Allen S, Lemieux A, Hatsukami D. Sex differences in hormonal responses to stress and smoking relapse: A prospective examination. Nicotine & Tobacco

[3] Maas AH, Appelman YE. Gender differences in coronary heart disease. Netherlands

[4] U.S. Department of Health and Human Services. Let's Make the Next Generation Tobacco-Free: Your Guide to the 50th Anniversary Surgeon General's Report on Smoking and Health. [PDF–795 KB]. Atlanta: U.S. Department of Health and Human Services, Centers for Disease Control and Prevention, National Center for Chronic Disease Prevention and Health Promotion, Office on Smoking and Health. 2014. https:// www.surgeongeneral.gov/library/reports/50-years-of-progress/consumer-guide.pdf.

[5] U.S. Department of Health and Human Services. The Health Consequences of Involuntary Exposure to Tobacco Smoke: A Report of the Surgeon General. Atlanta: U.S. Department of Health and Human Services, Centers for Disease Control and Prevention, National Center for Chronic Disease Prevention and Health Promotion, Office on Smoking and Health. 2006. https://www.ncbi.nlm.nih.gov/books/NBK44324/

[6] U.S. Department of Health and Human Services. A Report of the Surgeon General: How Tobacco Smoke Causes Disease: What It Means to You. Atlanta: U.S. Department of Health and Human Services, Centers for Disease Control and Prevention, National Center for Chronic Disease Prevention and Health Promotion, Office on Smoking and Health. 2010. https://www.surgeongeneral.gov/library/reports/secondhandsmoke/full-

[7] U.S.Department of Health and Human Services. The Health Consequences of Smoking—50 Years of Progress: A Report of the Surgeon General. Atlanta: U.S. Department of Health and Human Services, Centers for Disease Control and Prevention, National Center for Chronic Disease Prevention and Health Promotion, Office on Smoking and Health. 2014. https://www.cdc.gov/tobacco/data\_statistics/sgr/2010/consumer\_booklet/pdfs/con-

[8] Rodgman A, Perfetti TA. The Chemical Components of Tobacco and Tobacco Smoke.

[9] Saitoh F, Noma M, Kawashima N. The alkaloid contents of 60 Nicotiana species.

[10] Carvalho BL et al. Genetic parameters estimates associated to conversion of nicotine to nornicotine in Burley tobacco. American Journal of Plant Sciences. 2014;**5**:3380-3388 [11] Russell MA, Feyerabend C, Cole PV. Plasma nicotine levels after cigarette smoking and

chewing nicotine gum. British Medical Journal. 1976;**1**(6017):1043-1046

pdf/Bookshelf\_NBK44324.pdf. [Accessed: January 11, 2017]

44844/1/9789241564441\_eng.pdf

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[Accessed: January 11, 2016]

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Boca Raton, FL: CRC Press/Taylor & Francis Group; 2009

Heart Journal. 2010;**18**(12):598-602

## **5. Conclusion**

Several data have widely suggested nicotine as one of the factors responsible for the formation of atheromatous plaques in the vascular intima. Numerous studies so far, including our research, indicate that nicotine induces intracellular Ca2+ influx in HVSMCs via nAChRs and possibly via another nicotine-specific receptor. Consequently, HVSMCs are transformed from the contractile-type to the synthetic-like type, which occurs during the development of atheromatous plaques. Aside from cigarettes, nicotine is also contained in nicotine patches and gums used for smoking cessation. Thus, there is a possibility that these nicotine patches or gums promote atheromatous plaque formation. Therefore, we hypothesize that elucidating the mechanism of action of nicotine will lead to new means of preventing and treating atherosclerotic plaque formation and development of arteriosclerosis.

## **Acknowledgements**

I would like to thank my supervisor, Dr. Kazuhiro Kohama. I am grateful to all former members of Kohama's laboratory group, who contributed to this work: especially Dr. Shinji Yoshiyama, Zhenyi Chen, and Sheng Li. I also thank Miss. Azusa Inoue for her help with the illustrations. This study was supported by the grants from the Smoking Research Foundation, the Grantsin-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology in Japan (23590295 and 15K00809), and the Takeda Science Foundation.

## **Author details**

#### Akio Nakamura

Address all correspondence to: nakamura-akio@jissen.ac.jp

Department of Food and Health Science, Faculty of Human Life Sciences, Jissen Women's University, Tokyo, Japan

#### **References**

[1] World Health Organization. World Health Statistics 2016: Monitoring Health for the SDGs, Sustainable Development Goals. 2016. Available from: http://www.who.int/

gho/publications/world\_health\_statistics/en/. http://apps.who.int/iris/bitstream/10665/ 44844/1/9789241564441\_eng.pdf

[2] al'Absi M, Nakajima M, Allen S, Lemieux A, Hatsukami D. Sex differences in hormonal responses to stress and smoking relapse: A prospective examination. Nicotine & Tobacco Research. 2015;**17**(4):382-389

therapeutic nicotine patches and gums used for smoking cessation. Thus, there is a possibility that these nicotine patches or gums promote atheromatous plaque formation. Moreover, smokeless tobacco contains large amounts of sodium, which enhance nicotine absorption [56]. These problems should also be considered sufficiently because nicotine used even during smoking cessation treatment and avoidance of tobacco sidestream smoke induces arteriosclerosis.

Several data have widely suggested nicotine as one of the factors responsible for the formation of atheromatous plaques in the vascular intima. Numerous studies so far, including our research, indicate that nicotine induces intracellular Ca2+ influx in HVSMCs via nAChRs and possibly via another nicotine-specific receptor. Consequently, HVSMCs are transformed from the contractile-type to the synthetic-like type, which occurs during the development of atheromatous plaques. Aside from cigarettes, nicotine is also contained in nicotine patches and gums used for smoking cessation. Thus, there is a possibility that these nicotine patches or gums promote atheromatous plaque formation. Therefore, we hypothesize that elucidating the mechanism of action of nicotine will lead to new means of preventing and treating athero-

I would like to thank my supervisor, Dr. Kazuhiro Kohama. I am grateful to all former members of Kohama's laboratory group, who contributed to this work: especially Dr. Shinji Yoshiyama, Zhenyi Chen, and Sheng Li. I also thank Miss. Azusa Inoue for her help with the illustrations. This study was supported by the grants from the Smoking Research Foundation, the Grantsin-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and

Technology in Japan (23590295 and 15K00809), and the Takeda Science Foundation.

Department of Food and Health Science, Faculty of Human Life Sciences, Jissen Women's

[1] World Health Organization. World Health Statistics 2016: Monitoring Health for the SDGs, Sustainable Development Goals. 2016. Available from: http://www.who.int/

sclerotic plaque formation and development of arteriosclerosis.

Address all correspondence to: nakamura-akio@jissen.ac.jp

**5. Conclusion**

54 Atherosclerosis - Yesterday, Today and Tomorrow

**Acknowledgements**

**Author details**

Akio Nakamura

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University, Tokyo, Japan


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**Chapter 5**

**Provisional chapter**

**Food Restriction and Atherosclerotic Plaque**

**Food Restriction and Atherosclerotic Plaque** 

DOI: 10.5772/intechopen.76560

Food restriction is a promising therapy for many age-associated pathologies as it stimulates the health-supportive mechanism autophagy. Because atherosclerosis is an inflammatory, age-related disease, dietary modification can be an important strategy in preventing atherosclerotic plaque development. A cholesterol-supplemented diet, used to induce plaque formation in rabbits, induces a pronounced hypercholesterolemia, which can be reversed after 4 weeks of normal diet. However, food restriction induces a further increase in circulating LDL cholesterol. These elevated cholesterol levels are associated with the induction of autophagy. Although neither a short-term normal diet nor food restriction alters plaque size, rabbits fed a normal diet show signs of increased plaque stability such as elevated collagen content and decreased expression of vascular cell adhesion molecule (VCAM)-1. Surprisingly, these favorable effects are not present after 4 weeks of food restriction. On the contrary, atherosclerotic plaques of food-restricted rabbits displayed enhanced apoptosis, a process known to further undermine plaque stability. In conclusion, severe short-term food restriction in rabbits prevents stabilization of atherosclerotic plaques as

observed after regular cholesterol withdrawal via a normal diet.

**Keywords:** atherosclerosis, plaque stability, food restriction, cholesterol, autophagy

Atherosclerosis is an inflammatory disease characterized by the formation of plaques in the large- and medium-sized arteries. Despite current pharmacological therapies, atherosclerosis remains the leading cause of death and morbidity among adults in the Western world [1]. Because a diet rich in calories, together with a sedentary lifestyle, contributes to the development

> © 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.

Dorien G. De Munck, Guido R.Y. De Meyer and

Dorien G. De Munck, Guido R.Y. De Meyer and

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.76560

**Stabilization**

**Stabilization**

Wim Martinet

Wim Martinet

**Abstract**

**1. Introduction**

#### **Food Restriction and Atherosclerotic Plaque Stabilization Food Restriction and Atherosclerotic Plaque Stabilization**

DOI: 10.5772/intechopen.76560

Dorien G. De Munck, Guido R.Y. De Meyer and Wim Martinet Dorien G. De Munck, Guido R.Y. De Meyer and Wim Martinet

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.76560

#### **Abstract**

Food restriction is a promising therapy for many age-associated pathologies as it stimulates the health-supportive mechanism autophagy. Because atherosclerosis is an inflammatory, age-related disease, dietary modification can be an important strategy in preventing atherosclerotic plaque development. A cholesterol-supplemented diet, used to induce plaque formation in rabbits, induces a pronounced hypercholesterolemia, which can be reversed after 4 weeks of normal diet. However, food restriction induces a further increase in circulating LDL cholesterol. These elevated cholesterol levels are associated with the induction of autophagy. Although neither a short-term normal diet nor food restriction alters plaque size, rabbits fed a normal diet show signs of increased plaque stability such as elevated collagen content and decreased expression of vascular cell adhesion molecule (VCAM)-1. Surprisingly, these favorable effects are not present after 4 weeks of food restriction. On the contrary, atherosclerotic plaques of food-restricted rabbits displayed enhanced apoptosis, a process known to further undermine plaque stability. In conclusion, severe short-term food restriction in rabbits prevents stabilization of atherosclerotic plaques as observed after regular cholesterol withdrawal via a normal diet.

**Keywords:** atherosclerosis, plaque stability, food restriction, cholesterol, autophagy

#### **1. Introduction**

Atherosclerosis is an inflammatory disease characterized by the formation of plaques in the large- and medium-sized arteries. Despite current pharmacological therapies, atherosclerosis remains the leading cause of death and morbidity among adults in the Western world [1]. Because a diet rich in calories, together with a sedentary lifestyle, contributes to the development

© 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.

of atherosclerosis, dietary change is considered an important strategy in the prevention of atherosclerosis [2]. Moreover, dietary modification has shown to play an important role in several age-associated pathologies and in aging itself. Moderate calorie restriction results in a lifespan expansion of different species including yeast, fruit flies, nematodes, fish, rodents, and rhesus monkeys [3]. Besides favorable effects on longevity, long-term as well as short-term caloric restriction improves the cardiovascular disease risk profile in humans [4, 5]. Consistent with this finding, animal studies showed that dietary restriction attenuates atherosclerotic plaque development and decreases endothelial dysfunction [6, 7].

Despite increased levels of circulating LDL, there is no difference in lipid accumulation in the liver or aorta of rabbits undergoing severe food restriction. Both normal diet and food restric-

Food Restriction and Atherosclerotic Plaque Stabilization

http://dx.doi.org/10.5772/intechopen.76560

63

LDL cholesterol levels are negatively correlated with SQSTM1/p62 protein levels in the liver (**Figure 1**), suggesting stimulation of autophagy as an alternative mechanism for the increase

**Figure 1.** Induction of autophagy in liver of rabbits that were fed 0.3% cholesterol for 20 weeks (baseline), followed by cholesterol withdrawal for 4 weeks either via a normal diet or a restricted diet (20% of normal diet). Liver samples of ten rabbits per group were analyzed by Western blotting for the expression of autophagy marker proteins LC3-II (A) and p62 (B). \*\*P < 0.01, \*\*\*P < 0.001 (One-way ANOVA with post-hoc LSD, n = 10 in each group). (C) Serum LDL-levels show an

inverse correlation with liver p62 protein levels (Pearson Correlation Coefficient −0.44, P<0.05).

**3. Hypercholesterolemia induced by food restriction is associated** 

tion do not affect serum triglycerides (**Table 1**).

**with autophagy induction**

Starvation, as an extreme form of food restriction, is also one of the most important stimuli for autophagy induction [8]. Autophagy is a subcellular degradation pathway for long-lived proteins and damaged organelles. Under normal conditions, autophagy is a homeostatic process that is found in all cell types. However, under stress conditions, it functions as an important cell survival mechanism through nutrient recycling and the generation of energy [9]. Growing evidence indicates that autophagy deficiency plays a crucial role in plaque growth and destabilization [10–12]. Moreover, autophagy induction is suggested as a novel strategy for the prevention and treatment of atherosclerosis [13, 14].
