**Meet the editor**

Dr. Naofumi Shiomi studied recombinant yeast and its utilization as a researcher at the Laboratory of Production Technology of Kanena Corporation for 15 years until 1998 and earned his PhD in Engineering from Kyoto University. He now works as a professor at the School of Human Sciences of Kobe College in Japan, where he teaches applied microbiology, biotechnology,

and life science in his "Applied Life Science" laboratory. He has studied bioremediation and biomedical science for 17 years at Kobe College and has published more than 40 papers and several book chapters. His recent research has focused on the prevention of obesity and aging.

## Contents

#### **Preface XI**



## Preface

Why do living things gradually senesce? This question is an important theme for human beings, and numerous studies have sought its answer. Some 30 years ago, Brackbarn et al. discovered that telomeres functioned as a clock of cell life, remarkably advancing studies on the molecular mechanisms of aging. As a result, many genes and proteins related to aging have been discovered and their roles elucidated. However, we have not yet answered the main question because of the complicated mechanism of aging in cells. Regarding aging in tissues, many factors such as biologically active substances, signal proteins, hormones, the immune system, and the environment are involved in the process. As such, the complexities of the mechanism make understanding of the fine details difficult. Comprehensive discus‐ sion is necessary to clarify the mechanisms underlying aging and rejuvenation.

I therefore proposed to publish this book to obtain a comprehensive assessment of the recent advances in the fields of aging and rejuvenation. In the early parts of this book, the overall aspect of the aging and rejuvenation processes is introduced by the editor, with other researchers expanding on the role of the genes of the circadian clock and ion channel, which, respectively, control the rhythms of cells and calcium ions and decrease those functions due to aging. Techni‐ ques capable of improving these functions may provide a novel rejuvenation system.

In the latter chapters, the mechanisms underlying aging and rejuvenation in tissues are in‐ troduced. Researchers will describe the aging of skin, the molecular mechanism of skin ag‐ ing, and the role of certain compounds and oxidative stress in antiaging. We then move on to discussing the role of aging-related molecules in the blood. The results of attempts at reju‐ venation by parabiosis between young and old mice or injection of blood components are reviewed, and the possibility of the successful development of antiaging therapy is dis‐ cussed. Antiaging therapy using blood components may become a realizable and immedi‐ ately useful procedure for rejuvenation. Finally, we examine novel biomarkers for mild cognitive impairment and Alzheimer's disease, which play an important role in preventing aging in the brain.

I believe that this book will be useful for those studying or developing new drugs to counter the aging process and for students studying aging. Furthermore, it may provide novel ideas on aging and rejuvenation procedures, in addition to supplying important information on available techniques.

I would like to thank Ms. Andrea Koric, Ms. Ivona Lovric, the publishing process managers of the InTech Publishing Group for their great support and assistance throughout the writ‐ ing and publication process of this book.

> **Prof. Naofumi Shiomi** School of Human Sciences, Kobe College Japan

## **Introductory Chapter: Recent Studies on Cellular Aging and Rejuvenation**

Naofumi Shiomi

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/63897

#### **1. Introduction**

Certain creatures possess the properties of eternal youth and deathlessness. These include Turritopsis (a species of small jellyfish) and planarians. Old Turritopsis starts a degenera‐ tion to transform into polyps and thereby achieves a perpetual life cycle [1]. The old cells contained in an old imago change into young cells when the imago transforms into a polyp. The polyp then starts to grow until it reaches the imago stage. As the Turritopsis can repeat this cycle forever, it can be considered to exist in a state of deathlessness. Planarians possess a special property of not growing older. They contain numerous stem cells throughout their bodies and every portion of their body can reproduce [2]. Planarians may therefore be an ageless organism. The property of agelessness is also present in humans. The case of Brooke Greenberg, an American woman who could not grow older after developing highlander syndrome, was the subject of a number of news reports in Japan a few years ago [3]. More‐ over, there are several reported cases of female patients who developed highlander syn‐ drome; however, the veracity of these reports is unclear, and the gene that causes the syndrome has not been elucidated. The development of highlander syndrome in human patients suggests that humans might possess the genes that enable the property of eternal youth.

The actualization of eternal youth is a long-held dream, and numerous studies have been performed to elucidate the mechanisms of aging and to achieve eternal youth. Consequently, recent studies have partially elucidated the process of aging and proposed several anti-aging or rejuvenation procedures; however, the studies are currently in the middle stage, and the key elements of the aging process have yet to be elucidated. The purpose of this chapter is to present an overview of the recent topics on cellular aging and rejuvenation to provide an outline of the content in this book. This chapter also complements the chapters that follow; in which researchers introduce topics related to aging and rejuvenation.

© 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.

#### **2. The mechanisms of cellular aging**

#### **2.1. Aging by telomere shortening**

Cultured human cells become older through repeated cell division. Cells eventually stop dividing when they reach the critical passage number, the Hayflick limit [4]. Blackburn [5] and Greider discovered that the telomeres function as a clock by marking the passage of time in cells. The molecular mechanism of aging, which is described below [6–8], was elucidated through this discovery.

RNA primer is degraded and exchanged to DNA with DNA polymerase I followed by replication with DNA polymerase III. However, the RNA primer present at the terminal portion of telomeres cannot be exchanged to DNA. As a result, approximately 100 sequence bases are shortened in every replication. The shortening of telomeres acts as a fuse and decides the limit of cell division. The length limit (M1 period) in human fibroblasts is approximately 5 kb, but most cells in elderly humans do not reach the M1 period [9]. Moreover, mouse telomeres are of sufficient length, even in old mice, due to the expression of telomerase. Otherwise, cells in the M1 period can continue to undergo cell division by transforming with T antigen [10]. In such cells, the shortening of telomeres continues, and cell division is finally stopped again by the fusion of mutual terminals (the M2 period) or apoptosis. Telomeres are therefore protected from further shortening by a safe limit (the M1 period).

The pathway from telomere shortening to the cessation of cell division has also been elucidated [7]. A telomere is formed by both double-stranded DNA, which is made by repeated sequences ("TTAGGG" in the mammals), and single-strand DNA (G-tail) of similar sequences that exist at the terminal portion. A telomere has to construct the T-rope structure to avoid degradation by DNA degrading enzymes. This structure gives the first signal to initiate the progression of cell division. The shortening of telomeres causes the obstruction of the T-rope structure and signals certain proteins, including telomeric repeat-binding factor 2 (TRF2), AMP kinase and histone deacetylase, to delay or stop cell division. The detection of the signal by AMP kinase activates p53 and/or p21 proteins and inhibits the work of the cyclin-dependent kinase (CDK) complex. Finally, the inhibition of the CDK complex causes cell division to stop at the Go period, because CDK is a control switch that determines whether cell division should be promoted.

#### **2.2. The effects of mitochondria on the aging process**

Mitochondria also have a role in the aging process [11, 12]. Mitochondria produce ATP using the electron transport chain pathway where reactive oxygen species (hydrogen peroxide, hydroxyl radical and hydroperoxyl radical) are produced. Reactive oxygen species are often leaked through the mitochondrial inner membrane, damage DNA, proteins, and lipids. It has been confirmed by the experiments with nematodes [13] that reactive oxygen species promote aging. For instance, the mutation of the mev-1 gene in nematodes was found to result in a shorter life span, because the gene disruption caused a defect in the electron transport chain and an increase in the level of reactive oxygen species. Conversely, a mutation of the age-1 gene in nematodes caused the enhancement of catalase activity which degraded hydrogen peroxide and resulted in a prolonged life span.

Recent studies have suggested an interaction between mitochondrial dysfunction and telomere shortening because of the following process [12, 14, 15]. The shortening of telomeres causes the activation of the p53 protein, the activation of which inhibits the activities of PGC-1α and β, which induce the activation of mitochondria. This inhibition finally causes a decrease in many important mitochondrial activities and the progression of the aging process. On the contrary, an increase in the level of active oxygen species due to mitochondrial dysfunction often causes the oxidation of telomeres; the numerous guanine repeats on telomeres cause them to react easily with oxygen. In telomere DNA, oxidation disturbs the combination of TRF2 with telomere DNAs and the normal T-rope structure, which is the first signal that cell division cannot proceed. Thus, under high concentrations oxygen, the telomeres in human cells are rapidly shortened and cell growth is inhibited.

#### **2.3. The factors promoting the aging process**

**2. The mechanisms of cellular aging**

2 Molecular Mechanisms of the Aging Process and Rejuvenation

Cultured human cells become older through repeated cell division. Cells eventually stop dividing when they reach the critical passage number, the Hayflick limit [4]. Blackburn [5] and Greider discovered that the telomeres function as a clock by marking the passage of time in cells. The molecular mechanism of aging, which is described below [6–8], was elucidated

RNA primer is degraded and exchanged to DNA with DNA polymerase I followed by replication with DNA polymerase III. However, the RNA primer present at the terminal portion of telomeres cannot be exchanged to DNA. As a result, approximately 100 sequence bases are shortened in every replication. The shortening of telomeres acts as a fuse and decides the limit of cell division. The length limit (M1 period) in human fibroblasts is approximately 5 kb, but most cells in elderly humans do not reach the M1 period [9]. Moreover, mouse telomeres are of sufficient length, even in old mice, due to the expression of telomerase. Otherwise, cells in the M1 period can continue to undergo cell division by transforming with T antigen [10]. In such cells, the shortening of telomeres continues, and cell division is finally stopped again by the fusion of mutual terminals (the M2 period) or apoptosis. Telomeres are

The pathway from telomere shortening to the cessation of cell division has also been elucidated [7]. A telomere is formed by both double-stranded DNA, which is made by repeated sequences ("TTAGGG" in the mammals), and single-strand DNA (G-tail) of similar sequences that exist at the terminal portion. A telomere has to construct the T-rope structure to avoid degradation by DNA degrading enzymes. This structure gives the first signal to initiate the progression of cell division. The shortening of telomeres causes the obstruction of the T-rope structure and signals certain proteins, including telomeric repeat-binding factor 2 (TRF2), AMP kinase and histone deacetylase, to delay or stop cell division. The detection of the signal by AMP kinase activates p53 and/or p21 proteins and inhibits the work of the cyclin-dependent kinase (CDK) complex. Finally, the inhibition of the CDK complex causes cell division to stop at the Go period, because CDK is a control switch that determines whether cell division should be

Mitochondria also have a role in the aging process [11, 12]. Mitochondria produce ATP using the electron transport chain pathway where reactive oxygen species (hydrogen peroxide, hydroxyl radical and hydroperoxyl radical) are produced. Reactive oxygen species are often leaked through the mitochondrial inner membrane, damage DNA, proteins, and lipids. It has been confirmed by the experiments with nematodes [13] that reactive oxygen species promote aging. For instance, the mutation of the mev-1 gene in nematodes was found to result in a shorter life span, because the gene disruption caused a defect in the electron transport chain and an increase in the level of reactive oxygen species. Conversely, a mutation of the age-1

therefore protected from further shortening by a safe limit (the M1 period).

**2.2. The effects of mitochondria on the aging process**

**2.1. Aging by telomere shortening**

through this discovery.

promoted.

Studies using both old cells (M1 period) and young cells (Low PDL) have suggested other causes of the aging. When cell fusion occurs between a young cell and an old cell from which the nucleus has been removed, the cell division of the fused cell is inhibited. However, when old cells that were previously treated with a protein synthesis inhibitor are used, growth is not inhibited. Moreover, when the cell membrane of old cells or mRNAs of cells stopped at the Go period, respectively, are injected into younger cells, cell division stops or DNA replication is inhibited. These results suggest that some proteins, mRNAs and/or cell membranes that are present in older cells gradually accumulate with every cell division and promote the aging process. The genes corresponding to such compounds have also been screened [16, 17]. Some genes (gas, gadd, mot1, and hic-5) have been cloned. Unfortunately, they were not the most important genes for controlling the aging process. Recently screening has been performed using RNA, and some promising genes have been identified [18, 19].

#### **2.4. The genes associated with premature senility syndromes**

Information that is important for elucidating the aging process in humans can be obtained from the genes that cause premature senility syndrome. Five types of helicases (RecQL1, BLM, WRN RecQL4/RTS, and RecQL5) that untangle DNA chains exist in humans. The change of the proteins to abnormal sequences causes premature senility syndromes [20]. Werner, Bloom, and Rothmund–Thomson syndromes are caused by abnormal structures of the WRN, BLM, and RecQL4/RT proteins, respectively. The WRN protein, which is related to the replication, restoration, transcription, and stabilization of DNA or telomeres, is remarkable. In the case of patients of Werner syndrome, the onset of symptoms occurs after patients stop growing at approximately 10 years of age. In Werner syndrome, the aging process advances much faster than in normal individuals. Patients show normal nerve and immune systems but possess unusual chromosomes. The WRN gene was expected to become a target of aging in normal individuals, because with the exception of the speed at which aging advances, the symptoms are similar to the normal aging process. However, WRN knockout mice do not show premature senility syndrome, whereas WRN and TERC knockout mice do [21]. Further investigation is necessary to improve our understanding of the relationship between WRN and the aging process.

The other remarkable premature senility syndrome is Hutchinson–Gilford progeria syndrome (HGPS), which is caused by partial loss of the lamin A protein [22]. HGPS patients are normal at birth. HGPS develops at 6–18 months of age; the average life span of an HGPS patient is 13 years. Lamin A exists inside a nuclear membrane and supports the structure of the membrane. It is changed to a farnesylated version to perform nuclear translocation, and farnesylated lamin A is related to both the replication and transcription of DNA and signal transduction. The unusual farnesylated lamin A that is found in HGPS patients is called "progerin" [23]. Progerin accumulates and inhibits translocation, and the inhibition causes the aging of cells. In normal individuals, progerin gradually accumulates in the skin cells due to aging. Progerin is therefore a target of treatments to delay the aging process.

#### **3. Realizing cellular rejuvenation**

#### **3.1. Rejuvenation by telomerase activation**

In humans, although telomerase can make telomeres longer, most types of cells (including fibroblasts, spanchnic cells, and nerve cells) show very low telomerase activity [24]. In contrast, germ and cancer cells, which actively perform cell division, show high telomerase activity and long telomeres. This suggests that the cells may be rejuvenated if telomerase can be activated [25–27].

Studies on the fibroblasts and mice that express telomerase reverse transcriptase (TERT) gene by transformation have supported this hypothesis [28–30]. For example, a human OSMU36- T2 fibroblast showing high telomerase activity was obtained by transforming the TERT gene into an OSMU36 fibroblast. The telomere length and the telomerase activity of OSMU36-T2 were several times higher than those observed in OSMU36 fibroblast, and many characteristics including the cell size, growth rate, and gene expression were similar to those observed in young fibroblasts [28]. Moreover, the life span of a TERT knockout mouse, which was produced by Harvard University, was much shorter (only 6 months) than normal mice due to the rapid shortening of the telomeres. When telomerase was activated in the knockout mice, neurons were formed and rejuvenation was found in some portions [29, 30]. Thus, it may be possible to initiate cellular rejuvenation in individuals as well as cultured cells through the activation of telomerase.

Telomerase activity and telomere length are affected by lifestyle [31, 32]. Researchers at California University group examined the effects of food, exercise, and psychological stress on the telomere length in 35 males [31]. The members of one group continued to consume vegetables as a staple food, to perform adequate exercise and to decrease psychological stress through self-control for 5 years. As a result, their telomeres were longer than the members of the control group. Other researchers investigated the effects of exercise. The results suggested that the athletes who ran more than 40 km every day were 16 years younger in telomere length. Moreover, telomerase can be activated by some chemical compounds, which are expected to have applications as rejuvenation drugs. For instance, the rate at which telomeres shorten is accelerated by high serum concentrations of cholesterol, because cholesterol hastens cell division. Thus, mononucleosis patients who continuously took statin (an anticholesteremic agent) showed higher telomerase activity and longer telomeres than the patients who did not take statin [33]. Several years ago, a compound named TA-65, which was isolated from the root of the Hedysarum, was screened as a telomerase activating compound [34], and the rejuvenation effect of TA-65 was examined in mice.

As telomerase activity can be controlled by lifestyle and some compounds, the enhancement of telomerase activity may become an effective treatment to promote cellular rejuvenation. However, the enhancement of telomerase activity may also cause the activation or induction of cancer cells, because high telomerase activity is one of the characteristics of cancer cells [35– 37]. Further studies to determine whether the activation of telomerase can truly induce a rejuvenation condition without the risk of cancer cell activation will be necessary before it can be used in supplements and drugs.

#### **3.2. Rejuvenation by antioxidants**

senility syndrome, whereas WRN and TERC knockout mice do [21]. Further investigation is necessary to improve our understanding of the relationship between WRN and the aging

The other remarkable premature senility syndrome is Hutchinson–Gilford progeria syndrome (HGPS), which is caused by partial loss of the lamin A protein [22]. HGPS patients are normal at birth. HGPS develops at 6–18 months of age; the average life span of an HGPS patient is 13 years. Lamin A exists inside a nuclear membrane and supports the structure of the membrane. It is changed to a farnesylated version to perform nuclear translocation, and farnesylated lamin A is related to both the replication and transcription of DNA and signal transduction. The unusual farnesylated lamin A that is found in HGPS patients is called "progerin" [23]. Progerin accumulates and inhibits translocation, and the inhibition causes the aging of cells. In normal individuals, progerin gradually accumulates in the skin cells due to

In humans, although telomerase can make telomeres longer, most types of cells (including fibroblasts, spanchnic cells, and nerve cells) show very low telomerase activity [24]. In contrast, germ and cancer cells, which actively perform cell division, show high telomerase activity and long telomeres. This suggests that the cells may be rejuvenated if telomerase can be activated

Studies on the fibroblasts and mice that express telomerase reverse transcriptase (TERT) gene by transformation have supported this hypothesis [28–30]. For example, a human OSMU36- T2 fibroblast showing high telomerase activity was obtained by transforming the TERT gene into an OSMU36 fibroblast. The telomere length and the telomerase activity of OSMU36-T2 were several times higher than those observed in OSMU36 fibroblast, and many characteristics including the cell size, growth rate, and gene expression were similar to those observed in young fibroblasts [28]. Moreover, the life span of a TERT knockout mouse, which was produced by Harvard University, was much shorter (only 6 months) than normal mice due to the rapid shortening of the telomeres. When telomerase was activated in the knockout mice, neurons were formed and rejuvenation was found in some portions [29, 30]. Thus, it may be possible to initiate cellular rejuvenation in individuals as well as cultured cells through the

Telomerase activity and telomere length are affected by lifestyle [31, 32]. Researchers at California University group examined the effects of food, exercise, and psychological stress on the telomere length in 35 males [31]. The members of one group continued to consume vegetables as a staple food, to perform adequate exercise and to decrease psychological stress through self-control for 5 years. As a result, their telomeres were longer than the members of the control group. Other researchers investigated the effects of exercise. The results suggested that the athletes who ran more than 40 km every day were 16 years younger in telomere length.

aging. Progerin is therefore a target of treatments to delay the aging process.

**3. Realizing cellular rejuvenation**

4 Molecular Mechanisms of the Aging Process and Rejuvenation

**3.1. Rejuvenation by telomerase activation**

process.

[25–27].

activation of telomerase.

Leucocytes secrete reactive oxygen species to protect against psychological stress in social life or physical stresses including atmospheric pollutants, UV, and viruses. The excessive produc‐ tion of reactive oxygen species promotes aging (as described in Section 2.2.). Although catalase, superoxide dismutase (SOD), and glutathione peroxydase can remove such reactive oxygen species in humans, catalase, and SOD activities gradually decrease due to aging. Thus, reactive oxygen species cannot be sufficiently removed in elderly individuals. In other words, the aging process can be delayed or remediated if the excessive production of reactive oxygen species is prevented.

Antioxidants, such as vitamins and polyphenols, are effective in removing active oxygen species, and their anti-aging effects have been studied for many years [38, 39]. The studies suggest that the oxidation of cultured cells can be inhibited by antioxidants and that mice that continuously took antioxidants showed lower oxidation and a longer life span than controls. Recent studies in humans, however, suggested that the anti-aging effects are doubtful. For example, in experiments in which healthy human subjects took β-carotene or vitamins for a long period of time, the ratio of depth was not decreased. Certain amounts or components of antioxidants may be required to make the effects of antioxidants prominent in humans.

#### **3.3. Rejuvenation by anti-aging hormones**

Anti-aging hormones have remarkable potential as anti-aging or rejuvenation treatments. There are several candidate compounds, including klotho, sirtuin, Bach1, Clk-1, and polya‐ mines [40–46]. The klotho gene is mainly expressed in the nervous system to control the calcium concentration in blood; klotho works as a controller of homeostasis, although the precise work of the protein has not been sufficiently elucidated [40, 41]. Klotho knockout mice develop many symptoms related to aging, such as arteriosclerosis, osteoporosis, motor impairment and have a shortened life span. Conversely, klotho knock-in mice have a life span that is several years longer [42]. Thus, klotho is a promising target in rejuvenation.

Histone deacetylases (sirtuin) is another candidate protein [43, 44]. Sirtuin works as an energy economizing hormone to prolong the life span. Sirtuin 1, 6 and 7 knockout mice showed faster aging and a shorter life span. Hibernation, which represents a state of extreme energy limitation, extended the life spans of yeasts, rematodes, and mice. One reason for the prolon‐ gation of the life span is that the amount of excess active oxygen is decreased by the energy limitation; thus, the promotion of the aging process by undesirable oxidation is inhibited. Another cause is the activity of sirtuin. Sirtuin 2 is activated by the decrease of NAD, which is caused by energy limitation. The activation of sirtuin 2 increases the deacetylation of histones to silence the genes, and the economizing of energy prolongs life span.

The following monkey experiments were performed to estimate the action of sirtuin in relation to energy limitation. The Wisconsin National Primate Research Center examined the effects of calorie restriction on 76 rhesus monkeys. The monkeys that had were fed low-calorie food (a 30% calorie reduction) looked much younger and showed a longer life span than control members (100% calorie) [47]. However, a similar experiment by the NIA in the United States in 2012 suggested the reverse results [48]. Therefore, further discussion is necessary to confirm the relationship between anti-aging and energy limitation.

Resveratrol has been screened as a compound that is effective for enhancing the expression of the sirtuin gene [49]. When resveratrol was given to mice that had been fed a high-calorie diet, some of the factors of aging were inhibited, and the activities of AMPK and PGC-1α were enhanced; however, the anti-aging were insufficient. If compounds that can more effectively enhance the sirtuin gene are discovered, they will become a prominent anti-aging hormone.

Polyamines also show remarkable characteristics. Polyamines, such as spermine, spermidine, and putrescine, are essential for the growth of mammals and play important roles in cell division and differentiation; however, their precise action has not been sufficiently elucidated [50, 51]. In most cells of the human body, the concentrations are maintained at low levels. They are only found at high levels in cells that are actively working, such as testicular cells and cancer cells. The amounts of polyamines in those cells are especially increased before DNA synthesis; the growth rate in cancer cells increases in proportion to the amount of polyamine that is added to the medium. Polyamines are therefore related to the activation of cells.

Some studies suggest that polyamines function as an anti-aging hormone. The addition of polyamines to fibroblasts enhanced the expression of genes related to aging [20], and mice that were contentiously fed a diet containing a high concentration of polyamine appeared much younger and had a longer life span than mice that were fed a diet without polyamine [52]. Other researchers, who had studied the relationship between *Lactobacillus bifidus* and life span, suggested that the life span of mice was prolonged by the polyamines that were produced by *L. bifidus* cells [53, 54]. The amounts of spermine and spermidine in humans gradually decrease due to aging, and the concentration of polyamines in the blood can easily be increased by eating foods, such as soy beans. Thus, polyamines may also be useful as supplements for anti-aging or rejuvenation.

#### **4. Conclusion**

impairment and have a shortened life span. Conversely, klotho knock-in mice have a life span

Histone deacetylases (sirtuin) is another candidate protein [43, 44]. Sirtuin works as an energy economizing hormone to prolong the life span. Sirtuin 1, 6 and 7 knockout mice showed faster aging and a shorter life span. Hibernation, which represents a state of extreme energy limitation, extended the life spans of yeasts, rematodes, and mice. One reason for the prolon‐ gation of the life span is that the amount of excess active oxygen is decreased by the energy limitation; thus, the promotion of the aging process by undesirable oxidation is inhibited. Another cause is the activity of sirtuin. Sirtuin 2 is activated by the decrease of NAD, which is caused by energy limitation. The activation of sirtuin 2 increases the deacetylation of histones

The following monkey experiments were performed to estimate the action of sirtuin in relation to energy limitation. The Wisconsin National Primate Research Center examined the effects of calorie restriction on 76 rhesus monkeys. The monkeys that had were fed low-calorie food (a 30% calorie reduction) looked much younger and showed a longer life span than control members (100% calorie) [47]. However, a similar experiment by the NIA in the United States in 2012 suggested the reverse results [48]. Therefore, further discussion is necessary to confirm

Resveratrol has been screened as a compound that is effective for enhancing the expression of the sirtuin gene [49]. When resveratrol was given to mice that had been fed a high-calorie diet, some of the factors of aging were inhibited, and the activities of AMPK and PGC-1α were enhanced; however, the anti-aging were insufficient. If compounds that can more effectively enhance the sirtuin gene are discovered, they will become a prominent anti-aging hormone.

Polyamines also show remarkable characteristics. Polyamines, such as spermine, spermidine, and putrescine, are essential for the growth of mammals and play important roles in cell division and differentiation; however, their precise action has not been sufficiently elucidated [50, 51]. In most cells of the human body, the concentrations are maintained at low levels. They are only found at high levels in cells that are actively working, such as testicular cells and cancer cells. The amounts of polyamines in those cells are especially increased before DNA synthesis; the growth rate in cancer cells increases in proportion to the amount of polyamine that is added to the medium. Polyamines are therefore related to the activation of cells.

Some studies suggest that polyamines function as an anti-aging hormone. The addition of polyamines to fibroblasts enhanced the expression of genes related to aging [20], and mice that were contentiously fed a diet containing a high concentration of polyamine appeared much younger and had a longer life span than mice that were fed a diet without polyamine [52]. Other researchers, who had studied the relationship between *Lactobacillus bifidus* and life span, suggested that the life span of mice was prolonged by the polyamines that were produced by *L. bifidus* cells [53, 54]. The amounts of spermine and spermidine in humans gradually decrease due to aging, and the concentration of polyamines in the blood can easily be increased by eating foods, such as soy beans. Thus, polyamines may also be useful as supplements for anti-aging

that is several years longer [42]. Thus, klotho is a promising target in rejuvenation.

to silence the genes, and the economizing of energy prolongs life span.

the relationship between anti-aging and energy limitation.

6 Molecular Mechanisms of the Aging Process and Rejuvenation

or rejuvenation.

In this chapter, the author focused on cellular aging and rejuvenation. Recent studies have gradually elucidated the aging process and have suggested that telomerase, the mitochondria and other components (RNAs and proteins) are involved in the aging process. However, these studies are ongoing. Moreover, studies on the aging process have identified several anti-aging compounds that can activate telomerase (TA-65), inactivate reactive oxygen species (antioxidants) and work as anti-aging hormones (klotho, sirtuin, resveratrol, and polyamine). In the near future, these compounds and/or other compounds with greater effects may become prominent drugs for anti-aging and rejuvenation.

Research into regeneration with the use of iPS cells is recently showing remarkable progress. Although the topic was not introduced in this chapter, the regeneration of cutaneous and nerve tissues or internal organs using regeneration medicine will be another means of realizing rejuvenation. Researchers studying anti-aging will have to watch the progress of both regeneration medicine and cellular rejuvenation research.

#### **Author details**

Naofumi Shiomi\*

Address all correspondence to: shiomi@mail.kobe-c.ac.jp

Department of Human Sciences, Kobe College, Hyogo, Japan

#### **References**


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## **Circadian Clock Gene Regulation in Aging and Drug Discovery**

Yufeng Li, Yanqi Dang, Shuang Ling and Jin-Wen Xu

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/63184

#### **Abstract**

The circadian clock is an endogenous timer in prokaryotes and mammals. Resting and adjusting the internal clock can assist in pacing the daily routine. Growing evidence indicates that the circadian clock and aging process are closely associated. The disruption of the circadian clock leads to accelerated aging and increased incidence of various diseases. In particular, elderly people are more vulnerable and have a higher risk of diseases than do young people. In this study, we reviewed studies on aging and circadian rhythms over the last decade, with a focus on circadian clock gene regula‐ tion in aging and drug discovery for targeting the circadian clock in diseases.

**Keywords:** Bmal1, clock, circadian clock genes, aging, drug discovery

#### **1. Introduction**

Circadian rhythms affect almost all daily activity behavioral patterns, physiology, and gene expression. Circadian rhythms indicate the appropriate time for various activities, such as consuming food and mating, and gradually form the circadian clock [1]. The circadian clock is an internal timekeeping system, which facilitates adaptation to the external world in anticipa‐ tion of daily environmental changes. A 24-h circadian rhythm pattern has been observed in almost all cells [2]. Circadian clocks coordinate external day and night cycles with diverse environmental and metabolic stimuli. However, a disrupted circadian clock causes various diseases, such as insomnia, diabetes, cancer, and metabolic syndrome [3]. The ability of the brain timing system and function of circadian rhythm-regulating genes decline with age. The

© 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.

disruptionofcircadianrhythmsdisruptsthecoordinationamongbodysystems.Agingdamages body homeostasis and results in a subhealth status [4]. Consequently, the systems of elderly people aremore vulnerable thanthose of youngpeople [5].Thedisruptionof circadianrhythms adversely affects the aging process and increases the risk of diseases. A set of circadian clock genes form negative feedback loops that can regulate transcription, with a 24-h circadian oscillation [4, 6]. The circadian function significantly affects the aging process. Chronic destructions of the circadian function are associated with the occurrence of various agerelated diseases, such as cancer and premature aging. Therefore, studies are warranted to determine how the circadian clock genes orchestrate interactions between the internal physi‐ ology and the aging progress.

In this study, we reviewed studies within the last decade on aging and circadian rhythms and the mechanism underlying the maintenance of biological processes by the circadian clock, with a focus on circadian clock gene regulation in aging and antiaging drug discovery.

### **2. Circadian clock**

#### **2.1. Structure of the circadian system**

Living systems possess an exquisite internal biological clock, and the major function of which is to regulate the daily sleep–wake cycle [7]. The circadian clock follows a rhythm of approx‐ imately 24 h and ensures accurate adaptation to external daily rhythms through a powerful endogenous timing system [8]. The circadian clock also drives numerous molecular and cellular processes by generating oscillations. Virtually, all body cells have an autonomous circadian clock [9, 10], which is composed of a central clock existing in the suprachiasmatic nucleus (SCN) neuron and peripheral clocks. Clock, Bmal1 (brain and muscle ARNT-like protein-1), Pers, and Crys constitute a set of circadian oscillation genes in mammals [11].

#### *2.1.1. Suprachiasmatic nucleus*

The central circadian clock, located in the SCN of the anterior hypothalamus, is the primary circadian pacemaker [12]. The SCN comprises a network of approximately 20,000 neurons. Each neuron is considered to have an oscillator of the autonomous circadian clock. As the neurons are joined and oscillated in a consistent manner [13], the SCN neuron can generate an autonomous circadian clock similar to other cells [14, 15]. The SCN as the primary circadian pacemaker regulates independent gene expressions through neuronal firing [16].

The retina captures optical signals and transmits signals to the SCN (**Figure 1**) [17]. SCN neurons organize coupling mechanisms that ensure their synchronization even in darkness [18]. SCN neurons change the gene expression levels by converting electrical information into chemical information [16]. Neuronal firing frequency can synchronize the other cells of the body with rhythmic changes [19]. The central clock is controlled by external signals; food and light are the strongest signals affecting the clock (**Figure 1**). Once synchronized, the central clock consequently mediates the synchronization of the peripheral clocks through signaling [20].

Moreover, lability and plasticity of the phase in the intrinsic period are two critical functions of the central clock [21]. As the phase is labile, the length of the intrinsic period leads to different phases [22]. The waveform of the SCN amplitude is mainly related to the light cycle. The waveform is narrow with a high amplitude in short photoperiods, whereas it is broader with a low amplitude in long photoperiods. The circadian waveform in SCN oscillation is strong correlated with the SCN and behavioral rhythms [23].

#### *2.1.2. Peripheral clocks*

disruptionofcircadianrhythmsdisruptsthecoordinationamongbodysystems.Agingdamages body homeostasis and results in a subhealth status [4]. Consequently, the systems of elderly people aremore vulnerable thanthose of youngpeople [5].Thedisruptionof circadianrhythms adversely affects the aging process and increases the risk of diseases. A set of circadian clock genes form negative feedback loops that can regulate transcription, with a 24-h circadian oscillation [4, 6]. The circadian function significantly affects the aging process. Chronic destructions of the circadian function are associated with the occurrence of various agerelated diseases, such as cancer and premature aging. Therefore, studies are warranted to determine how the circadian clock genes orchestrate interactions between the internal physi‐

In this study, we reviewed studies within the last decade on aging and circadian rhythms and the mechanism underlying the maintenance of biological processes by the circadian clock, with

Living systems possess an exquisite internal biological clock, and the major function of which is to regulate the daily sleep–wake cycle [7]. The circadian clock follows a rhythm of approx‐ imately 24 h and ensures accurate adaptation to external daily rhythms through a powerful endogenous timing system [8]. The circadian clock also drives numerous molecular and cellular processes by generating oscillations. Virtually, all body cells have an autonomous circadian clock [9, 10], which is composed of a central clock existing in the suprachiasmatic nucleus (SCN) neuron and peripheral clocks. Clock, Bmal1 (brain and muscle ARNT-like protein-1), Pers, and Crys constitute a set of circadian oscillation genes in mammals [11].

The central circadian clock, located in the SCN of the anterior hypothalamus, is the primary circadian pacemaker [12]. The SCN comprises a network of approximately 20,000 neurons. Each neuron is considered to have an oscillator of the autonomous circadian clock. As the neurons are joined and oscillated in a consistent manner [13], the SCN neuron can generate an autonomous circadian clock similar to other cells [14, 15]. The SCN as the primary circadian

The retina captures optical signals and transmits signals to the SCN (**Figure 1**) [17]. SCN neurons organize coupling mechanisms that ensure their synchronization even in darkness [18]. SCN neurons change the gene expression levels by converting electrical information into chemical information [16]. Neuronal firing frequency can synchronize the other cells of the body with rhythmic changes [19]. The central clock is controlled by external signals; food and light are the strongest signals affecting the clock (**Figure 1**). Once synchronized, the central clock consequently mediates the synchronization of the peripheral clocks through signaling

pacemaker regulates independent gene expressions through neuronal firing [16].

a focus on circadian clock gene regulation in aging and antiaging drug discovery.

ology and the aging progress.

14 Molecular Mechanisms of the Aging Process and Rejuvenation

**2. Circadian clock**

*2.1.1. Suprachiasmatic nucleus*

[20].

**2.1. Structure of the circadian system**

Peripheral and central clocks have been discovered in various tissues. One study reported the ubiquity of peripheral clock and its mechanism in both SCN and other cells [24]. Another study reported that cultured SCN cells maintained a firm rhythmic pattern through photoreception, also expressed in many organs, such as the liver, lungs, and kidneys [25]. In addition, numer‐ ous mammalian peripheral tissues exhibit circadian oscillation; consequently, oscillations are suppressed when the SCN is absent [9]. Thus, a delayed feedback loop originally associating the same components is considered to be composed of the rhythm-generating molecular circuitry, which is constructed by both SCN and peripheral cells [16]. Several pivotal physio‐ logical functions are influenced by light–dark oscillations in peripheral organs (**Figure 1**), including the heart, liver, lung, kidney, and skeletal muscles [26].

**Figure 1.** Structure of the circadian system. The retina captures photic information and transmits signals to the SCN. Food and light transmit signals to the peripheral clock.

Local peripheral oscillators can be synchronized by neuronal signals and stimulating hor‐ mones. The SCN sends signals to all body systems, coordinating the feeding–fasting cycles [27, 28]. Although the SCN functions as the master synchronizer of the entire system, food intake can disrupt the control in peripheral clocks. A change in the feeding schedule alters the phase in the central and peripheral clocks in the liver [29]. Moreover, light information is transmitted to the adrenal gland, liver, and pancreas by the SCN, which distributes a rhythmic signal to all tissues of the peripheral organs [30]. The central neural and peripheral tissues maintain the normal neurological and metabolic homeostasis in the sleep–wake cycle [31]. The endogenous mechanism of oscillation in peripheral cells is a gene regulatory network to generate sustained oscillations. A group of genes forms the core network of the mammalian circadian clock, which can function even in the absence of external inputs in individual cells [32]. Numerous signaling pathways participate in the phase entrainment of peripheral clocks and warrant additional studies.

#### **2.2. Circadian clock and diseases**

The circadian clock regulates the sleep–wake cycle, metabolism, hormone secretion, and other physiological processes. A recent study suggested that chronic circadian disruption is deleterious to health and causes myocardial infarction, sudden cardiac death, and aortic aneurysm rupture risk [33]. Circadian dysregulation has extensive consequences not only on glucose control but also on inflammation. REV–ERBα and retinoid-related orphan receptors (RORs), which are downstream proteins of the signaling pathway, can affect adipogenesis and thrombosis through circadian clock regulation. This study indicates that metabolic and circadian pathways, which involve the nuclear receptor superfamily, are associated to the central node [34].

Circadian rhythm and sleep disorders lead to an increased incidence of metabolic syndrome [35]. Chronic sleep curtailment may affect the increase in the prevalence of diabetes and obesity. The effect may change glucose metabolism by reducing energy consumption [36]. Hypothalamic–pituitary–thyroid axis rhythms are endocrine rhythms and are accurately governed by the circadian system [37]. Evidence suggests that a seasonal change in thyroid hormone availability in the hypothalamus, and pituitary gland is a crucial element [38]. Sexdifferentiated circadian timing systems exist in the hypothalamic–pituitary–gonadal (HPG) axis, the hypothalamic–adrenal–pituitary (HPA) axis, and sleep-arousal systems [39]. When various stressors appear, the HPA axis adjusts the circadian rhythms of the peripheral clocks through the glucocorticoid receptor [40]. In addition, the female HPG axis is regulated by a molecular clock in gonadotropin secretion because it relates to the timing of gonadotropin secretion in ovulation and parturition [41]. The circadian timing system and estradiol sensitive neural circuits driving the HPG axis functioning include gonadotropin-releasing hormone secretion and preovulatory luteinizing hormone [42].

Moreover, a strong association exists between the circadian clock and hormone secretion. The human placenta synthesizes the melatonin-regulating circadian system of many tissues, and the unusual secretion adversely affects fetal and maternal health [43]. The circadian system, which is composed of a family of clock genes, also regulates hormonal production and activity [44]. Maternal melatonin is a circadian signal for oscillating the fetal SCN clock. However, maternal melatonin probably cannot control the adrenal gland [45]; therefore, the circadian clock system affects the fetus during embryonic development.

Adequate sleep is crucial for maintaining the function of circadian rhythms. The lack of sleep disrupts circadian rhythms. Impaired endocrine and physiological circadian rhythms affect the quantity of immune cells [46]. Circadian oscillations can mediate cognitive performance through sleep. However, it cannot work correctly in case of the environmental disturbance of the clock, including shift work and schedules [47]. Circadian systems are disrupted through inadequate melatonin secretion, and the altered clock gene expression can cause human metabolic syndrome and cardiovascular diseases [35].

#### **3. Circadian clock gene regulation in aging**

to the adrenal gland, liver, and pancreas by the SCN, which distributes a rhythmic signal to all tissues of the peripheral organs [30]. The central neural and peripheral tissues maintain the normal neurological and metabolic homeostasis in the sleep–wake cycle [31]. The endogenous mechanism of oscillation in peripheral cells is a gene regulatory network to generate sustained oscillations. A group of genes forms the core network of the mammalian circadian clock, which can function even in the absence of external inputs in individual cells [32]. Numerous signaling pathways participate in the phase entrainment of peripheral clocks and warrant additional

The circadian clock regulates the sleep–wake cycle, metabolism, hormone secretion, and other physiological processes. A recent study suggested that chronic circadian disruption is deleterious to health and causes myocardial infarction, sudden cardiac death, and aortic aneurysm rupture risk [33]. Circadian dysregulation has extensive consequences not only on glucose control but also on inflammation. REV–ERBα and retinoid-related orphan receptors (RORs), which are downstream proteins of the signaling pathway, can affect adipogenesis and thrombosis through circadian clock regulation. This study indicates that metabolic and circadian pathways, which involve the nuclear receptor superfamily, are associated to the

Circadian rhythm and sleep disorders lead to an increased incidence of metabolic syndrome [35]. Chronic sleep curtailment may affect the increase in the prevalence of diabetes and obesity. The effect may change glucose metabolism by reducing energy consumption [36]. Hypothalamic–pituitary–thyroid axis rhythms are endocrine rhythms and are accurately governed by the circadian system [37]. Evidence suggests that a seasonal change in thyroid hormone availability in the hypothalamus, and pituitary gland is a crucial element [38]. Sexdifferentiated circadian timing systems exist in the hypothalamic–pituitary–gonadal (HPG) axis, the hypothalamic–adrenal–pituitary (HPA) axis, and sleep-arousal systems [39]. When various stressors appear, the HPA axis adjusts the circadian rhythms of the peripheral clocks through the glucocorticoid receptor [40]. In addition, the female HPG axis is regulated by a molecular clock in gonadotropin secretion because it relates to the timing of gonadotropin secretion in ovulation and parturition [41]. The circadian timing system and estradiol sensitive neural circuits driving the HPG axis functioning include gonadotropin-releasing hormone

Moreover, a strong association exists between the circadian clock and hormone secretion. The human placenta synthesizes the melatonin-regulating circadian system of many tissues, and the unusual secretion adversely affects fetal and maternal health [43]. The circadian system, which is composed of a family of clock genes, also regulates hormonal production and activity [44]. Maternal melatonin is a circadian signal for oscillating the fetal SCN clock. However, maternal melatonin probably cannot control the adrenal gland [45]; therefore, the circadian

Adequate sleep is crucial for maintaining the function of circadian rhythms. The lack of sleep disrupts circadian rhythms. Impaired endocrine and physiological circadian rhythms affect

studies.

central node [34].

**2.2. Circadian clock and diseases**

16 Molecular Mechanisms of the Aging Process and Rejuvenation

secretion and preovulatory luteinizing hormone [42].

clock system affects the fetus during embryonic development.

Circadian oscillations regulate transcription by using a set of genes, thus establishing an autoregulatory feedback loop. A study reported that circadian gene expression is widespread through the body [19]. Animal studies have revealed that a disrupted circadian clock function accelerated the development of aging phenotypes. The temporal precision of circadian system is lost with advancing age. Growing evidence indicates that aging is affected by circadian clocks. Circadian clocks can change gene expression, physiological functions, and daily cycles. The circadian system leads to various age-related pathologies and exhibits a weak precision with advancing age. The aged SCN shows changes in the expression of vasoactive intestinal polypeptide [48]. Circadian rhythms of the electrical activity are decreased [49]. When a young SCN is transplanted into aged animals, the behavioral rhythms functioning in locomotion is improved [50]. These studies indicate that the SCN is crucial to improve the age-related circadian system, which may be deteriorated in aged individuals.

**Figure 2.** Circadian clock gene regulation in aging.

Bmal1 and clock activate the expression of period (Per), cryptochrome (Cry), RORα, and REV– ERBα. The negative elements of the clock PER and CRY proteins, which are associated with Clock–Bmal1 at E-box sites, enter the nucleus. REV–ERBα competes with each other for their binding DNA in the Bmal1 promoter, and the expression of Bmal1 is repressed.

#### **3.1. Effects of Bmal1 genes on the aging process**

Bmal1 belongs to the family of the basic helix–loop–helix (bHLH)–PAS domain containing transcription factors [51]. Bmal1 is a transcriptional factor and major component of the circadian clock; it plays a critical role in accelerating aging and the development of age-related pathologies. Bmal1 as the core clock gene regulates the expression of other circadian clock genes, which affects the physiological circadian rhythm [52]. Bmal1 deficiency has been shown to significantly shorten lifespan and accelerate senescence. *Bmal1*-/- mice have a shorter lifespan and exhibit cataracts, organ shrinkage, and other symptoms of premature aging [53].

Bmal1 may be directly associated with premature aging and reduced lifespan. Recent studies have reported a significantly weakened function of circadian rhythms in aged animals. The disturbances in circadian rhythms may cause thrombosis, a critical result of age-related cardiovascular diseases. Genetic ablation of the gene Bmal1 gene in mice significantly elevates the circulating von Willebrand factor, fibrinogen, and PAI-1 [54]. Bmal1 regulates BDH1 and PIK3R1; thus, Bmal1 affects metabolism, cell signaling, and the contractile function of the heart. Bmal1 predicts impairments in ketone body metabolism and depressing glucose utilization in the hearts of cardiomyocyte-specific Bmal1-knockout mice [55].

Bmal1 is crucial for regulating oxidative stress. Oxidative stress maintains reactive oxygen species (ROS) homeostasis and senescence through the hypoxia-inducible factor-mediated pathway. *Bmal1*-/- cells do not induce replicative senescence but rather stress-induced senes‐ cence. Stress-induced senescence causes cell and organ aging [56]. A recent study revealed a significantly decreased rhythmic function in aged animals. The study reported that the activity of the mammalian target of rapamycin complex 1 pathway is controlled by the circadian clock through Bmal1-dependent mechanisms. This regulation evidenced an association between aging and metabolism [57]. Bmal1 in the heart also determines the pathological consequences of the chronic disruption of the circadian clock. *Bmal1*-/- mice develop dilated cardiomyopathy with aging and decreased cardiac performance with changes in titin [58]. In addition, Bmal1 controls the blood coagulation pathway by changing platelet numbers and altering the vascular function. This control results from arterial and venous thrombogenicity by attenuat‐ ing nitric oxide and anticoagulant factor synthesis [59].

#### **3.2. Effects of Clock on the aging process**

Clock is a circadian clock protein similar to Bmal1 and is a transcription factor of the circadian system. Bmal1 and Clock have different functions in modulating the biological function. The deficiency of Clock does not induce any significant age-related changes in organs and tissues; however, they are affected in *Bmal1*-/- mice [60]. The results of Clock and Bmal1 deficiency in physiology are different. Clock encodes a novel bHLH–PAS domain protein of transcription factors [61, 62]. Clock is a unique gene with various features, including DNA binding, protein dimerization, and domain activation [62]. Clock is widely recognized to have circadian functions. The deficiency of the Clock protein significantly affects longevity. The aging of Clock-deficient mice results in a higher rate of pathologies, cataracts, and dermatitis than that of wild-type mice [60].

When Clock is a mutant gene, the pattern of circadian gene expression loses circadian behavior and is disrupted [61, 62]. While Clock in mice is knocked out, the robust circadian rhythms in spontaneous activity are still expressed [63]. Bmal1 forms a heterodimer as the transcription‐ ally active complex to drive circadian rhythmicity in Clock-deficient animals [64]. In addition, neuronal PAS domain protein 2 functionally replaces the Clock activity in Clock-deficient mice [65]. Age-related circadian changes are caused by both wild-type and heterozygous Clock mutants [66]. Clock–Bmal1 complex activate the transcription of other genes with E-box elements in their promoters [67]. The dimers translocate to the nucleus and obstruct the Clock– Bmal1 complex, thereby hindering the additional transcription of the other genes [68] and ultimately leading Clock and Bmal1 to increase Per transcription and restart the cycle [69]. Clock, as the core component of circadian transcription, operates in complex with another protein. Clock deficiency was shown to cause age-related cataracts [60]. Clock-deficient mice lose body weight, relative organ weight, or ectopic calcification during aging. These mice maintain behavioral rhythms, indicating that peripheral circadian oscillators require Clock [70].

#### **3.3. Effects of the Clock–Bmal1 complex on the aging process**

**3.1. Effects of Bmal1 genes on the aging process**

18 Molecular Mechanisms of the Aging Process and Rejuvenation

Bmal1 belongs to the family of the basic helix–loop–helix (bHLH)–PAS domain containing transcription factors [51]. Bmal1 is a transcriptional factor and major component of the circadian clock; it plays a critical role in accelerating aging and the development of age-related pathologies. Bmal1 as the core clock gene regulates the expression of other circadian clock genes, which affects the physiological circadian rhythm [52]. Bmal1 deficiency has been shown to significantly shorten lifespan and accelerate senescence. *Bmal1*-/- mice have a shorter lifespan

Bmal1 may be directly associated with premature aging and reduced lifespan. Recent studies have reported a significantly weakened function of circadian rhythms in aged animals. The disturbances in circadian rhythms may cause thrombosis, a critical result of age-related cardiovascular diseases. Genetic ablation of the gene Bmal1 gene in mice significantly elevates the circulating von Willebrand factor, fibrinogen, and PAI-1 [54]. Bmal1 regulates BDH1 and PIK3R1; thus, Bmal1 affects metabolism, cell signaling, and the contractile function of the heart. Bmal1 predicts impairments in ketone body metabolism and depressing glucose utilization in

Bmal1 is crucial for regulating oxidative stress. Oxidative stress maintains reactive oxygen species (ROS) homeostasis and senescence through the hypoxia-inducible factor-mediated pathway. *Bmal1*-/- cells do not induce replicative senescence but rather stress-induced senes‐ cence. Stress-induced senescence causes cell and organ aging [56]. A recent study revealed a significantly decreased rhythmic function in aged animals. The study reported that the activity of the mammalian target of rapamycin complex 1 pathway is controlled by the circadian clock through Bmal1-dependent mechanisms. This regulation evidenced an association between aging and metabolism [57]. Bmal1 in the heart also determines the pathological consequences of the chronic disruption of the circadian clock. *Bmal1*-/- mice develop dilated cardiomyopathy with aging and decreased cardiac performance with changes in titin [58]. In addition, Bmal1 controls the blood coagulation pathway by changing platelet numbers and altering the vascular function. This control results from arterial and venous thrombogenicity by attenuat‐

Clock is a circadian clock protein similar to Bmal1 and is a transcription factor of the circadian system. Bmal1 and Clock have different functions in modulating the biological function. The deficiency of Clock does not induce any significant age-related changes in organs and tissues; however, they are affected in *Bmal1*-/- mice [60]. The results of Clock and Bmal1 deficiency in physiology are different. Clock encodes a novel bHLH–PAS domain protein of transcription factors [61, 62]. Clock is a unique gene with various features, including DNA binding, protein dimerization, and domain activation [62]. Clock is widely recognized to have circadian functions. The deficiency of the Clock protein significantly affects longevity. The aging of Clock-deficient mice results in a higher rate of pathologies, cataracts, and dermatitis than that

and exhibit cataracts, organ shrinkage, and other symptoms of premature aging [53].

the hearts of cardiomyocyte-specific Bmal1-knockout mice [55].

ing nitric oxide and anticoagulant factor synthesis [59].

**3.2. Effects of Clock on the aging process**

of wild-type mice [60].

In animals, a transcription–translation loop that revolves around the transcription factors Clock and Bmal1 forms circadian oscillators. The core positive element of the clock is Clock– Bmal1, which is the heterodimeric transcription factor output of circadian locomotor cycles [71]. The negative elements of the clock PER and CRY proteins, associated with Clock–Bmal1 at E-box sites, enter the nucleus [64, 72] (**Figure 2**). The transcriptional activity of Clock–Bmal1 is suppressed by recruiting the SIN3–histone deacetylase (HDAC) complex and preventing transcriptional termination [72, 73]. Histone acetyltransferases (HATs) and HDACs generate rhythms in histone acetylation and the circadian rhythms [74]. Mixed-lineage leukemia protein-1, a H3K4 methyltransferase, has a nonredundant role in the circadian oscillator [75]. The Jumonji C domain-containing H3K4me3 demethylase family and AT-rich interaction domain-containing histone lysine demethylase 1a form a Clock–Bmal1 complex; the complex is then recruited to the Per2 promotor, simultaneously enhancing transcription by Clock– Bmal1 [76]. The transcription factors Clock and Bmal1, which form heterodimers and bind to an E-box enhancer element, regulate gene transcription [77, 78]. Bmal1 and Clock activate the genes Per and Cry. Once a certain concentration of PER and CRY proteins accumulate, they form BMAL1–CLOCK complex, consequently inhibiting gene transcription (**Figure 2**) [79, 80].

Clock–Bmal1 is not simply a passive consequence of negative feedback protein but rather induces proactive regulation [81]. As the core component of molecular clock, the Clock–Bmal1 complex controls numerous clock genes [82]. This complex may activate as well as repress transcription, and the switch depends on the interaction of Clock–Bmal1 with Cry [83]. The Clock–Bmal1-dependent recruitment of HATs promotes the periodic disruption of Clock– Bmal1 [84]. Clock–Bmal1 as a signaling molecule resets the clock through the Ca2+-dependent protein kinase C pathway [85, 86]. The cAMP response element-binding protein activates the Clock–Bmal1 complex that rapidly resets phase and mediates the acute transactivation of Clock–Bmal1, consequently inducing immediate-early Per1 transcription [87]. Clock–Bmal1 DNA binding promotes rhythmic chromatin opening and remodeling. It mediates the rhythmic transcription factors binding to Clock–Bmal1 and the transcriptional output, suggesting that Clock–Bmal1 drives rhythmic gene expression and biological functions [88].

#### **3.4. Effects of PER and CRY on the aging process**

The Per and Cry genes can combine with the E-box domain promoters using Clock and Bmal1, thus driving the transcription of messenger RNA [79, 80]. Per and Cry genes play a negative transcriptional feedback loop in mouse [89]. When PER and CRY proteins reach adequately high levels, they form dimer feedback to the nucleus. The Clock–Bmal1 complex is then binded to turn off transcription [4]. Regulatory kinases Case in Kinase I epsilon (CKIε) of rodents phosphorylates PER and degrades it to feed back to the cell nucleus [90, 91]. CKIε masks the mPER1 nuclear localization signal, and mPer2 causes mPer1–mPer2 heterodimer formation in the cytoplasm. Phosphorylation-dependent cytoplasmic retention may be the reason for CKIε regulating the mammalian circadian rhythm [91]. In aged animals, Per1 transcription is induced by light and reduced with a significantly longer delay to resynchronization [92], whereas in young animals, the disruption of the Per genes results in insensitivity to light [89, 93].

Advancing age reduces retinal sensitivity, which causes various age-related diseases. In elderly people, homeostatic sleep is association with the circadian clock gene Per3 in coding regions. The Per3 gene associates with a phase advance in the melatonin profile; therefore, elderly people experience more nocturnal wakefulness [5]. In situ hybridization for Per2 mRNA revealed that the age-related decrease in the diurnal rhythm amplitude in the hippo‐ campus may aggravate cognitive deficits [94]. Pattern differences in clock gene expression can be associated with a depressive state. Under abnormal light–dark conditions, Per1 and Per2 genes may result in a depressive state [95]. The expression of rPer1, rPer2, or rCry1 mRNA is similar in both young and old SCN; however, when stimulated by light, aging reduces the gene expression [89]. A decreased Per gene expression suggests an impaired clock regulatory network and stress defense pathways may accelerate aging [96].

#### **3.5. Effects of the SIRT1 gene on the aging process**

Sirtuins belong to the silent information regulator (SIR)2 family of proteins [97, 98]. SIR2 and its orthologs regulate senescence in yeast, worms, and flies [99–101]. SIR2 retards senescence and extends the lifespan of diverse species through caloric restriction [101, 102]. Sirtuins are nicotinamide adenine dinucleotide (NAD+ )-dependent deacylases, which promote cell survival by suppressing apoptosis or senescence [103]. Sirtuins play key roles in expediting the resistance by increasing antioxidant pathways and facilitating DNA damage repair [104]. Seven mammalian sirtuins exist, and SIRT1 is one of the most crucial mammalian SIR2 orthologs [98]. SIRT1 is involved in cellular metabolism and circadian core clockwork machi‐ nery in biological systems [105]. The direct deacetylation activity and NAD+ salvage pathway were found to correlate with SIRT1 in the circadian rhythm system [106]. In addition, SIRT1 regulates the Clock–Bmal1 complex, and deacetylates and degrades Per2 [79]. The SIRT1 activity contributes to disturbances in the acetylation of H3 and Bmal1 and transduces cellular metabolic signals [105]. Moreover, SIRT1 has multiple downstream targets, including p53, Ku70, nuclear factor-kappaB, forkhead box O factors, peroxisome proliferator-activated receptor (PPAR) gamma coactivator 1-alpha (PCG-1α), PPARγ [98].

The central pacemaker is activated by SIRT1 to maintain robust circadian, and its decaying may accelerate aging [107]. SIRT1 can produce moderate changes in the intrinsic circadian period and is associated with age-related decline in the central clock. Some studies have reported a possible association between aging, sirtuins, and clock genes. SIRT1 activates the transcription of Bmal1 through PGC-1α to increase the amplitude of expression of BMAL1 and other proteins to control circadian rhythms in the SCN [108]. Recent studies have suggested that caloric restriction is a nongenetic manipulation that extends the lifespan; it is beneficial because SIRT1 has led to the search for sirtuin activators [109]. SIRT1 avoids mice getting dietinduced obesity by deacetylating and activating PGC-1α in the skeletal muscles [110]. In addition, the activity of the adipogenic nuclear receptor PPARγ is repressed by SIRT1 activation, thus reducing fat accumulation and adipocyte differentiation [98]. Moreover, SIRT1 mediates the lifespan extension through caloric restriction, affecting metabolism and lifespan in humans.

#### **3.6. Effects of the ROR gene on the aging process**

DNA binding promotes rhythmic chromatin opening and remodeling. It mediates the rhythmic transcription factors binding to Clock–Bmal1 and the transcriptional output, suggesting that Clock–Bmal1 drives rhythmic gene expression and biological functions [88].

The Per and Cry genes can combine with the E-box domain promoters using Clock and Bmal1, thus driving the transcription of messenger RNA [79, 80]. Per and Cry genes play a negative transcriptional feedback loop in mouse [89]. When PER and CRY proteins reach adequately high levels, they form dimer feedback to the nucleus. The Clock–Bmal1 complex is then binded to turn off transcription [4]. Regulatory kinases Case in Kinase I epsilon (CKIε) of rodents phosphorylates PER and degrades it to feed back to the cell nucleus [90, 91]. CKIε masks the mPER1 nuclear localization signal, and mPer2 causes mPer1–mPer2 heterodimer formation in the cytoplasm. Phosphorylation-dependent cytoplasmic retention may be the reason for CKIε regulating the mammalian circadian rhythm [91]. In aged animals, Per1 transcription is induced by light and reduced with a significantly longer delay to resynchronization [92], whereas in young animals, the disruption of the Per genes results in insensitivity to light [89,

Advancing age reduces retinal sensitivity, which causes various age-related diseases. In elderly people, homeostatic sleep is association with the circadian clock gene Per3 in coding regions. The Per3 gene associates with a phase advance in the melatonin profile; therefore, elderly people experience more nocturnal wakefulness [5]. In situ hybridization for Per2 mRNA revealed that the age-related decrease in the diurnal rhythm amplitude in the hippo‐ campus may aggravate cognitive deficits [94]. Pattern differences in clock gene expression can be associated with a depressive state. Under abnormal light–dark conditions, Per1 and Per2 genes may result in a depressive state [95]. The expression of rPer1, rPer2, or rCry1 mRNA is similar in both young and old SCN; however, when stimulated by light, aging reduces the gene expression [89]. A decreased Per gene expression suggests an impaired clock regulatory

Sirtuins belong to the silent information regulator (SIR)2 family of proteins [97, 98]. SIR2 and its orthologs regulate senescence in yeast, worms, and flies [99–101]. SIR2 retards senescence and extends the lifespan of diverse species through caloric restriction [101, 102]. Sirtuins are

survival by suppressing apoptosis or senescence [103]. Sirtuins play key roles in expediting the resistance by increasing antioxidant pathways and facilitating DNA damage repair [104]. Seven mammalian sirtuins exist, and SIRT1 is one of the most crucial mammalian SIR2 orthologs [98]. SIRT1 is involved in cellular metabolism and circadian core clockwork machi‐

were found to correlate with SIRT1 in the circadian rhythm system [106]. In addition, SIRT1 regulates the Clock–Bmal1 complex, and deacetylates and degrades Per2 [79]. The SIRT1 activity contributes to disturbances in the acetylation of H3 and Bmal1 and transduces cellular

nery in biological systems [105]. The direct deacetylation activity and NAD+

)-dependent deacylases, which promote cell

salvage pathway

network and stress defense pathways may accelerate aging [96].

**3.5. Effects of the SIRT1 gene on the aging process**

nicotinamide adenine dinucleotide (NAD+

**3.4. Effects of PER and CRY on the aging process**

20 Molecular Mechanisms of the Aging Process and Rejuvenation

93].

RORs are considered a core clock machinery because they regulate the cyclic expression of Bmal1 and Clock, thus providing an essential link between the positive and negative loops of the circadian clock [111]. RORs can be classified into three types: RORα, RORβ, and RORγ. RORα is the first member of the ROR subfamily and resembles the retinoic acid receptors and RXRs [112]. Both RORα and RORβ are required for the maturation of photoreceptors in the retina, and RORγ affects the development of several secondary lymphoid tissues [113].

RORα, an orphan nuclear receptor, plays a crucial role in integrating the circadian clock and regulating the cardiovascular function. Four RORα isoforms exist in humans, namely RORα1– 4, whereas only two isoforms α1 and α4 are present in mouse [114]. These isoforms participate in the different physiological processes and exhibit a distinct pattern of tissue-specific expres‐ sion. For example, the expression of RORα1 and RORα4 was significantly higher in mouse cerebellum than in other tissues, whereas RORα4 is predominantly expressed in liver tissues [115]. RORα and REV–ERBα compete for the binding of their shared DNA-binding elements in the Bmal1 promoter (**Figure 2**) [116, 117]. RORα displays rhythmic expression patterns during the circadian cycle in some tissues. RORα expression shows a weak circadian oscillation in the liver, kidneys, retina, and lungs [114, 118, 119]. Moreover, RORα promotes Bmal1 transcription [116] through two RORα autonomous response elements [118]. RORα1 enhances the circadian amplitude of Bmal1 mRNA expression and regulates the downstream clock genes after serum shock [118]. In addition, the reduced expression of RORα is closely associated with aging in genetic hypertensive rats [120]. The enhancement of the RORα expression was shown to antiaging in human endothelial cells by facilitating Bmal1 transcription, [121]. The defi‐ ciency of RORα causes cerebellar hypoplasia and affects Purkinje cell survival and differen‐ tiation in aging [122]. RORα has been shown to restraint age-related degenerative, including muscular atrophy, immune deficiencies, osteoporosis, atherosclerosis, and inflammation [123, 124]. RORα plays a neuroprotective role during development and provides protection against this injury [124].

#### **3.7. Effects of the REV–ERBα gene on the aging process**

REV–ERB nuclear receptors are considered to be essential core clock components and serve as pivotal regulators of rhythmic metabolism [125]. REV–ERB has α and β isoforms, which coexpression in adipose tissues and the liver and brain [126, 127]. REV–ERBα, an orphan nuclear receptor, negatively regulates the activity of the Clock–Bmal1 complex. Its transcrip‐ tion is controlled through Per and Cry transcription [128]. REV–ERBα competes with activators of the ROR family of nuclear receptors for binding to specific ROR-responsive elements to ensure the rhythmic transcription of Bmal1 and Clock [118, 129]. The development of metabolic disorders indicates disrupted circadian rhythms [111]. REV–ERBA is associated with circadian rhythms and metabolism and is pivotal in regulating the core mammalian molecular clock (**Figure 2**). Furthermore, REV–ERBα controls the transcription of metabolic pathways while serving as a crucial output regulator.

Age is the essential risk factor for metabolic syndrome. REV–ERBα connects the core clock and numerous physiological processes. The deficiency of REV–ERBα results in asynchronous circadian rhythms, with a tendency for diet-induced obesity, impaired glucose, and lipid utilization leading to an increased risk of diabetes [130]. Evidence suggests that REV–ERB has opened new avenues for treating metabolic syndrome by affecting the circadian rhythm and metabolism [111]: for example, targeting REV–ERBα as a critical component of the peripheral clock to treat bipolar disorder by affecting glycogen synthase kinase 3 [131]. REV–ERB agonists can reduce dyslipidemia, hyperglycemia syndromes, and obesity [130, 132].

#### **4. Drug discovery**

New biological targets are being developed for discovering novel drugs. People can identify with aging, thus antiaging drugs constitute a lucrative market. Medical therapy contributes in delaying the aging process in humans. Researchers focus not only on the compounds having a validated target but also on herbs used as antiaging products in traditional medicine. Some drugs used to treat chronic diseases can be examined; these diseases are associated with senescence, such as cardiovascular diseases, type 2 diabetes mellitus, and cancer. Some studies on the compounds have been conducted, whereas studies on herbs are necessary. Many lead compounds and approved drugs are derived from herbs. In China, traditional Chinese medicine (TCM) has been investigated in large-scale clinical trials, with extensive research on aging concerns. With the development of longevity drugs, Chinese herbs have been demon‐ strated to retard aging, such as *Polygonum multiflorum*, Ginseng. Meanwhile, TCM is gradually attracting worldwide attention.

#### **4.1. Compounds as antiaging drugs**

#### *4.1.1. Rapamycin*

124]. RORα plays a neuroprotective role during development and provides protection against

REV–ERB nuclear receptors are considered to be essential core clock components and serve as pivotal regulators of rhythmic metabolism [125]. REV–ERB has α and β isoforms, which coexpression in adipose tissues and the liver and brain [126, 127]. REV–ERBα, an orphan nuclear receptor, negatively regulates the activity of the Clock–Bmal1 complex. Its transcrip‐ tion is controlled through Per and Cry transcription [128]. REV–ERBα competes with activators of the ROR family of nuclear receptors for binding to specific ROR-responsive elements to ensure the rhythmic transcription of Bmal1 and Clock [118, 129]. The development of metabolic disorders indicates disrupted circadian rhythms [111]. REV–ERBA is associated with circadian rhythms and metabolism and is pivotal in regulating the core mammalian molecular clock (**Figure 2**). Furthermore, REV–ERBα controls the transcription of metabolic pathways while

Age is the essential risk factor for metabolic syndrome. REV–ERBα connects the core clock and numerous physiological processes. The deficiency of REV–ERBα results in asynchronous circadian rhythms, with a tendency for diet-induced obesity, impaired glucose, and lipid utilization leading to an increased risk of diabetes [130]. Evidence suggests that REV–ERB has opened new avenues for treating metabolic syndrome by affecting the circadian rhythm and metabolism [111]: for example, targeting REV–ERBα as a critical component of the peripheral clock to treat bipolar disorder by affecting glycogen synthase kinase 3 [131]. REV–ERB agonists

New biological targets are being developed for discovering novel drugs. People can identify with aging, thus antiaging drugs constitute a lucrative market. Medical therapy contributes in delaying the aging process in humans. Researchers focus not only on the compounds having a validated target but also on herbs used as antiaging products in traditional medicine. Some drugs used to treat chronic diseases can be examined; these diseases are associated with senescence, such as cardiovascular diseases, type 2 diabetes mellitus, and cancer. Some studies on the compounds have been conducted, whereas studies on herbs are necessary. Many lead compounds and approved drugs are derived from herbs. In China, traditional Chinese medicine (TCM) has been investigated in large-scale clinical trials, with extensive research on aging concerns. With the development of longevity drugs, Chinese herbs have been demon‐ strated to retard aging, such as *Polygonum multiflorum*, Ginseng. Meanwhile, TCM is gradually

can reduce dyslipidemia, hyperglycemia syndromes, and obesity [130, 132].

**3.7. Effects of the REV–ERBα gene on the aging process**

22 Molecular Mechanisms of the Aging Process and Rejuvenation

serving as a crucial output regulator.

**4. Drug discovery**

attracting worldwide attention.

this injury [124].

Rapamycin, used as an immunosuppressive drug, has been recently considered for antiaging treatments. Rapamycin retards multiple aspects of aging, including extending the lifespan and slowing aging in mice [133]. Rapamycin evidences an association between metabolism and longevity by controlling the target of rapamycin kinases to regulate cell growth and prolifer‐ ation through mitogenic signals [134]. In addition, rapamycin extends mouse lifespan princi‐ pally by inhibiting many aspects of cancer development [135]. Rapamycin appears to retard the effects of age on the liver, endometrium [133], and bone marrow [136]. Rapamycin was shown to reduce the effects of aging in mice [137]; it can retard clinically relevant β-amyloid and tau accumulation in aged CNS tissues [138].

#### *4.1.2. Resveratrol*

Resveratrol, a natural phenol, retards aging by selective SIRT1 activation. Resveratrol as an established antioxidant has multiple beneficial activities and delays the onset of age-associated diseases. Resveratrol can reduce the expression of SIRT1 by using an acetylated substrate and NAD+ [109]. In yeast, resveratrol extends the lifespan by upregulating Sir2 or increasing DNA stability [99]. Resveratrol has antioxidant, antiaging, and antiangiogenic properties. Further‐ more, resveratrol plays a dual role on the vasculature; it maintains vascular fitness through its anti-inflammatory and anticoagulant activities, whereas it inhibits angiogenesis to suppress tumor growth [139].

#### **4.2. Herbs as antiaging drugs**

#### *4.2.1. Polygonum multiflorumygonum multiflorum*

*Polygonum multiflorum*, a crucial TCM, is extensively used for health maintenance and disease treatment, particularly as an antiaging drug in China. An active component extracted from the roots of P. *multiflorum* Thunb is called 2,3,5,4'-tetrahydroxystilbene-2-O-β-d-glucoside (THSG). THSG, the major bioactive compound of *P. multiflorum*, has antioxidant [140], anti-inflamma‐ tory [141], and endothelial-protective activities [142]. Our previous study revealed that THSG prevents vascular senescence by increasing eNOS expression and SIRT1 activity and reducing the acetylation of p53 at the K373 site [143]. In another study, we indicated that THSG extends the lifespan by promoting the expression of the longevity gene Klotho [144]. The antioxidant capacity of THSG is similar to resveratrol or even stronger. Moreover, THSG possesses a potent antioxidant capacity and extends the lifetime of the nematode *Caenorhabditis elegans* [145]. THSG is a potent antiaging drug and has potential as a pharmaceutical antiaging drug.

#### *4.2.2. Ginseng*

Ginseng is a highly valued herb in Asia and is recorded in Chinese Pharmacopoeia. The major active components of ginseng are ginsenosides. The beneficial effects of ginseng have been investigated; ginseng and its constituents have antianemic, antiaging, antioxidant, antineo‐ plastic, and anti-stress activities. Rg1 and Rb1 improve the spatial learning ability by increasing the hippocampal synaptic density [109, 110]. Ginseng could play a crucial role in enhancing the defense system during the aging process. The mechanisms of ginsenosides warrant further investigation.

#### **5. Conclusion**

Considerable progress has been made to unravel the association between the circadian clock and aging. The role of circadian clocks in governing many other physiological systems has been reported; however, sufficient data are lacking. The circadian clock represents a nearly ubiquitous aspect of cellular regulation and molecular regulatory process, which exerts effects on organismal behavior. The researches on circadian clock genes regulation in aging have emerged recently, making the issue become attractive and outstanding.

The association between the circadian clock genes and aging might affect age-related diseases. The circadian clock is altered in normal aging, resulting in a decreased amplitude of rhythms in daily life, subsequently posing difficulties for humans to adjust to temporal changes. The study has major implications in determining why circadian rhythms diminish during aging and whether this decline could be reversed to tissue homeostasis and age-related diseases. However, additional studies are required to comprehensively understand the circadian clock effects on aging and obtain the target drug for extending the lifespan. Circadian genes must be extensively studied, including their different signaling pathways that are unknown, particularly the association between circadian genes and diseases. Focus on the effects of gene expression, translation, and signaling alterations on the biological clock would increase our knowledge regarding individualized treatment program and drug discovery.

Till date, studies have revealed several circadian clock genes that are crucial in regulating the lifespan. The manipulation of circadian clock genes would largely benefit the study of aging. This study not only explains circadian rhythm affects aging but also presents drugs for determining the therapeutic potential of targeting the circadian clock in diseases.

#### **Author details**

Yufeng Li, Yanqi Dang, Shuang Ling and Jin-Wen Xu\*

\*Address all correspondence to: jinwen.xu88@gmail.com

Murad Research Institute for Modernized Chinese Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai, China

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Considerable progress has been made to unravel the association between the circadian clock and aging. The role of circadian clocks in governing many other physiological systems has been reported; however, sufficient data are lacking. The circadian clock represents a nearly ubiquitous aspect of cellular regulation and molecular regulatory process, which exerts effects on organismal behavior. The researches on circadian clock genes regulation in aging have

The association between the circadian clock genes and aging might affect age-related diseases. The circadian clock is altered in normal aging, resulting in a decreased amplitude of rhythms in daily life, subsequently posing difficulties for humans to adjust to temporal changes. The study has major implications in determining why circadian rhythms diminish during aging and whether this decline could be reversed to tissue homeostasis and age-related diseases. However, additional studies are required to comprehensively understand the circadian clock effects on aging and obtain the target drug for extending the lifespan. Circadian genes must be extensively studied, including their different signaling pathways that are unknown, particularly the association between circadian genes and diseases. Focus on the effects of gene expression, translation, and signaling alterations on the biological clock would increase our

Till date, studies have revealed several circadian clock genes that are crucial in regulating the lifespan. The manipulation of circadian clock genes would largely benefit the study of aging. This study not only explains circadian rhythm affects aging but also presents drugs for

emerged recently, making the issue become attractive and outstanding.

knowledge regarding individualized treatment program and drug discovery.

determining the therapeutic potential of targeting the circadian clock in diseases.

Murad Research Institute for Modernized Chinese Medicine, Shanghai University of

Yufeng Li, Yanqi Dang, Shuang Ling and Jin-Wen Xu\*

Traditional Chinese Medicine, Shanghai, China

\*Address all correspondence to: jinwen.xu88@gmail.com

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24 Molecular Mechanisms of the Aging Process and Rejuvenation

**5. Conclusion**

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## **Ion Channels in Aging and Aging-Related Diseases**

Vidhya Rao, Simon Kaja and Saverio Gentile

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/63951

#### **Abstract**

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Aging in humans is the decline over function of time of biological processes that include the capacity to grow, to reproduce, to interact, and to adapt, resulting in progressive organ malfunctions, illnesses, and ultimately death. As the average life expectancy is estimat‐ ed to be above 60 years for about 25% of the world's population by 2050, understanding the causes of and designing treatments for aging-related disease is a compelling priority. Although every organ and tissue undergoes the process of aging, it appears that only few pathogeneses are typically detected with high frequency in elderly individuals. These include cardiovascular disease, neurodegeneration, vision loss, and cancer. Therefore, aging could be measured by monitoring the occurrence and progression of these diseases. However, each of these medical conditions alone is not a good marker for aging as elder patients present comorbid chronic conditions. In addition, treatment of one disease does not significantly prolong life expectancy. Therefore, it appears that a possible antiaging therapeutic strategy should consider simultaneous treatment of several diseases or move toward identification of a common target among the biological processes involved in aging. In this chapter, we will discuss some of the basic concepts of the role of ion channels in aging and will present an overview of the function of ion channels in some of the most common aging-related diseases.

**Keywords:** ion channels, aging, aging-related diseases, sinoatrial node, neurodegener‐ ation, glaucoma, cancer

#### **1. Introduction**

Ion channels are pore membrane-associated proteins that allow movement of ionic fluxes between intracellular and extracellular fluids and within intracellular compartments. About

© 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.

1.5% of the human genome encodes more than 400 ion channels. In addition, heteromeriza‐ tion and alternative splicing further increase functional diversity of ion channels [1–3].

Most ion channels are selective as they can discriminate between electrical charge and size of ions and they can allow unidirectional movement of only a specific ion (mostly K+ , Na+ , Ca2+, or Cl<sup>−</sup> ) through the pore. Changes of ionic fluxes and, consequently, variations of electrical charge across membranes control virtually every cellular event including contraction, neuro‐ nal conductance, secretion, proliferation, and cell death. Therefore, abnormal changes of ionic gradients can underlie age-dependent decline of physiological functions. In addition, mal‐ function of ion channels is often associated with organ failure [4] during the process of aging.

In a resting state, ATP-driven pumps generate and maintain ionic gradients resulting in high intracellular K+ (the most abundant intracellular ion) and low Na+ and Ca2+. In addition, the cytosolic surface membrane of all living cells is characterized by the accumulation of a net negative electrical charge than the extracellular surface of the membrane (membrane potential) that can range from −20 mV in epithelial nonexcitable cells to −100 mV in neurons. Because of this separation of electric charge and ionic concentrations across the membrane, opening of a Na+ ion channel produces a flow of a positive charge (current, *i*) caused by the movement of Na+ ions from the extracellular environment to the cytosol (Na+ current, *i*Na+ ). The augmented concentration of positive charges in the cytosol decreases the net negative charge with a process called depolarization. In plants, depolarization is achieved by an outward Cl flux.

In neurons and muscle cells, such as cardiac myocytes, depolarization is a critical event that underlies neuronal signaling and contraction. Depolarization activates more Na+ (via a positive feedback), K+ , and Ca2+ channels and the respective ions will cross the membrane according to their electrochemical gradients. Therefore, K+ leaving the cells will counteract depolarization, an event called repolarization. Inactivation of Na+ and Ca2+ channels in combination with the outward fluxes of K+ will produce the falling phase of the action potential which will continue until the ionic balances across the membranes are reestablished (resting phase). Each cell type presents a collection of ion channels that shape amplitude and duration of the action potential differently. For example, in neurons, changes of ionic fluxes occur very rapidly producing action potentials with fast depolarization and repolarization (lasting less than 1 ms) that propagates as unidirectional waves along the axon. Frequencies, duration, and amplitudes of action potentials produce neuronal signaling that guarantees transfer and elaboration of information from different body compartments, external to internal environment, and vice versa [5].

In the heart, action potentials are much slower (400 ms) than in neurons and they mostly serve the purpose to control contraction. This event is regulated by a collaborative effort between surface membrane and intracellular Ca2+ channels (ICCs) (e.g., ryanodine receptors, RyRs) in which the initial small amount of Ca2+ ions entering the cell from the extracellular environment bind and activate RyRs channels located on intracellular Ca2+ stores. This event, called Ca2+ induced Ca2+ release, produces large changes of cytosolic Ca2+ concentration that is used by contracting fibers to produce motion and guarantee a conversion of an electrical stimulus into a mechanical response [6].

#### **2. Ion channels in the aging mosaic of the sinoatrial node (SAN)**

1.5% of the human genome encodes more than 400 ion channels. In addition, heteromeriza‐

Most ion channels are selective as they can discriminate between electrical charge and size of

In a resting state, ATP-driven pumps generate and maintain ionic gradients resulting in high

cytosolic surface membrane of all living cells is characterized by the accumulation of a net negative electrical charge than the extracellular surface of the membrane (membrane potential) that can range from −20 mV in epithelial nonexcitable cells to −100 mV in neurons. Because of this separation of electric charge and ionic concentrations across the membrane, opening of a Na+ ion channel produces a flow of a positive charge (current, *i*) caused by the movement of

concentration of positive charges in the cytosol decreases the net negative charge with a process

In neurons and muscle cells, such as cardiac myocytes, depolarization is a critical event that

their electrochemical gradients. Therefore, K+ leaving the cells will counteract depolarization,

until the ionic balances across the membranes are reestablished (resting phase). Each cell type presents a collection of ion channels that shape amplitude and duration of the action potential differently. For example, in neurons, changes of ionic fluxes occur very rapidly producing action potentials with fast depolarization and repolarization (lasting less than 1 ms) that propagates as unidirectional waves along the axon. Frequencies, duration, and amplitudes of action potentials produce neuronal signaling that guarantees transfer and elaboration of information from different body compartments, external to internal environment, and vice

In the heart, action potentials are much slower (400 ms) than in neurons and they mostly serve the purpose to control contraction. This event is regulated by a collaborative effort between surface membrane and intracellular Ca2+ channels (ICCs) (e.g., ryanodine receptors, RyRs) in which the initial small amount of Ca2+ ions entering the cell from the extracellular environment bind and activate RyRs channels located on intracellular Ca2+ stores. This event, called Ca2+ induced Ca2+ release, produces large changes of cytosolic Ca2+ concentration that is used by contracting fibers to produce motion and guarantee a conversion of an electrical stimulus into

, and Ca2+ channels and the respective ions will cross the membrane according to

will produce the falling phase of the action potential which will continue

) through the pore. Changes of ionic fluxes and, consequently, variations of electrical charge across membranes control virtually every cellular event including contraction, neuro‐ nal conductance, secretion, proliferation, and cell death. Therefore, abnormal changes of ionic gradients can underlie age-dependent decline of physiological functions. In addition, mal‐ function of ion channels is often associated with organ failure [4] during the process of aging.

, Na+

and Ca2+. In addition, the

flux.

and Ca2+ channels in combination with the

). The augmented

(via a positive

, Ca2+,

tion and alternative splicing further increase functional diversity of ion channels [1–3].

ions and they can allow unidirectional movement of only a specific ion (mostly K+

(the most abundant intracellular ion) and low Na+

ions from the extracellular environment to the cytosol (Na+ current, *i*Na+

called depolarization. In plants, depolarization is achieved by an outward Cl-

underlies neuronal signaling and contraction. Depolarization activates more Na+

an event called repolarization. Inactivation of Na+

38 Molecular Mechanisms of the Aging Process and Rejuvenation

or Cl<sup>−</sup>

Na+

feedback), K+

versa [5].

outward fluxes of K+

a mechanical response [6].

intracellular K+

The sinoatrial node is a specialized bundle of neurons that innervate the heart and act as a "pacemaker" by generating electrical impulses at regular intervals that allow the heart to contract rhythmically. Typically, SAN dysfunction (SND) can be related to the use of specific medications (e.g., beta blockers) [7]; however, SAN function is known to decline during aging [8], resulting in pacemaker diseases in senior people. Unfortunately, SND can be corrected only by treating the extrinsic causes. Clinical manifestation of aging-dependent SND can be associated with a dramatic alteration of a series of parameters that can range from an increased action potential duration of the neurons composing the SAN to a reduction of the intrinsic heart rate [8] (IHR; defined as the rate at which the heart contracts without the contribution of the SAN and hormones). These phenomena are directly associated with changes in the activity of several ion channels that fail to control outward and inward ionic fluxes properly in the SAN and cardiac muscle. Interestingly, several study focusing on understanding age-depend‐ ent changes of gene expression revealed that expression of several channels are altered in the SAN and cardiac myocytes of aged animals. Perhaps counterintuitively, it was observed that in aged animals Na+ , Ca2+, and K+ channels increased their expression level in the SAN and atrial muscle [9–12].

It is predictable that an increased expression of Na+ and Ca2+ channels would produce larger Na+ and Ca2+ fluxes. The consequent increased cytosolic concentration of these two ions in neurons could explain the elongation of the aging-dependent action potential duration in the SAN because, for example, more time is required to remove Ca2+. In contrast, increased K+ currents would repolarize the cells faster and therefore increased K+ efflux should accelerate the action potential duration.

Age-dependent changes of ion channel expression can also be specific to cardiac myocytes. For example, a significantly decreased RyR expression level has been found only in atrial cells that may be responsible for the age-dependent reduction of the IHR [9]. Furthermore, recent studies showed that an abnormal cytosolic Ca2+ concentration due to upregulation of the transient receptor potential vanilloid type 2 (TRPV2) calcium channel could cause alteration of posttranslational modification of progerin, which can contribute to the phenotype of premature aging linked to Hutchinson-Gilford progeria syndrome (HGPS) [13, 14].

#### **3. Ion channels in the aging of the nervous system**

The human nervous system is composed of two parts, the central nervous system (CNS) and the peripheral nervous system (PNS). Cells of the human nervous system mostly comprise neuronal cells, whose axons bundle in the PNS and nonneuronal cells such as glia cells, microglia, astrocytes, and oligodendrocytes. As any other cell, all cells of the nervous system are susceptible to aging and although aging dramatically increases the risk of developing cognitive disorders that are typical of the CNS such as Alzheimer's disease (AD) or PNS such as amyotrophic lateral sclerosis, only few elders contract these pathologies (www.alz.org; www.pdf.org; www.alsa.org). This suggests that a purely genetic origin of these diseases linked to aging is unlikely as individuals can live for over 100 years without any overt behavioral sign of neurodegeneration. In addition, these diseases affect particular populations of cells in specific areas of the brain suggesting that perhaps aging is not the trigger of these diseases but only worsens preexisting conditions.

Changes in Ca2+ homeostasis have been linked to aging-dependent deterioration of neuronal activity [15–20]. However, it is not yet clear whether it is a decreased or increased cytosolic Ca2+ concentration that mediates its noxious effects on brain performance during aging. For example, Ca2+ channels have been found to be reduced in genetically modified mice with accelerated age-dependent decay in learning and memory [21, 22]. However, this conclusion is contradicted by another study in which it is demonstrated that a long-lasting intracellular Ca2+ might render neurons vulnerable to age [22]. Furthermore, elegant work by the Stutzmann laboratory reported an increase in type 2 RyR transcripts in brains with mild cognitive impairment compared to those with no cognitive impairment. In addition, they found a reduction in a specific type 2 RyR splice variant that is associated with antiapoptotic function in brains of patients with mild cognitive impairment and Alzheimer's disease [23].

#### **3.1. Reactive oxygen species (ROS) and ion channels in aging**

Although there is a considerably large amount of studies on aging-related neurodegenerative diseases, very little is known about the process of aging in normal neurons. Nevertheless, it is generally accepted that neurons of an aging brain are characterized by accumulation of reactive oxygen species over time that are probably generated by an altered target of rapamycin (TOR) pathway [24]. However, ROS has also a protective role in neurons but the mechanism that control the threshold to which ROS become toxic is still heavily debated.

The deleterious effects of free radicals on neuronal homeostasis can be attributed to the ability of these chemical species to interact with a large variety of targets such as DNA and lipids. However, recent research has brought to light that activity of several neuronal ion channels can significantly change upon chemical interaction with free radicals and that this event can be related to aberrant cognitive functions during aging. For example, oxidizing agents dramatically inhibit Na+ channels activity without affecting the concentration of the channels at the surface membrane suggesting a direct effect of the agent on the Na+ channel [25]. Inhibition of Na+ channel activity dampens the ability to produce enough depolarization to generate an action potential and therefore affects the overall brain function.

Oxidation of K+ channels that are expressed in brain neurons has been reported. For example, the activity of K+ channels expressed in the hippocampus (which is the part of the brain that controls memory formation) can be increased by ROS [26] resulting in an inhibition of neuronal excitability and possibly incapacity to form or retain memory during aging. Interestingly, several studies have shown that ROS can increase or decrease cytosolic Ca2+ according to the type of Ca2+ channel analyzed. For example, oxidation of the "L-type" Ca2+ channel decreased Ca2+ currents [27]. However, the oxidative activity of *β*-amyloid protein (A*β*) produced in the brains of Alzheimer's patients increased the activity of L-type Ca2+ [28, 29], suggesting that distinct oxidative agents can exert different effects on Ca2+ homeostasis. In contrast, reducing agents have been reported to increase activity of the "T-type" calcium channel [30]. This suggests that ROS agents can increase cytosolic Ca2+ concentration and that this event can contribute to loss of neuronal function in aging. Inositol 1, 4, 5-trisphosphate (IP3) receptors (IP3Rs) and RyRs are the two major intracellular Ca2+ channels that release Ca2+ from neuronal intracellular stores such as the endoplasmic reticulum (ER).

www.pdf.org; www.alsa.org). This suggests that a purely genetic origin of these diseases linked to aging is unlikely as individuals can live for over 100 years without any overt behavioral sign of neurodegeneration. In addition, these diseases affect particular populations of cells in specific areas of the brain suggesting that perhaps aging is not the trigger of these

Changes in Ca2+ homeostasis have been linked to aging-dependent deterioration of neuronal activity [15–20]. However, it is not yet clear whether it is a decreased or increased cytosolic Ca2+ concentration that mediates its noxious effects on brain performance during aging. For example, Ca2+ channels have been found to be reduced in genetically modified mice with accelerated age-dependent decay in learning and memory [21, 22]. However, this conclusion is contradicted by another study in which it is demonstrated that a long-lasting intracellular Ca2+ might render neurons vulnerable to age [22]. Furthermore, elegant work by the Stutzmann laboratory reported an increase in type 2 RyR transcripts in brains with mild cognitive impairment compared to those with no cognitive impairment. In addition, they found a reduction in a specific type 2 RyR splice variant that is associated with antiapoptotic function

in brains of patients with mild cognitive impairment and Alzheimer's disease [23].

Although there is a considerably large amount of studies on aging-related neurodegenerative diseases, very little is known about the process of aging in normal neurons. Nevertheless, it is generally accepted that neurons of an aging brain are characterized by accumulation of reactive oxygen species over time that are probably generated by an altered target of rapamycin (TOR) pathway [24]. However, ROS has also a protective role in neurons but the mechanism that

The deleterious effects of free radicals on neuronal homeostasis can be attributed to the ability of these chemical species to interact with a large variety of targets such as DNA and lipids. However, recent research has brought to light that activity of several neuronal ion channels can significantly change upon chemical interaction with free radicals and that this event can be related to aberrant cognitive functions during aging. For example, oxidizing agents dramatically inhibit Na+ channels activity without affecting the concentration of the channels

Oxidation of K+ channels that are expressed in brain neurons has been reported. For example, the activity of K+ channels expressed in the hippocampus (which is the part of the brain that controls memory formation) can be increased by ROS [26] resulting in an inhibition of neuronal excitability and possibly incapacity to form or retain memory during aging. Interestingly, several studies have shown that ROS can increase or decrease cytosolic Ca2+ according to the type of Ca2+ channel analyzed. For example, oxidation of the "L-type" Ca2+ channel decreased Ca2+ currents [27]. However, the oxidative activity of *β*-amyloid protein (A*β*) produced in the brains of Alzheimer's patients increased the activity of L-type Ca2+ [28, 29], suggesting that distinct oxidative agents can exert different effects on Ca2+ homeostasis. In contrast, reducing

channel activity dampens the ability to produce enough depolarization to

channel [25].

**3.1. Reactive oxygen species (ROS) and ion channels in aging**

control the threshold to which ROS become toxic is still heavily debated.

at the surface membrane suggesting a direct effect of the agent on the Na+

generate an action potential and therefore affects the overall brain function.

Inhibition of Na+

diseases but only worsens preexisting conditions.

40 Molecular Mechanisms of the Aging Process and Rejuvenation

Intracellular Ca2+ channels mediate numerous Ca2+-dependent processes, including cellular growth and development, gene expression, and neurotransmission [31–40]. Therefore, it is not surprising that aberrant ICC function has been implicated in aging and several age-related pathologies including Alzheimer's disease, Huntington's disease (HD), and glaucoma [41– 48]. Phosphatidylinositol and IP3 levels are reduced in brains of patients with AD and the ensuing smaller number of IP3 binding sites correlates with the number of amyloid plaques and neurofibrillary tangles [19–24]. Importantly, similar IP3R dysfunction was found in in vitro models for AD including primary neuronal cultures from mouse models of AD [44, 49, 50] and cortical neurons exposed to β-amyloid protein [49]. These findings are particularly interesting, given the selective response of IP3R to elevated levels of oxidative stress. IP3R-mediated Ca2+ release was increased following activation of M3 muscarinic receptors under conditions of elevated oxidative stress [51]. Similarly, nonlethal oxidative stress in neuronal cells resulted in a selective upregulation at both the transcriptional and translational levels of type 2 IP3Rs [52]. These channels exhibit the strongest affinity for the endogenous ligand, IP3, and are preferentially expressed in the membranes of the nuclear envelope, where they mediate nuclear Ca2+ release [52–55]. This nuclear Ca2+ release is thought to control gene expression responsible for cellular survival and death pathways, and therefore, represents a promising drug target for neurodegenerative diseases [56]. This is exemplified by the recent finding that homocysteine-inducible, endoplasmic reticulum stress-inducible, ubiquitin-like domain member 1 (HERPUD1) is cytoprotective by preventing IP3R-mediated Ca2+ transfer from the ER to mitochondria [57]. Analogously to IP3Rs, several neurodegenerative and age-related disorders show RyR dysfunction contributing to disease pathology and progression. AD patients exhibit changes in neuronal RyR expression and in ryanodine binding that correlates with cognitive decline and A*β* deposition [42].

#### **3.2. Aging of the nonneuronal cells in the nervous system**

The vast amount of cells in the brain is nonneuronal cells such as glia, microglia, astrocytes, and oligodendrocytes. These cells maintain homeostasis, form myelin, and, importantly, they can provide protection to the CNS and PNS as they act as the first and main form of neuronal immune defense. Microglia acts as the resident innate immune system in the brain as they respond to and propagate inflammatory signals (e.g., impending from PNS) by producing proinflammatory cytokines that ultimately can generate cognitive consequences. Several clinical and experimental studies reported that both increased oxidative stress and increased inflammation are (among others) hallmarks of brain aging [30, 58]. Remarkably, it has been found that activation of microglia is dependent on the expression of the Kv1.5 potassium channel and *β*-amyloid peptide induces expression of the Kv1.5 potassium channels in microglia [59–61]. This suggests that increased oxidative stress in aging could produce inflammation by upregulating K+ channels in microglia and that alteration of ion channels could be one of the possible causes of aging-dependent decline of neuronal functions.

#### **4. Ion channels and glaucoma**

Glaucoma is an age-related, progressive optic neuropathy that manifests with pathological changes in the optic nerve, activation and remodeling of optic nerve head astrocytes (ONHAs), and slow progressive death of retinal ganglion cells, often leading to blindness. Although the exact pathophysiology is still not completely understood, there is evidence to support ischemic, mechanical, and inflammatory components. In an effort to protect the integrity of the optic nerve and prevent the loss of retinal ganglion cells, research efforts have recently concentrated on glioprotection approaches targeting ONHAs [62, 63]. Glia and neuronal cells alike utilize fine-tuned calcium signaling pathways to control physiological functions. Recent studies have revealed complex intracellular Ca2+ signaling pathways in primary ONHA culture [55] and for the first time described a differential distribution of type 2 IP3Rs and type 2 RyRs in the ER and the membranes of the nuclear envelope. Hence, it is likely that these receptor subtypes activate specific Ca2+-sensitive genes that determine cellular fate. Of particular interest for the role of aberrant Ca2+ signaling in glaucoma is the finding that exposure of ONHAs to elevated hydrostatic pressure, as a model for increased intraocular pressure in glaucoma, results in the differential upregulation of type 2 RyRs [64]. Given the nuclear membrane localization of type 2 RyRs in ONHAs [55], it is likely that RyR-mediated Ca2+ release differentially activates gene expression. Similar pathways of calcium signaling have been identified in astrocytes from other parts of the CNS and recently been reviewed in detail [65].

#### **5. Ion channels and cancer**

Cancer is a group of diseases that claim about 8.4 million lives every year (www.cancer.org). Although in the past decades, medical research has dramatically improved prevention, diagnosis, and treatment of cancer and despite being among the most preventable diseases, cancer remains a leading cause of death worldwide.

Virtually, cancer can develop in any tissue and although each cancer type can be characterized by its unique features, the basic mechanisms that generate cancers are similar in all forms of the diseases. In a normal and healthy tissue, cell proliferation and cell death are controlled by a very complex, timely, and integrated signaling network that includes a series of checkpoints to ensure proper division factors of the cell or death. Cancer originates from an uncontrolled proliferation of cells that evade cell death and can eventually invade and/or outspread into other body compartments (metastasis).

Cancer can be caused by a multitude of environmental factors that can be external such as smoking, sun exposure, and/or internal such as inherited faulty genes and/or infections. Although there can be a significant difference in the prevalence of cancers among different societies, overall cancer can affect every human being. Accordingly, aging is the highest single risk factor for developing cancer [66]. Recently, several ion channels have been found to play a major role in maintaining homeostasis of nonexcitable cells (**Figure 1**).

inflammation by upregulating K+

42 Molecular Mechanisms of the Aging Process and Rejuvenation

**4. Ion channels and glaucoma**

detail [65].

**5. Ion channels and cancer**

other body compartments (metastasis).

cancer remains a leading cause of death worldwide.

channels in microglia and that alteration of ion channels

could be one of the possible causes of aging-dependent decline of neuronal functions.

Glaucoma is an age-related, progressive optic neuropathy that manifests with pathological changes in the optic nerve, activation and remodeling of optic nerve head astrocytes (ONHAs), and slow progressive death of retinal ganglion cells, often leading to blindness. Although the exact pathophysiology is still not completely understood, there is evidence to support ischemic, mechanical, and inflammatory components. In an effort to protect the integrity of the optic nerve and prevent the loss of retinal ganglion cells, research efforts have recently concentrated on glioprotection approaches targeting ONHAs [62, 63]. Glia and neuronal cells alike utilize fine-tuned calcium signaling pathways to control physiological functions. Recent studies have revealed complex intracellular Ca2+ signaling pathways in primary ONHA culture [55] and for the first time described a differential distribution of type 2 IP3Rs and type 2 RyRs in the ER and the membranes of the nuclear envelope. Hence, it is likely that these receptor subtypes activate specific Ca2+-sensitive genes that determine cellular fate. Of particular interest for the role of aberrant Ca2+ signaling in glaucoma is the finding that exposure of ONHAs to elevated hydrostatic pressure, as a model for increased intraocular pressure in glaucoma, results in the differential upregulation of type 2 RyRs [64]. Given the nuclear membrane localization of type 2 RyRs in ONHAs [55], it is likely that RyR-mediated Ca2+ release differentially activates gene expression. Similar pathways of calcium signaling have been identified in astrocytes from other parts of the CNS and recently been reviewed in

Cancer is a group of diseases that claim about 8.4 million lives every year (www.cancer.org). Although in the past decades, medical research has dramatically improved prevention, diagnosis, and treatment of cancer and despite being among the most preventable diseases,

Virtually, cancer can develop in any tissue and although each cancer type can be characterized by its unique features, the basic mechanisms that generate cancers are similar in all forms of the diseases. In a normal and healthy tissue, cell proliferation and cell death are controlled by a very complex, timely, and integrated signaling network that includes a series of checkpoints to ensure proper division factors of the cell or death. Cancer originates from an uncontrolled proliferation of cells that evade cell death and can eventually invade and/or outspread into

Cancer can be caused by a multitude of environmental factors that can be external such as smoking, sun exposure, and/or internal such as inherited faulty genes and/or infections.

**Figure 1.** Schematic representation of the contribution of different ion channels to the membrane potential in function of time (e.g., nonexcitable cell vs. neuronal action potential). Overexpression and/or upregulation (e.g., via hormonedependent regulation) of certain ion channels can contribute to suppress differentiation and increase duplication rate resulting in the generation of a cancerogenic phenotype.

In addition to the traditional role of allowing movements of ions across membranes, ion channels can also control mechanisms of transport, secretion, cell volume, and protein synthesis. For example, glucose transport is controlled by gradients of Na+ [67, 68]. Furthermore, several transcription factors or proteins involved in secretory mechanism are activated by Ca2+ [69, 70]. Therefore, changes of intracellular ionic concentrations can regulate a variety of cellular event ranging from production of energy to protein synthesis, which are necessary for the ultimate process of cellular duplication. Several studies have reported that cancer cells of different histogenesis can express specific ion channels that can play an important role during proliferation [71].

One of the better characterized ion channels in cancer is the Kv11.1 (hERG1) potassium channel. This potassium channel is encoded by the human ether-a-go-go related gene 1 (hERG1), which has been found typically expressed in the mammalian heart in which it play a fundamental role in controlling repolarization and duration of action potential [72]. Re‐ markably, hERG1 channel has also been found expressed in different nonexcitable cancer cells but not in the organ from which the tumor has originated [73]. This suggests that the presence of this channel might provide a selective advantage to proliferation. Blockade or stimulation of hERG1 channel activity determined a strong inhibitory effect on cancer cell proliferation [74–78]. In addition, complete removal of the hERG1 protein from breast cancer cells deter‐ mined death by activation of apoptosis [78]. These events indicate that the hERG1 is very important for cancer biology and its activity is kept under strict control.

Interestingly, the inhibitory effect on cell proliferation as a consequence of chronic stimulation of hERG1 channel was characterized by activation of a "cellular senescent program" [76, 79]. Senescent cells were initially described by Hayflick and Moorhead [80] as cells that have lost the ability to duplicate, though they may not die. Today, cellular senescence is defined as a permanent arrest of the cell cycle induced by a progressive increase of stresses [81–83]. At this time, it is not known what kind of stresses hERG1 agonists produce on cancer cells but their effect is mediated by permanent arrest of the cell cycle, increased expression of tumor suppressors (e.g., p21waf/cif and p16INK4A) and decreased level of tumor markers (e.g., cyclins) resulting in a potent inhibition of cell proliferation [84–86]. This suggests that, by taking advantage of the ability to accelerate aging in cancer cells, hERG1 agonists could be used as an anticancer therapeutic strategy.

Other ion channels have been found playing fundamental roles in regulating biochemical signaling that underline important events in cancer biology which includes metastasis. Overtime, cancer cells acquire the ability to move and invade surrounding tissues by protrud‐ ing membrane structures (invadopodia and pseudopodia) [87] through the intracellular space of the host organ. Remarkably, it has been discovered that ion channels are fundamental factors for regulation of invasion and migration of cancer cells. For example, the concerted activity of Ca2+, K+ , and Cl channels that can exquisitely colocalize on the glioma surface membrane generates fluxes of ions and water that creates shrinkage of the membrane with consequent formation of invadopodia [88]. A direct consequence of this event is that cancer cells can move across tissue barriers (e.g., blood vessel) and colonize other body compartments. In addition, it has been shown that overexpression of Kv10 channels in which ion flux has been obstructed by a specific mutation did not lose the ability to promote cell proliferation [89, 90]. Although the mechanism through which this event occurs is not clearly understood, it appears that these channels can regulate activities of proteins that control proliferative cell signaling also when ion fluxes are not involved.

As hormones control most of the major organ functions by activating a variety of cellular signaling, it is not surprising that ion channels can be downstream effectors of hormone receptors. Growth of many cancers can depend on altered expression of hormone receptors. For example, high percentage of breast cancers are very sensitive to the action of insulin and/ or sex hormones such as estrogen [91] or prostate cancer to testosterone [92]. Hormones can control ion channel activity by increasing their synthesis or by activating membrane signaling pathways (**Figure 1**). For example, hormones that bind G protein-coupled receptors (GPCR) produce release of the active *βγ*subunit of the heteromeric GTPase complex that ultimately binds and activates K+ channels (e.g., GIRK). Alternatively, soluble hormone receptors can activate nongenomic signaling resulting in stimulation of kinases or phosphatases that modulate activity of ion channels by directly targeting these proteins [93–95]. Furthermore, as secretion is vastly controlled by intracellular changes in Ca2+ concentrations (e.g., Ca2+ dependent insulin secretion), hormones must rely on ion channel function to be released in the body environment [96]. It is well established that with aging, organs become less sensitive to hormones. Although several examples of hormone-regulated ion channel activities have been proposed, knowledge on the role of these signalings in pathological conditions such as cancer, age-related disease, and/or aging is very limited. Therefore, it appears that there is a compelling need to study the role of hormonal regulation of ion channels to better understand both aging and cancer.

#### **6. Modulation of ion channels**

Interestingly, the inhibitory effect on cell proliferation as a consequence of chronic stimulation of hERG1 channel was characterized by activation of a "cellular senescent program" [76, 79]. Senescent cells were initially described by Hayflick and Moorhead [80] as cells that have lost the ability to duplicate, though they may not die. Today, cellular senescence is defined as a permanent arrest of the cell cycle induced by a progressive increase of stresses [81–83]. At this time, it is not known what kind of stresses hERG1 agonists produce on cancer cells but their effect is mediated by permanent arrest of the cell cycle, increased expression of tumor suppressors (e.g., p21waf/cif and p16INK4A) and decreased level of tumor markers (e.g., cyclins) resulting in a potent inhibition of cell proliferation [84–86]. This suggests that, by taking advantage of the ability to accelerate aging in cancer cells, hERG1 agonists could be used as

Other ion channels have been found playing fundamental roles in regulating biochemical signaling that underline important events in cancer biology which includes metastasis. Overtime, cancer cells acquire the ability to move and invade surrounding tissues by protrud‐ ing membrane structures (invadopodia and pseudopodia) [87] through the intracellular space of the host organ. Remarkably, it has been discovered that ion channels are fundamental factors for regulation of invasion and migration of cancer cells. For example, the concerted activity of

generates fluxes of ions and water that creates shrinkage of the membrane with consequent formation of invadopodia [88]. A direct consequence of this event is that cancer cells can move across tissue barriers (e.g., blood vessel) and colonize other body compartments. In addition, it has been shown that overexpression of Kv10 channels in which ion flux has been obstructed by a specific mutation did not lose the ability to promote cell proliferation [89, 90]. Although the mechanism through which this event occurs is not clearly understood, it appears that these channels can regulate activities of proteins that control proliferative cell signaling also when

As hormones control most of the major organ functions by activating a variety of cellular signaling, it is not surprising that ion channels can be downstream effectors of hormone receptors. Growth of many cancers can depend on altered expression of hormone receptors. For example, high percentage of breast cancers are very sensitive to the action of insulin and/ or sex hormones such as estrogen [91] or prostate cancer to testosterone [92]. Hormones can control ion channel activity by increasing their synthesis or by activating membrane signaling pathways (**Figure 1**). For example, hormones that bind G protein-coupled receptors (GPCR) produce release of the active *βγ*subunit of the heteromeric GTPase complex that ultimately

activate nongenomic signaling resulting in stimulation of kinases or phosphatases that modulate activity of ion channels by directly targeting these proteins [93–95]. Furthermore, as secretion is vastly controlled by intracellular changes in Ca2+ concentrations (e.g., Ca2+ dependent insulin secretion), hormones must rely on ion channel function to be released in the body environment [96]. It is well established that with aging, organs become less sensitive to hormones. Although several examples of hormone-regulated ion channel activities have been proposed, knowledge on the role of these signalings in pathological conditions such as

channels (e.g., GIRK). Alternatively, soluble hormone receptors can

, and Cl channels that can exquisitely colocalize on the glioma surface membrane

an anticancer therapeutic strategy.

44 Molecular Mechanisms of the Aging Process and Rejuvenation

ion fluxes are not involved.

binds and activates K+

Ca2+, K+

Another level of complexity of regulation of ion channels is added by a number of modulatory proteins that have been shown to bind channels and alter their biophysical properties.

**Figure 2.** Presenilins differentially regulate RyR-mediated Ca2+ release.Representations of an individual RyR and its in‐ teraction with presenilin are shown in the top panels. Corresponding graphs below illustrate characteristics of Ca2+ transients mediated by RyR activity. Whole-cell cytosolic calcium concentrations (ordinate) are plotted over time (ab‐ scissa) to show the changes in the kinetics of Ca2+ transients dependent on presenilin binding to the RyR. Seesaws de‐ pict a predominant effect of presenilin 1 (PS1) over presenilin 2 PS2 or PS2 over PS1, as seen in young and aged animals, respectively [48]. (A) Binding of the PS1 N-terminal fragment to RyR increases open probability and results in heightened calcium release and fast channel inhibition by calcium at the RyR's inhibitory low affinity Ca2+ binding site. (B) Binding of PS2 to the RyR blocks inhibition at the low affinity Ca2+ binding site resulting in an increased duration of the Ca2+ transient.This figure was modified from reference [103], which was published under Open Access licence (®2015 by Andrew J. Payne *et al.*).

One of the best examples of involvement of ion channel modulation in aging includes Homer/ Vesl proteins and the group of presenilins. The group of Homer proteins (reviewed in [97]) is a family of ubiquitously expressed scaffolding molecules. Through a conserved binding motif, Homer proteins interact with a number of synaptic proteins. Homer 1 proteins directly interact with IP3Rs, RyRs, the group of transient receptor potential canonical (TRPC) channels, as well as mGluRs and some voltage-gated Ca2+ channels (reviewed in [97]). Intriguingly, in addition to enhancing synaptic transmission and providing a means of regulating excitability through tethering plasma membrane proteins to receptors and channels in the ER by formation of Homer tetramers, Homer proteins can alter the biophysical properties of their binding partners [98–100]. These interactions, especially with intracellular Ca2+ channels, have recently attracted increased interest due to their alterations in age-related diseases in the nervous system. For instance, in the aging brain, loss of the short isoform, Homer 1a, correlated with the loss of cognitive and motor function in mice [46]. Similarly, upregulation of the long isoform, Homer 1c, in the retina of glaucomatous mice showed a statistically significant association with severity of the disease phenotype and disease progression [47]. Furthermore, loss of Homer 1c immunoreactivity at glutamatergic synapses after experimental stroke was identified as a potential biomarker for early neurodegenerative processes, prior to initiation of apoptotic pathways [101]. Similarly, binding of presenilin proteins to RyRs results in a functional change of intracellular Ca2+ release [102–105]. Recent studies have demonstrated that altered levels of presenilin proteins in the aging brain correlate with the presence and severity of impairments in cognitive and motor function [48]; **Figure 2**), identifying the group of presenilins as putative drug target for neurodegeneration. In summary, intracellular Ca2+ channels are critical mediators of intracellular Ca2+ homeostasis and respond differentially to aging and patholog‐ ical stimuli including oxidative stress. Furthermore, intracellular Ca2+ signaling is differentially regulated in various cell types and tissues and by a large number of modulators, providing a multitude of targets for pharmaceutical intervention in conditions characterized by neurode‐ generation and aging.

#### **7. Perspective**

Aging is a process common to all living organisms that is associated with a progressive failure to adapt to changes in the environment. As ion channels are evolutionary conserved proteins that all cells of all living creatures utilize to sense and adapt to variations of both extracellular and intracellular environments, it is not surprising that malfunction of ion channels increases disease susceptibility that often simulates ailments of getting older. This suggests that drugs targeting ion channels can hold promise for treating aging. However, in consideration of the fact that more than 400 genes encoding for ion channels subunits have been identified so far, the role of ion channels in aging and aging-related diseases remains significantly underex‐ plored. In addition, aging-dependent alteration of a particular ion channel appears to be organ and/or tissue-specific indicating that pharmacologic therapies targeting a specific ion channel should be tailored to a particular organ.

The ultimate consequence of all diseases is pain which appears to get worse with age and can have serious negative impact on quality of life. In recent time, a substantial increased aware‐ ness on the critical role of ion channels in diseases and pain has been achieved so that ion channels are emerging as novel therapeutic targets in the treatment of pain.

#### **Acknowledgements**

This material is the result of work supported with resources and the use of facilities at the Edward Hines Jr. VA Hospital, Hines, IL. The contents do not represent the views of the U.S. Department of Veterans Affairs or the United States Government. Additional support by the Dr. John P. and Therese E. Mulcahy Endowed Professorship in Ophthalmology (SK) and by the Cronk Family Foundation is gratefully acknowledged.

To Fiore e Rosa

to enhancing synaptic transmission and providing a means of regulating excitability through tethering plasma membrane proteins to receptors and channels in the ER by formation of Homer tetramers, Homer proteins can alter the biophysical properties of their binding partners [98–100]. These interactions, especially with intracellular Ca2+ channels, have recently attracted increased interest due to their alterations in age-related diseases in the nervous system. For instance, in the aging brain, loss of the short isoform, Homer 1a, correlated with the loss of cognitive and motor function in mice [46]. Similarly, upregulation of the long isoform, Homer 1c, in the retina of glaucomatous mice showed a statistically significant association with severity of the disease phenotype and disease progression [47]. Furthermore, loss of Homer 1c immunoreactivity at glutamatergic synapses after experimental stroke was identified as a potential biomarker for early neurodegenerative processes, prior to initiation of apoptotic pathways [101]. Similarly, binding of presenilin proteins to RyRs results in a functional change of intracellular Ca2+ release [102–105]. Recent studies have demonstrated that altered levels of presenilin proteins in the aging brain correlate with the presence and severity of impairments in cognitive and motor function [48]; **Figure 2**), identifying the group of presenilins as putative drug target for neurodegeneration. In summary, intracellular Ca2+ channels are critical mediators of intracellular Ca2+ homeostasis and respond differentially to aging and patholog‐ ical stimuli including oxidative stress. Furthermore, intracellular Ca2+ signaling is differentially regulated in various cell types and tissues and by a large number of modulators, providing a multitude of targets for pharmaceutical intervention in conditions characterized by neurode‐

46 Molecular Mechanisms of the Aging Process and Rejuvenation

Aging is a process common to all living organisms that is associated with a progressive failure to adapt to changes in the environment. As ion channels are evolutionary conserved proteins that all cells of all living creatures utilize to sense and adapt to variations of both extracellular and intracellular environments, it is not surprising that malfunction of ion channels increases disease susceptibility that often simulates ailments of getting older. This suggests that drugs targeting ion channels can hold promise for treating aging. However, in consideration of the fact that more than 400 genes encoding for ion channels subunits have been identified so far, the role of ion channels in aging and aging-related diseases remains significantly underex‐ plored. In addition, aging-dependent alteration of a particular ion channel appears to be organ and/or tissue-specific indicating that pharmacologic therapies targeting a specific ion channel

The ultimate consequence of all diseases is pain which appears to get worse with age and can have serious negative impact on quality of life. In recent time, a substantial increased aware‐ ness on the critical role of ion channels in diseases and pain has been achieved so that ion

channels are emerging as novel therapeutic targets in the treatment of pain.

generation and aging.

should be tailored to a particular organ.

**7. Perspective**

#### **Author details**

Vidhya Rao1 , Simon Kaja1,2,3 and Saverio Gentile1\*

\*Address all correspondence to: sagentile@lumc.edu

1 Department of Molecular Pharmacology and Therapeutics, Stritch School of Medicine, Loyola University Chicago, Maywood, Illinois, USA

2 Department of Ophthalmology, Stritch School of Medicine, Loyola University Chicago, Maywood, Illinois, USA

3 Research Service, Edward Hines Jr. Veterans Administration Hospital, Hines, Illinois, USA

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54 Molecular Mechanisms of the Aging Process and Rejuvenation

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### **Molecular Mechanisms of Skin Aging and Rejuvenation**

Miri Kim and Hyun Jeong Park

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/62983

#### **Abstract**

The aging process in the skin is complex and influenced by more intrinsic and extrinsic factors than any other body organ. The effects of these two types of factors overlap for the most part. The combined effects of these two aging processes also affect dermal matrix alterations. The main clinical signs of skin aging include wrinkling and irregular pigmentation, which are influenced by a combination of intrinsic and extrinsic (e.g., UV radiation, heat, smoking, and pollutants) factors. Histologically, collagen decreases, and the dermis is replaced by abnormal elastic fibers as a cause of wrinkle formation through the loss of skin elasticity. There have been numerous studies of skin aging performed to elucidate the underlying molecular mechanisms and to develop various antiaging therapeutics and preventive strategies. We summarized the molecular mechanisms and treatments of skin aging. Mainly UV radiation induces ROS formation and DNA damage, leading to increased production of MMPs and decreased production of collagen in keratinocytes and fibroblasts, which reflect the central aspects of skin aging. Besides UV radiation exposure, extrinsic factors including tobacco smoking, exposure to environ‐ mental pollutants, infrared radiation, and heat contribute to premature skin aging. Like UV radiation, these factors cause ROS formation and increase expression of MMPs, thus accelerating skin aging by inducing extracellular matrix (ECM) degradation. Accumu‐ lated collagen fibrils inhibit the new collagen synthesis and account for the further degradation of the ECM through this positive feedback loop. Accumulating evidence for molecular mechanisms of skin aging should provide clinicians with an expanding spectrum of therapeutic targets in the treatment of skin aging.

**Keywords:** skin aging, photoaging, molecular mechanisms, antiaging treatments, re‐ juvenation

© 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.

#### **1. Introduction**

Skin aging is a complex process affected by both genetic and environmental factors, and it is largely influenced by the cumulative damage from exposure to ultraviolet (UV) radiation. Chronic exposure of UV radiation on human skin leads to solar elastosis, degradation of the extracellular matrix (ECM), and wrinkle formation. Skin aging is affected by both intrinsic and extrinsic factors. Intrinsic or chronological skin aging results from the passage of time and is influenced by genetic factors. Extrinsic skin aging mainly results from UV irradiation, which is called photoaging. These two types of aging processes are superimposed in sun-exposed skin, and they have common clinical features caused by dermal matrix alterations that mainly contribute towrinkle formation,laxity,andfragilityofagedskin[1].Thedermalmatrixcontains ECM proteins such as collagen, elastin, and proteoglycans which is responsible for conferring strength and resiliency of the skin. Skin aging associated with dermal matrix alterations and atrophycanbe causedbysenescenceofdermal cells suchas fibroblasts, anddecreasedsynthesis andacceleratedbreakdownofdermal collagenfibers [2].Mousemodels of skinaginghave been developed extensively to elucidate intrinsic aging and photoaging processes,to validate *in vitro* biochemicaldata,andtotesttheeffectsofpharmacologicaltools forretardingskinagingbecause they have the advantages of being genetically similar to humans and are easily available. This review is focused on the molecular mechanisms of skin aging and antiaging treatment with a brief summary of the clinical and histological features of skin aging (**Figure 1**).

**Figure 1.** Histology of photoaged skin. The predominant histological finding of photodamaged skin is solar elastosis, which is basophilic degeneration of elastotic fibers in the dermis. Solar elastosis separates from the epidermis by a nar‐ row band of normal-appearing collagen (grenz zone) with collagen fibers arranged horizontally (H&E stating. Original magnification ×400).

### **2. Clinical manifestations of chronological aging and photoaging**

**1. Introduction**

58 Molecular Mechanisms of the Aging Process and Rejuvenation

magnification ×400).

Skin aging is a complex process affected by both genetic and environmental factors, and it is largely influenced by the cumulative damage from exposure to ultraviolet (UV) radiation. Chronic exposure of UV radiation on human skin leads to solar elastosis, degradation of the extracellular matrix (ECM), and wrinkle formation. Skin aging is affected by both intrinsic and extrinsic factors. Intrinsic or chronological skin aging results from the passage of time and is influenced by genetic factors. Extrinsic skin aging mainly results from UV irradiation, which is called photoaging. These two types of aging processes are superimposed in sun-exposed skin, and they have common clinical features caused by dermal matrix alterations that mainly contribute towrinkle formation,laxity,andfragilityofagedskin[1].Thedermalmatrixcontains ECM proteins such as collagen, elastin, and proteoglycans which is responsible for conferring strength and resiliency of the skin. Skin aging associated with dermal matrix alterations and atrophycanbe causedbysenescenceofdermal cells suchas fibroblasts, anddecreasedsynthesis andacceleratedbreakdownofdermal collagenfibers [2].Mousemodels of skinaginghave been developed extensively to elucidate intrinsic aging and photoaging processes,to validate *in vitro* biochemicaldata,andtotesttheeffectsofpharmacologicaltools forretardingskinagingbecause they have the advantages of being genetically similar to humans and are easily available. This review is focused on the molecular mechanisms of skin aging and antiaging treatment with a

brief summary of the clinical and histological features of skin aging (**Figure 1**).

**Figure 1.** Histology of photoaged skin. The predominant histological finding of photodamaged skin is solar elastosis, which is basophilic degeneration of elastotic fibers in the dermis. Solar elastosis separates from the epidermis by a nar‐ row band of normal-appearing collagen (grenz zone) with collagen fibers arranged horizontally (H&E stating. Original

Clinical features of skin aging vary among individuals with differences in both genetic factors and lifestyles, which result in various degrees of cutaneous outcomes. Clinical signs of chronological aging include thinning of the skin, cigarette paper-like wrinkles, xerosis, loss of elasticity, and development of benign vascular formations such as cherry angiomas and benign overgrowths such as seborrheic keratosis [3]. Chronological aging is mainly the result of loss of soft tissue volume from fat atrophy, gravity-induced soft tissue redistribution, and weak‐ ened facial skeletal support related to bone resorption [3]. Clinical signs of photoaging include wrinkling, laxity, and a leather-like appearance, which are mainly the result of structural changes in the connective tissue of the dermis. These changes include both enzymatic degra‐ dation and reduced de novo synthesis of collagen, which cause wrinkling of the skin. The cumulative UV irradiation dose and Fitzpatrick skin type are used to assign the degree of photoaging. Individuals with Fitzpatrick skin types I and II show atrophic skin changes with fewer wrinkles, focal depigmentation, dysplastic premalignant changes such as actinic keratosis and malignant skin cancer. By contrast, skin types III and IV skin display hypertro‐ phic features with deep wrinkles, leathery appearance, and lentigines [4]. In addition, photo‐ aging includes changes in the dermal vascular structure, which appear clinically as telangiectasia (**Figure 2**).

**Figure 2.** Schematic representation of pathogenesis of premature/extrinsic skin aging. ROS: reactive oxygen species, AhR: arylhydrocarbon receptor, NF-kB: nuclear factor kappa‐B, IL-1: interleukin‐1, TNF-α: tumor necrosis factor, CCN1: cysteine-rich protein 61, MAPK: mitogen‐activated protein kinase, AP‐1: activator protein 1, and MMPs: matrix metalloproteinases.

### **3. Histology of chronological aging and photoaging**

Histological changes in chronologically aged skin are characterized by epidermal atrophy with reduced amounts of fibroblasts and collagen content in the dermis. The epidermal atrophy of intrinsically aged skin, which particularly affects the stratum spinosum, is related to lower epidermal turnover rate because of prolonged cell cycles [5]. Several studies of skin aging have reported that the epidermis is hypocellular with decreased melanocytes, mast cells, and Langerhans cells [6]. After the age of 30 years, the number of melanocytes decreases by 8–20% per decade [7]. The number of Langerhans cells in the epidermis becomes markedly decreased with noticeable morphological alterations and functional impairment. In dermis of chrono‐ logically aged skin, the collagen fibers are loose, thin, and disorganized compared with those in the sun-protected skin of young people [8]. The dermis of chronologically aged skin shows fewer mast cells and fibroblasts than that of young skin, and the amounts of collagen and elastic fibers are decreased [9]. In a study of the chronological changes in collagen fibers, the synthesis of collagen was found to be decreased by 30% in the first 4 years of menopause, then by 2% per year [10].

Photoaging causes several histological changes in the skin that are distinct from histological alterations that occur intrinsically during aging. In photoaging, the thickness of the epidermis and the morphology of epidermal is heterogeneous [11]. The epidermis of photodamaged skin is thicker than that of intrinsically aged skin, whereas the epidermis of severely photodamaged skin elicits epidermal atrophy [12]. Furthermore, increased numbers of atypical melanocytes and keratinocytes may be seen [4]. Melanogenesis is also upregulated and participates in the neutralization of free radicals induced by UV radiation exposure, which may act as a mecha‐ nism for protection from photodamage [13]. Major alterations of photoaged skin and their molecular effects occur primarily within the dermis and the dermoepidermal junction. There is an increased amount of glycosaminoglycans and proteoglycans within the aged dermis, possibly because of a rise in the level of matrix metalloproteinases (MMPs) with increased numbers of hyperplastic fibroblasts [14]. Unlike the hypocellular feature of chronologically aged skin, photoaged skin can present an increased number of inflammatory cells such as mast cells, eosinophils, and mononuclear cells [15]. In addition, the amount of extracellular matrix is decreased and breakdown of collagen fibers is increased, which may appear as wrinkles [16]. Meanwhile, the most pronounced histological feature of photoaging is the disintegration of elastic fibers (solar elastosis), which results in accumulation of amorphous, thickened, curled, and fragmented elastic fibers [17]. Elastotic material consists of elastin, fibrillin, glycosamino‐ glycans, particularly hyaluronic acid and versican, a large chondroitin sulfate proteoglycan. The pathogenesis of solar elastosis is assumed to be a result of both degradation and de novo synthesis, although it is not yet fully understood [18].

#### **4. Molecular mechanisms of skin aging**

#### **4.1. Telomere shortening**

Telomeres are tandem repeats of TTAGGG located at the distal ends of most eukaryotic organisms to protect chromosomes from degradation and from fusion with neighboring chromosomes [19]. Telomere shortening prevents aberrant cellular proliferation by limiting cellular division. A consequence of this protection is cellular senescence and aging [20]. Telomerase, also called telomere terminal transferase, is capable of adding the telomeric sequence TTAGGG to the 3′ end of telomeres [21]. In humans, telomerase plays a significant role in the maintenance of skin aging and oncogenesis [22]. The regulation of telomerase activity may therefore have significant role in antiaging and anticancer therapy. Gradual shortening of telomeres can explain cellular senescence that is essentially caused by intrinsic aging because it results from cell division. However, UV radiation exposure may induce telomere shortening by producing reactive oxygen species (ROS) in the skin [23]. Because UV radiation induces the formation of ROS in the skin and telomere shortening is accelerated by ROS, it has been postulated that skin exposed to UV radiation may have shorter telomeres compared with skin protected from UV radiation [1, 23]. Oikawa et al. [23] demonstrated that UVA irradiation accelerated telomere shortening in human cultured fibroblast cell lines by site-specific DNA damage at the GGG sequence in the telomere sequence. By contrast, Sugimoto et al. reported that telomere length in the epidermis and dermis was reduced with age and telomere length was not significantly different between epidermis from sun-exposed and sun-protected areas. They could not confirm that telomere shortening was associated with photoaging [19]. Telomeres are metabolically active and possess a set of characteristics *in vitro* and *in vivo*, which are known as biomarkers of aging and cellular senescence. Among biomarkers of cellular senescence, telomere shortening is a rather elegant and frequently used biomarker. The validity of telomere shortening as a marker for cellular senescence is based on the theoretical and experimental data. Further studies to determine the relationships between telomere restriction fragment length in skin cells and lifestyle/genetic background are neces‐ sary to confirm the validity of this marker. The details of molecular events and especially the function of telomere shortening in skin aging are not understood completely. However, interesting and important research progress is expected because advances in this field of research are mostly recent (**Table 1**).

reported that the epidermis is hypocellular with decreased melanocytes, mast cells, and Langerhans cells [6]. After the age of 30 years, the number of melanocytes decreases by 8–20% per decade [7]. The number of Langerhans cells in the epidermis becomes markedly decreased with noticeable morphological alterations and functional impairment. In dermis of chrono‐ logically aged skin, the collagen fibers are loose, thin, and disorganized compared with those in the sun-protected skin of young people [8]. The dermis of chronologically aged skin shows fewer mast cells and fibroblasts than that of young skin, and the amounts of collagen and elastic fibers are decreased [9]. In a study of the chronological changes in collagen fibers, the synthesis of collagen was found to be decreased by 30% in the first 4 years of menopause, then by 2%

Photoaging causes several histological changes in the skin that are distinct from histological alterations that occur intrinsically during aging. In photoaging, the thickness of the epidermis and the morphology of epidermal is heterogeneous [11]. The epidermis of photodamaged skin is thicker than that of intrinsically aged skin, whereas the epidermis of severely photodamaged skin elicits epidermal atrophy [12]. Furthermore, increased numbers of atypical melanocytes and keratinocytes may be seen [4]. Melanogenesis is also upregulated and participates in the neutralization of free radicals induced by UV radiation exposure, which may act as a mecha‐ nism for protection from photodamage [13]. Major alterations of photoaged skin and their molecular effects occur primarily within the dermis and the dermoepidermal junction. There is an increased amount of glycosaminoglycans and proteoglycans within the aged dermis, possibly because of a rise in the level of matrix metalloproteinases (MMPs) with increased numbers of hyperplastic fibroblasts [14]. Unlike the hypocellular feature of chronologically aged skin, photoaged skin can present an increased number of inflammatory cells such as mast cells, eosinophils, and mononuclear cells [15]. In addition, the amount of extracellular matrix is decreased and breakdown of collagen fibers is increased, which may appear as wrinkles [16]. Meanwhile, the most pronounced histological feature of photoaging is the disintegration of elastic fibers (solar elastosis), which results in accumulation of amorphous, thickened, curled, and fragmented elastic fibers [17]. Elastotic material consists of elastin, fibrillin, glycosamino‐ glycans, particularly hyaluronic acid and versican, a large chondroitin sulfate proteoglycan. The pathogenesis of solar elastosis is assumed to be a result of both degradation and de novo

Telomeres are tandem repeats of TTAGGG located at the distal ends of most eukaryotic organisms to protect chromosomes from degradation and from fusion with neighboring chromosomes [19]. Telomere shortening prevents aberrant cellular proliferation by limiting cellular division. A consequence of this protection is cellular senescence and aging [20]. Telomerase, also called telomere terminal transferase, is capable of adding the telomeric sequence TTAGGG to the 3′ end of telomeres [21]. In humans, telomerase plays a significant

synthesis, although it is not yet fully understood [18].

**4. Molecular mechanisms of skin aging**

**4.1. Telomere shortening**

per year [10].

60 Molecular Mechanisms of the Aging Process and Rejuvenation




**Table 1.** Clinical and histological features of photoaged skin and related molecular mechanisms.

#### **4.2. Matrix metalloproteinases and signal transduction pathways**

**Clinical Histological Molecular mechanism**

oxidase-positive, KIT+, and melanocyte sand

Decreased dopa

62 Molecular Mechanisms of the Aging Process and Rejuvenation

Wrinkles Thinned epidermis as

Sagging Loss of elastic tissue

Inelasticity Accumulated large

in the dermis and the

remaining fibers were disorganized, shortened, and fragmented

amounts of homogenization and a dark blue amorphous elastotic

material in the dermis, so-called solar elastosis

VII when compared to the

reduction in melanocytes

well as less elastotic changes, tropoelastin, and collagen

surrounding photoaged skin

Guttate hypomelanosis to be responsible for the development of solar lentigine

and heat

produce and increase inflammatory cytokines, such as IL-1α, IL-6, and TNFα,

to produce matrix

the degradation of

and induction of

extracellular matrix proteins

UVB radiation, infrared radiation,

metalloproteinases (MMPs), leading to

wrinkles destruction of collagen and elastic fibers, and formation of wrinkles in sun-exposed skin. The expression of MMP-1 and

MMP3 mRNA and protein levels is increased in dermal fibroblasts by the activation of ERK and JNK

to secrete GM-CSF, which triggers

MMP-1 to a greater extent than neprilysin

the secretion of IL-6, leading predominantly

neutrophil elastase) that participate in extracellular

the degradation of collagen I fibers by the enhanced activity of MMP-1

UV radiation, infrared radiation,

lead to an influx of neutrophils, which are packed with proteolytic enzymes (such as

matrix damage processes,

UV radiation exposure causes keratinocytes

fibroblasts and stimulate the gene expression of

by dermal fibroblasts and

to sagging of the skin via

and heat

which stimulate keratinocytes and dermal fibroblasts

Matrix metalloproteinases (MMPs) are an important family of zinc-containing proteinases that have the capacity to degrade most of the extracellular matrix proteins that comprise the structure of the skin dermal connective tissue [24, 25]. During the past 20 years, a remarkable amount of research has been conducted into both skin aging and MMPs, which can play a significant role in the aging process in the skin. In 1996, Fisher et al. [26] observed that MMP expression was induced and activated after UVB irradiation of the human skin. The molecular model proposed by Fisher et al. suggests that UV radiation can activate various growth factor and cytokine receptors on the cell surface and stimulate mitogen-activated protein kinase (MAPK) signal transduction, which then upregulates transcription factors activator protein-1 (AP-1) composed of c-Jun and c-Fos proteins, and nuclear factor kappa B (NF-κB) in the nucleus. The induction of AP-1 elevates MMP expression, including MMP-1 (collagenase), MMP-3 (stromelysin-1), and MMP-9 (92 kDa gelatinase), and results in the degradation of extracellular matrix components in human skin *in vivo* [1, 27]. This degradation results in accumulation of fragmented, disorganized collagen fibrils, and these damaged collagen products downregulate new collagen synthesis, which suggests that collagen synthesis is negatively regulated by collagen breakdown [28, 29]. The combined actions of MMP-1, MMP-3, and MMP-9 degrade most of type I and III dermal collagen. Furthermore, AP-1 inhibits procollagen biosynthesis by suppressing gene expression of type I and III procollagen in the dermis, which results in a reduced collagen content [25]. The data indicate that epidermal keratinocytes are a major cellular source of MMPs that are produced in response to exposure of human skin to solar UV irradiation. However, dermal cells may also play a role in epidermal production of MMPs, through indirect paracrine mechanisms, by release of growth factors or cytokines, which in turn modulate MMP production by epidermal keratinocytes.

#### **4.3. Oxidative stress**

Free radicals are considered the major contributors to the aging process of skin through the accumulation of ROS. The free radical theory postulates that extremely reactive chemical molecules are a major cause of the aging process [30]. The status of oxidant–antioxidant imbalance is referred to as oxidative stress. Oxidative stress occurring in cells can lead to the oxidation of cell membrane phospholipids, which results in the distortion of the transmem‐ brane signaling pathway [31]. Generally, increased ROS production leads to the activation of MAPK. MAPK induces AP-1, which consequently increases the expression of MMPs, resulting in a decrease of collagen in aged skin [32]. Oxidative stress also contributes to the increased oxidation of macromolecules, such as cellular lipids, proteins, and DNA, which causes cellular dysfunction with age. Age-related accumulation of damaged, oxidized, and aggregated altered proteins might lead to aging [31, 33]. Oxidative protein damage is the most common molecular sign of aging and is observed in photodamaged skin through ROS-mediated protein damage in the upper dermis [33]. Cellular accumulation of lipofuscin, a large protein–lipid aggregate, gradually increases with age, eventually inhibiting proteasome function [31]. Disruption of the epidermal calcium gradient is observed in aged skin because of changes in the composition of the cornified envelope, which results in reduced epidermal barrier function [31].

#### **4.4. Vascular alterations**

Human skin is affected by various environmental conditions such as solar UV radiation, infrared (IR) radiation, and heat, and these stimuli can contribute to skin angiogenesis. Interestingly, although the exposure of acute UV radiation stimulates skin angiogenesis, blood vessels of skin are decreased in chronically photodamaged skin. These differential effects of acute and chronic UV radiation on skin angiogenesis remain unknown. Acute and chronic UV irradiation of skin influences angiogenesis. UV irradiation induces angiogenesis via the upregulation of vascular endothelial growth factor and inhibition of thrombospondin-1, a potent inhibitor of angiogenesis [34].

#### **4.5. Cytokines in skin aging**

Cytokines play a central role in the visible clinical signs of aging [35]. Tumor necrosis factor α (TNF-α), which has a key role in proinflammatory process in skin, inhibits collagen synthesis and induces the production of MMP-9 [35, 36]. 3-Deoxysappanchalcone inhibits MMP-9 expression through the suppression of AP-1 and NF-κB in human skin keratinocytes [36]. Furthermore, high concentrations of TNF-α are correlated with a decrease of collagen pro‐ duction via induction of collagenase activity in fibroblasts [37]. Levels of interleukin (IL)-1 and IL-18 increase with age and promote skin inflammation, which causes age-related processes [38]. UV radiation exposure stimulated IL-1 receptor antagonist (IL-1ra), a competitive inhibitor of IL-1, although IL-1ra production in the skin decreased with age. IL-1ra has a regulatory role in the IL-1 related proinflammatory response, and may play a role in the regulation of IL-1-induced inflammatory responses, and maintain an appropriate balance between IL-1 and IL-1ra [38]. IL-18, an IL-1 superfamily cytokine, is a pleiotropic immune regulator that functions as an angiogenic mediator in inflammation [39]. IL-18 has been implicated as a strong proinflammatory mediator in the pathogenesis of age-related diseases by inducing interferon-γ [39]. In addition, another proinflammatory cytokine, IL-6, increases after menopause and is associated with the formation of skin wrinkles [40]. IL-6 levels are upregulated on exposure to UV radiation [41]. Increased levels of cysteine-rich protein 61 (CCN1) are observed in dermal fibroblasts with age, and CCN1 contributes to the skin connective tissue aging by collagen reduction and degradation in aged human skin *in vivo* [42]. Furthermore, CCN1 induced age-associated secretory proteins, including various proinflam‐ matory cytokines and MMPs, which results in aging of connective tissue [42].

#### **4.6. Other environmental stressors in skin aging**

#### *4.6.1. Tobacco smoke*

dermis, which results in a reduced collagen content [25]. The data indicate that epidermal keratinocytes are a major cellular source of MMPs that are produced in response to exposure of human skin to solar UV irradiation. However, dermal cells may also play a role in epidermal production of MMPs, through indirect paracrine mechanisms, by release of growth factors or

Free radicals are considered the major contributors to the aging process of skin through the accumulation of ROS. The free radical theory postulates that extremely reactive chemical molecules are a major cause of the aging process [30]. The status of oxidant–antioxidant imbalance is referred to as oxidative stress. Oxidative stress occurring in cells can lead to the oxidation of cell membrane phospholipids, which results in the distortion of the transmem‐ brane signaling pathway [31]. Generally, increased ROS production leads to the activation of MAPK. MAPK induces AP-1, which consequently increases the expression of MMPs, resulting in a decrease of collagen in aged skin [32]. Oxidative stress also contributes to the increased oxidation of macromolecules, such as cellular lipids, proteins, and DNA, which causes cellular dysfunction with age. Age-related accumulation of damaged, oxidized, and aggregated altered proteins might lead to aging [31, 33]. Oxidative protein damage is the most common molecular sign of aging and is observed in photodamaged skin through ROS-mediated protein damage in the upper dermis [33]. Cellular accumulation of lipofuscin, a large protein–lipid aggregate, gradually increases with age, eventually inhibiting proteasome function [31]. Disruption of the epidermal calcium gradient is observed in aged skin because of changes in the composition of the cornified envelope, which results in reduced epidermal barrier function

Human skin is affected by various environmental conditions such as solar UV radiation, infrared (IR) radiation, and heat, and these stimuli can contribute to skin angiogenesis. Interestingly, although the exposure of acute UV radiation stimulates skin angiogenesis, blood vessels of skin are decreased in chronically photodamaged skin. These differential effects of acute and chronic UV radiation on skin angiogenesis remain unknown. Acute and chronic UV irradiation of skin influences angiogenesis. UV irradiation induces angiogenesis via the upregulation of vascular endothelial growth factor and inhibition of thrombospondin-1, a

Cytokines play a central role in the visible clinical signs of aging [35]. Tumor necrosis factor α (TNF-α), which has a key role in proinflammatory process in skin, inhibits collagen synthesis and induces the production of MMP-9 [35, 36]. 3-Deoxysappanchalcone inhibits MMP-9 expression through the suppression of AP-1 and NF-κB in human skin keratinocytes [36]. Furthermore, high concentrations of TNF-α are correlated with a decrease of collagen pro‐ duction via induction of collagenase activity in fibroblasts [37]. Levels of interleukin (IL)-1 and

cytokines, which in turn modulate MMP production by epidermal keratinocytes.

**4.3. Oxidative stress**

64 Molecular Mechanisms of the Aging Process and Rejuvenation

[31].

**4.4. Vascular alterations**

potent inhibitor of angiogenesis [34].

**4.5. Cytokines in skin aging**

Like UV radiation exposure, smoking can result in extrinsic skin aging. Findings from large epidemiological studies imply that there is a link between tobacco smoking and premature skin aging [43–45]. Skin damage from long-term smoking can result in a "smoker's face" and can cause facial skin to appear grayish and lines to develop around the eyes and mouth, through damage to collagen fibers and elastin in the dermis [46]. Significantly increased levels of MMP-1 mRNA are observed in the dermal connective tissue of smokers compared with nonsmokers [47]. Increased MMP-1 leads to the degradation of collagen and elastic fibers, which are major extracellular matrix proteins in the dermis. To confirm the pathogenic role of tobacco in skin aging, Morita et al. [46] showed that topical application of water-soluble tobacco smoke extract to the backs of mice led to a loss of collagen bundles and a concomitant increase of damaged collagen in the upper dermis, which mimicked age-related skin. Furthermore, several *in vitro* studies provided possible pathomechanisms for the association between tobacco smoke extract and skin aging. Tobacco smoke extract reduces procollagen types I and III and induced the production of MMP-1 and 3, which degrades extracellular matrix proteins, and also results in abnormal regulation of extracellular matrix deposition in human cultured skin fibroblasts [48]. Tobacco smoke extract also inhibited cellular responsiveness to trans‐ forming growth factor-β (TGF-β), a key mediator of collagen synthesis through the induction of a nonfunctional form of TGF-β, and downregulation of the TGF-β receptor in supernatants of cultured skin fibroblasts, leading to decreased synthesis of extracellular matrix [49]. Moreover, tobacco smoke is a major source of polycyclic aromatic hydrocarbon exposure in humans. In this regard, it has been shown that tobacco smoke extract induced MMP-1 expression via activation of the aryl hydrocarbon receptor (AhR) signaling pathway in human fibroblasts and keratinocytes [50].

#### *4.6.2. Infrared radiation and heat*

Heat energy may be transmitted by IR radiation, which increases skin temperature. Lee et al. [51] found that the temperature of human skin can increase to about 40°C under IR irradiation following the conversion of absorbed IR radiation into heat energy. Chronic exposure of skin to heat may cause premature skin aging, just like UV radiation. The expression of MMP-1 and MMP-3 is induced by heat shock in cultured normal human skin fibroblasts through ERK and JNK activation [52]. Decreased type I procollagen levels and increased MMP-1 expression were observed in human skin exposed to IR radiation, which suggests that chronic IR irradiation can cause skin wrinkling [53]. MMP-12, which is the most active MMP against elastic fiber network in human skin, was induced after heat treatment *in vivo* and thereby contributed to the development of solar elastosis in photoaged skin, thereby contributing to premature skin aging [54]. IR irradiation induces an angiogenic switch by the upregulation of vascular endothelial growth factor and downregulation of thrombospondin-2, which leads to angio‐ genesis and inflammation in human skin *in vivo* [55].

#### *4.6.3. Environmental pollutants*

Exposure to outdoor air pollution from traffic and industry is associated with an increased risk of signs of extrinsic skin aging, in particular pigmented spots and wrinkles in white women of European ancestry [56]. Polycyclic aromatic hydrocarbons are the major participants and trigger the AhR signaling pathway, because their lipophilicity enables them to penetrate the skin easily. Activation of the AhR pathway increases MMP-1 expression in the normal human keratinocytes [57]. In addition, AhR signaling pathway could contribute to the modulate melanogenesis by upregulating tyrosinase enzyme activity [58]. These findings suggest that polycyclic aromatic hydrocarbon-induced AhR activation may play a significant role in the formation of dark pigmentation and coarse wrinkles, which are clinical hallmarks of extrinsic aging. Indoor air pollution released by the combustion of solid fuels for heating is a major environmental and public health challenge in developing countries [59]. Recently, Li et al. [60] reported that indoor air pollution from cooking with solid fuels was significantly associated with 5–8% deeper facial wrinkles and folds and an increased risk of developing fine wrinkles on the dorsum of hands in Chinese women. It is thus likely that exposure to indoor combustion of solid fuels might induce the same molecular pathways in skin cells as outdoor pollution and thereby cause wrinkle formation.

#### **5. Molecular aspects of antiaging treatments**

#### **5.1. Topical retinoids**

Topical retinoids (vitamin A derivatives, also known as retinol) are the main stay of treatment of patients gradually photoaging [61]. Tretinoin and tazarotene are the only two topical retinoids that are currently U.S. Food and Drug Administration (FDA) approved for the treatment of photoaging. The detailed mechanism of action of retinoids has been elucidated. They bind to and activate the retinoic acid receptors (RARs), which are present in a nuclear and work in pairs. These nuclear receptors have two binding sites, which is one for the ligands (retinoid), and one for specific DNA sequences of a target gene. In the presence of the ligands, those heterodimers can recognize a DNA sequence and regulate transcription. Their signal transduction is believed to be implicated in the synthesis of procollagen, thus increasing the formation of type I and III collagen, and inhibiting MMPs [1, 26]. Although the precise mechanisms are unknown, the induction of dermal collagen appears to be a crucial factor. Retinoic acid stimulated production of type VII collagen (anchoring fibrils) and type I collagen [4]. Clinically, retinoids reduce the appearance of fine lines, improve skin texture, correct tone and elasticity, and slow the progression of photoaging. The clinical improvements of topical retinoid therapy are typically seen after several weeks of therapy [62, 63]. A 24-month, doubleblind, vehicle-controlled, multicenter study of tretinoin cream versus vehicle in the treatment of photoaged facial skin was conducted. Topical administration of tretinoin cream for 24 months has proved to be effective in treating clinical signs including fine wrinkling at 2 months, hyperpigmentation at 4 months, coarse wrinkling at 1 month, and sallowness at 4 months [64]. In addition to these clinical benefits, the following histopathological effects are well documented: epidermal hyperplasia and thickening, a decrease of epidermal melanin content, an increase of collagen synthesis, and a decrease of dermal collagen breakdown by inhibiting MMPs and a decrease of p53 expression [65]. Topical retinoids may cause an irritant reaction such as erythema, scaling, burning sensation, and dermatitis, and these reactions cause to decrease patient compliance. Therefore, retinoids should be initiated at the lowest effective dose to minimize adverse reactions [66]. Although tazarotene is the only topical retinoid with a category X designation, topical retinoids are not recommended during pregnancy or lactation [67].

#### **5.2. Cosmeceuticals**

*4.6.2. Infrared radiation and heat*

66 Molecular Mechanisms of the Aging Process and Rejuvenation

*4.6.3. Environmental pollutants*

and thereby cause wrinkle formation.

**5.1. Topical retinoids**

**5. Molecular aspects of antiaging treatments**

genesis and inflammation in human skin *in vivo* [55].

Heat energy may be transmitted by IR radiation, which increases skin temperature. Lee et al. [51] found that the temperature of human skin can increase to about 40°C under IR irradiation following the conversion of absorbed IR radiation into heat energy. Chronic exposure of skin to heat may cause premature skin aging, just like UV radiation. The expression of MMP-1 and MMP-3 is induced by heat shock in cultured normal human skin fibroblasts through ERK and JNK activation [52]. Decreased type I procollagen levels and increased MMP-1 expression were observed in human skin exposed to IR radiation, which suggests that chronic IR irradiation can cause skin wrinkling [53]. MMP-12, which is the most active MMP against elastic fiber network in human skin, was induced after heat treatment *in vivo* and thereby contributed to the development of solar elastosis in photoaged skin, thereby contributing to premature skin aging [54]. IR irradiation induces an angiogenic switch by the upregulation of vascular endothelial growth factor and downregulation of thrombospondin-2, which leads to angio‐

Exposure to outdoor air pollution from traffic and industry is associated with an increased risk of signs of extrinsic skin aging, in particular pigmented spots and wrinkles in white women of European ancestry [56]. Polycyclic aromatic hydrocarbons are the major participants and trigger the AhR signaling pathway, because their lipophilicity enables them to penetrate the skin easily. Activation of the AhR pathway increases MMP-1 expression in the normal human keratinocytes [57]. In addition, AhR signaling pathway could contribute to the modulate melanogenesis by upregulating tyrosinase enzyme activity [58]. These findings suggest that polycyclic aromatic hydrocarbon-induced AhR activation may play a significant role in the formation of dark pigmentation and coarse wrinkles, which are clinical hallmarks of extrinsic aging. Indoor air pollution released by the combustion of solid fuels for heating is a major environmental and public health challenge in developing countries [59]. Recently, Li et al. [60] reported that indoor air pollution from cooking with solid fuels was significantly associated with 5–8% deeper facial wrinkles and folds and an increased risk of developing fine wrinkles on the dorsum of hands in Chinese women. It is thus likely that exposure to indoor combustion of solid fuels might induce the same molecular pathways in skin cells as outdoor pollution

Topical retinoids (vitamin A derivatives, also known as retinol) are the main stay of treatment of patients gradually photoaging [61]. Tretinoin and tazarotene are the only two topical retinoids that are currently U.S. Food and Drug Administration (FDA) approved for the treatment of photoaging. The detailed mechanism of action of retinoids has been elucidated.

Cosmeceuticals encompass a heterogeneous category of nonprescription topical products, including antioxidants, vitamins, hydroxy acids, and plant extracts [68]. Cosmeceuticals are marketed to the consumer based on the claims of their antiaging effects. Although some products may have scientific rationale and produce visible results in the treatment of photo‐ aging, they are not classified as drugs. Most have not been studied thoroughly, especially on human skin, and these products are not subject to the rigorous testing or regulation by agencies such as the FDA. However, the most cosmeceuticals serve a role in keeping the skin moistur‐ ized, maintain the homeostasis of the skin, and many are combined with a topical retinol to enhance their antiaging benefits. As mentioned above, UV and IR irradiation of skin leads to free radical formation. It has been hypothesized that antioxidants scavenge these free radicals and thus protect cells from damage. The antioxidants include coenzyme Q, lipoic acid, vitamin C, and vitamin E [69–71]. Among the many cosmeceuticals, there is increasing interest in botanical antioxidants. Representatively, green tea polyphenols (GTPs) have gained attention because of their potent antioxidant activities. In animal models, UV radiation-induced cutaneous edema and cyclooxygenase activity could be significantly inhibited by feeding GTPs to the animals. After topical application of creams containing GTPs, GTPs on mouse skin decreased the UV-induced hyperplasia, edema, and myeloperoxidase activity. Epigallocate‐ chin-3-gallate (EGCG), which is a polyphenolic constituent of GTP, has been reported to have the inhibitory effect on the expression and activity of MMPs and leukocyte elastase, which has an important role in tumor invasion and metastasis. In addition, GTPs have been shown to decrease the levels of oxidative stress, prevent UV radiation-induced DNA damage, their potential immunosuppressive effects, and skin cancers [72, 73]. Like GTPs, other botanic extracts have been described to contain potent antioxidant components, including curcumin (turmeric), silymarin (milk thistle), apigenin, resveratrol (red grape), and genistein (soy bean) [74]. In fact, recently, it has been demonstrated that these components can inhibit the inflam‐ matory response by downregulation of various proinflammatory mediators, inhibition of activated immune cells, or inhibiting inducible nitric oxide synthase (iNOS) and cyclooxyge‐ nase-2 (COX-2) via its inhibitory effects on NF-κB or AP-1 [75]. Further investigations are needed to focus on the potential application of cosmeceuticals and determine how they can contribute to improvements in skin aging at the maximum.

#### **5.3. Cytokines and growth factors**

Cytokines play a crucial role in the development of clinical features of skin aging. Numerous proinflammatory cytokines including TNF-α, IL-1, and IL-18 can affect the aging process by triggering collagen breakdown via upregulation of MMP-9. These inflammatory cytokines can lower the skin immunity and thus increases the risk of skin infections in old age [35]. Many growth factors are involved in wound healing and have the ability to increase fibroblast and keratinocyte proliferation within the dermis, thus inducing extracellular matrix formation [76]. Growth factors are produced and secreted by many skin cell types, including fibroblasts, keratinocytes, and melanocytes. These secreted growth factors are those that regulate the immune system, also known as cytokines [77, 78]. It is important to combine growth factors and cytokines (referred to collectively as GFs) because they work together and regulate each other via inter- and intracellular signaling pathways. The defined role of GFs in healing of skin wounds allows a parallel model to be developed for the role of GFs in skin rejuvenation. Like the skin wound-healing process, topical and injectable GFs have the potential to modify complex cellular network leading to the increase of collagen synthesis and decrease of collagen degradation. Therefore, they have emerged as an intriguing therapeutic modality to address skin aging through the stimulation of cell regeneration [79, 80]. In fact, topical GFs can cross the skin barrier and bind to cell surface receptors, which trigger a signaling cascade and lead to stimulate keratinocyte proliferation [81]. However, a limited number of controlled studies demonstrate that topically applied GFs can stimulate collagen synthesis and epidermal thickening, which is associated with clinical improvement in signs of photoaging. Because the use of GFs is still in an early stage, continued study to determine their efficacy is needed.

#### **5.4. Neurotoxins and dermal fillers**

Over the past 10 years, the use of botulinum toxin and dermal fillers for aesthetic purposes has risen sharply. Botulinum toxin is an injectable neuromodulator used to reduce fine lines and wrinkles. The toxin functions by inhibiting acetylcholine release at the neuromuscular junction, resulting in flaccid paralysis of targeted muscles [82]. It has been demonstrated that botulinum toxin decreased both collagen synthesis and the expression of MMP-9 in human dermal fibroblasts *in vitro*, leading to decreases collagen degradation [83]. Furthermore, botulinum toxin can also stimulate the expression of type I collagen in human dermal fibro‐ blasts, suggesting that botulinum toxin can stimulate remodeling of ECM, an essential for the rejuvenation of skin aging [83].

Dermal filler injections are used to improve coarse wrinkles and gradual loss of tissue volume [84]. Regardless of trade name, products with smaller particles (softer consistency) are the most useful for superficial injection to address fine lines, whereas larger particle (stiffer) hyaluronic fillers (HA fillers) are the best suited for deeper injection to treat volume loss and deep rhytids [85]. In general, superficial "fine-line" products last approximately 6 months or more, whereas larger particle products last for 6–12 months. In recent years, combination treatment with botulinum toxin and fillers was recommended to achieve superior clinical outcomes and greater patient satisfaction [86, 87].

Several studies demonstrated that fibroblasts in aged skin could be "rejuvenated" by enhancing structural support and mechanical force by injection of dermal filler, cross-linked hyaluronic acid, into the skin [88]. In addition, injection of dermal HA filler can stimulate fibroblasts to produce type I collagen associated with increase in mechanical forces. Enhanced mechanical support of the ECM also stimulates the fibroblast, endothelial cells, and keratinocytes proliferation, resulting in increase of vasculature and epidermal thickness mediated by upregulation of TGF-β receptor and various growth factors. Consistent with these observations in human skin, injection of dermal filler into derma**l** equivalent cultures *in vitro* induces elongation of fibroblasts and type I collagen synthesis by enhancing structural support of the ECM [88].

#### **5.5. Lasers and other novel devices**

chin-3-gallate (EGCG), which is a polyphenolic constituent of GTP, has been reported to have the inhibitory effect on the expression and activity of MMPs and leukocyte elastase, which has an important role in tumor invasion and metastasis. In addition, GTPs have been shown to decrease the levels of oxidative stress, prevent UV radiation-induced DNA damage, their potential immunosuppressive effects, and skin cancers [72, 73]. Like GTPs, other botanic extracts have been described to contain potent antioxidant components, including curcumin (turmeric), silymarin (milk thistle), apigenin, resveratrol (red grape), and genistein (soy bean) [74]. In fact, recently, it has been demonstrated that these components can inhibit the inflam‐ matory response by downregulation of various proinflammatory mediators, inhibition of activated immune cells, or inhibiting inducible nitric oxide synthase (iNOS) and cyclooxyge‐ nase-2 (COX-2) via its inhibitory effects on NF-κB or AP-1 [75]. Further investigations are needed to focus on the potential application of cosmeceuticals and determine how they can

Cytokines play a crucial role in the development of clinical features of skin aging. Numerous proinflammatory cytokines including TNF-α, IL-1, and IL-18 can affect the aging process by triggering collagen breakdown via upregulation of MMP-9. These inflammatory cytokines can lower the skin immunity and thus increases the risk of skin infections in old age [35]. Many growth factors are involved in wound healing and have the ability to increase fibroblast and keratinocyte proliferation within the dermis, thus inducing extracellular matrix formation [76]. Growth factors are produced and secreted by many skin cell types, including fibroblasts, keratinocytes, and melanocytes. These secreted growth factors are those that regulate the immune system, also known as cytokines [77, 78]. It is important to combine growth factors and cytokines (referred to collectively as GFs) because they work together and regulate each other via inter- and intracellular signaling pathways. The defined role of GFs in healing of skin wounds allows a parallel model to be developed for the role of GFs in skin rejuvenation. Like the skin wound-healing process, topical and injectable GFs have the potential to modify complex cellular network leading to the increase of collagen synthesis and decrease of collagen degradation. Therefore, they have emerged as an intriguing therapeutic modality to address skin aging through the stimulation of cell regeneration [79, 80]. In fact, topical GFs can cross the skin barrier and bind to cell surface receptors, which trigger a signaling cascade and lead to stimulate keratinocyte proliferation [81]. However, a limited number of controlled studies demonstrate that topically applied GFs can stimulate collagen synthesis and epidermal thickening, which is associated with clinical improvement in signs of photoaging. Because the use of GFs is still in an early stage, continued study to determine their efficacy is needed.

Over the past 10 years, the use of botulinum toxin and dermal fillers for aesthetic purposes has risen sharply. Botulinum toxin is an injectable neuromodulator used to reduce fine lines and wrinkles. The toxin functions by inhibiting acetylcholine release at the neuromuscular junction, resulting in flaccid paralysis of targeted muscles [82]. It has been demonstrated that

contribute to improvements in skin aging at the maximum.

**5.3. Cytokines and growth factors**

68 Molecular Mechanisms of the Aging Process and Rejuvenation

**5.4. Neurotoxins and dermal fillers**

Laser resurfacing is a skin rejuvenation technology developed to target the cutaneous signs of photodamage. Lasers and other light source procedures are divided into ablative and nona‐ blative resurfacing. Laser procedures are based on the theory of selective photothermolysis [89]. Ablative lasers were first used in the 1980s and markedly improved rhytids, dyspigmen‐ tation, skin laxity, and other signs of photoaging by inducing dermal collagen remodeling. The traditional ablative lasers consisted of the 10,600-nm CO2 and 2940-nm erbium-doped yttrium aluminum garnet (Er:YAG) lasers. Ablative laser resurfacing employs CO2 and Er:YAG lasers, which both target water as their chromophore. CO2 lasers emit light at 10,600 nm in the far-IR electromagnetic spectrum [90, 91]. Er:YAG lasers, introduced in 1996, emit light at 2940 nm, closer to the absorption peak of water, which results in very superficial absorption of laser light [92]. When compared with a CO2 laser, the absorption coefficient for the Er:YAG laser is 16 times higher [93]. Ablative laser resurfacing provides significant improvement of photo‐ damaged skin, photoinduced rhytids, dyschromias, and scars. However, because of the relatively high risk of adverse effects and long recovery period, fractional ablative and other nonablative lasers have since been developed. Nonablative lasers have been developed as an alternative to traditional ablative resurfacing with the aim to comply with patients' new demands for short healing time and reduced discomfort and are widely used for dermal collagen remodeling [94, 95]. Long-pulsed 1064-nm neodymium-doped (Nd): YAG laser treatment increased dermal collagen and decreased the expression of MMP-1 associated with the increased expression of TGF-β [96]. Fractional radiofrequency therapy is a novel, nonin‐ vasive method of tissue tightening. Radiowave energy is delivered deep into the skin, which causes water in the skin cells to heat up, stimulating the production of heat-shock proteins and promoting the wound-healing response [97]. Side effects including dyspigmentation, keloids, or thermal injury can occur and need to be considered. Therefore, in managing photoaging, treatments should be tailored to target the specific clinical features in different skin types and ethnic origin of the patients, and sufficient epidermal cooling after the treatment is required [98].

#### **Acknowledgements**

This work was supported by the Basic Science Research program through the National Research Foundation of Korea (NRF), which is funded by the Ministry of Education, Science and Technology (2015R1C1A2A01055073).

#### **Author details**

Miri Kim and Hyun Jeong Park\*

\*Address all correspondence to: hjpark@catholic.ac.kr

Department of Dermatology, Yeouido St. Mary's Hospital, The Catholic University of Korea, Seoul, Korea

#### **References**


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collagen remodeling [94, 95]. Long-pulsed 1064-nm neodymium-doped (Nd): YAG laser treatment increased dermal collagen and decreased the expression of MMP-1 associated with the increased expression of TGF-β [96]. Fractional radiofrequency therapy is a novel, nonin‐ vasive method of tissue tightening. Radiowave energy is delivered deep into the skin, which causes water in the skin cells to heat up, stimulating the production of heat-shock proteins and promoting the wound-healing response [97]. Side effects including dyspigmentation, keloids, or thermal injury can occur and need to be considered. Therefore, in managing photoaging, treatments should be tailored to target the specific clinical features in different skin types and ethnic origin of the patients, and sufficient epidermal cooling after the treatment is required

This work was supported by the Basic Science Research program through the National Research Foundation of Korea (NRF), which is funded by the Ministry of Education, Science

Department of Dermatology, Yeouido St. Mary's Hospital, The Catholic University of Korea,

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## **The Rejuvenation of the Immune System: Physiological, Cellular, and Molecular Mechanisms**

Iryna Pishel and Gennadij Butenko

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/64046

#### **Abstract**

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Age-related immune dysfunction has been confirmed by many studies. All of these changes result in increased susceptibility to infectious diseases and pathological conditions associated with inflammation or autoreactivity. To prevent these changes, we used various model approaches as follows: (i) the models of hematopoietic stem cells (HSCs) transplantation in irradiated chimeras, (ii) the model of heterochronic parabio‐ sis that provides regular physiological exchange by blood factors between partners, and (iii) cocultivation of lymphoid cells and niche-forming cells in vitro to determine its intercellular communication mechanisms. It was shown that the old HSCs equally effectively restore the immune system of young animals and their own. But, the young hematopoietic cells behaved like old in the old organism. Parabiosis model demon‐ strated that regular exchange by blood factors between heterochronic partners does not lead to the old system rejuvenation. And we observed impaired capacity of splenic CD11c+ DC and macrophages from young heterochronic parabionts to co-stimulate proliferation of T-cells in vitro with statistically significant decrease in nuclear factorkappa B (NFκB) p65 and increased expression of IκB*α* during early activation events. These findings suggest that age-related changes in the immune system are multifactor process, and whole-system environment of the organism plays a crucial role in the occurrence of age-related immune system alterations.

**Keywords:** immune system, T-cell, heterochronic chimeras, aging, parabiosis

#### **1. Introduction**

The immune system is a powerful barrier that protects the organism from the damaging factors of various origins: microorganisms, viruses, transformed cells, and tissues. Tumors, as well as

© 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.

damaged and aged cells, are damaging factors that appear throughout the life. It is known that the incidence of various infectious diseases, autoimmune diseases, and cancer increases with age. It is expected that these changes can be caused by age-related disorders of the immune system. The relationship between the occurrence of age-related diseases with defects in the immune system has led to the hypothesis that the aging of the immune system can trigger development of most age-related diseases. For this reason, great emphasis is paid to study aging processes of the immune system.

The aging of the immune system is the most striking example of the changes that occur in the aging. Age-related changes in the immune system begin to develop very early, still in puberty [1, 2]. Age-related disorders are observed in the functioning of many types of lymphoid cells, but the most significant changes occur in the population of T-lymphocytes. It is known that the T-cell response in the elderly is accompanied by breach of lymphocyte's ability to transmit intracellular signals, reduction in diversity repertoire of T-cell receptor, decrease the prolifer‐ ative response on antigen stimulation (response to antigens, mitogens, lectins, antibodies to CD3, etc.), and changes in the repertoire of produced cytokines [1].

The study of age-related changes in the immune system certainly gives rise to the question: due to what parts of the immune system are experiencing these violations? It is known that immune function is carried out by cells that are continuously updated in the body, passing through the stages of hematopoietic stem cells of bone marrow (BM) to highly differentiated immunocompetent cells: different subpopulations of T-lymphocytes, B-lymphocytes, and monocyte-macrophages. The lifespan of most of these cells depending upon the type and a functional feature ranges from several days to several months or even years. But, the duration of life is relatively short when compared with the duration of life of the organism in which they function [3].

Investigation of age-related disorders in the function of hematopoietic stem cells has shown that with aging the amount of B-lymphocyte progenitor cells reduces [4], such as diversity of B-cell repertoire in the mouse bone marrow [5]. For T-cell, the main factor that contributes to the reduction in T-lymphopoiesis with age is the thymic atrophy [1]. At the same time, more recent studies have shown that reducing the production of naïve lymphocytes with age is also dependent on intracellular changes in hematopoietic stem cells. It is known that old HSCs after transplantation to the young lethally irradiated mice can restore hematopoiesis with "aged" properties, including a reduced ability to generate T-cells [4, 6]. Reducing the number of Band T-lymphocyte progenitor cells changes the cellular composition of hematopoietic system, which leads to the relative dominance of myeloid cells, often called age-dependent myeloid skewed or offset [4, 6, 7].

It is assumed that trigger of the immune system dysfunction is age-related involution of the thymus. T-progenitor cells that migrated to the thymus from the bone marrow pass difficult way of "training" with a tight positive and negative selection. Only a small proportion of Tlymphocytes differentiates and migrates from the thymus to peripheral lymphoid organs. With aging, there is a loss of thymic epithelium, and as a result, defects occur in the maturation and differentiation of progenitors of T-lymphocytes. Additionally, the thymus is a target for various pathogens that utilize it for their persistence and affect editing repertoire of naïve Tcells [8].

damaged and aged cells, are damaging factors that appear throughout the life. It is known that the incidence of various infectious diseases, autoimmune diseases, and cancer increases with age. It is expected that these changes can be caused by age-related disorders of the immune system. The relationship between the occurrence of age-related diseases with defects in the immune system has led to the hypothesis that the aging of the immune system can trigger development of most age-related diseases. For this reason, great emphasis is paid to study aging

The aging of the immune system is the most striking example of the changes that occur in the aging. Age-related changes in the immune system begin to develop very early, still in puberty [1, 2]. Age-related disorders are observed in the functioning of many types of lymphoid cells, but the most significant changes occur in the population of T-lymphocytes. It is known that the T-cell response in the elderly is accompanied by breach of lymphocyte's ability to transmit intracellular signals, reduction in diversity repertoire of T-cell receptor, decrease the prolifer‐ ative response on antigen stimulation (response to antigens, mitogens, lectins, antibodies to

The study of age-related changes in the immune system certainly gives rise to the question: due to what parts of the immune system are experiencing these violations? It is known that immune function is carried out by cells that are continuously updated in the body, passing through the stages of hematopoietic stem cells of bone marrow (BM) to highly differentiated immunocompetent cells: different subpopulations of T-lymphocytes, B-lymphocytes, and monocyte-macrophages. The lifespan of most of these cells depending upon the type and a functional feature ranges from several days to several months or even years. But, the duration of life is relatively short when compared with the duration of life of the organism in which

Investigation of age-related disorders in the function of hematopoietic stem cells has shown that with aging the amount of B-lymphocyte progenitor cells reduces [4], such as diversity of B-cell repertoire in the mouse bone marrow [5]. For T-cell, the main factor that contributes to the reduction in T-lymphopoiesis with age is the thymic atrophy [1]. At the same time, more recent studies have shown that reducing the production of naïve lymphocytes with age is also dependent on intracellular changes in hematopoietic stem cells. It is known that old HSCs after transplantation to the young lethally irradiated mice can restore hematopoiesis with "aged" properties, including a reduced ability to generate T-cells [4, 6]. Reducing the number of Band T-lymphocyte progenitor cells changes the cellular composition of hematopoietic system, which leads to the relative dominance of myeloid cells, often called age-dependent myeloid

It is assumed that trigger of the immune system dysfunction is age-related involution of the thymus. T-progenitor cells that migrated to the thymus from the bone marrow pass difficult way of "training" with a tight positive and negative selection. Only a small proportion of Tlymphocytes differentiates and migrates from the thymus to peripheral lymphoid organs. With aging, there is a loss of thymic epithelium, and as a result, defects occur in the maturation and differentiation of progenitors of T-lymphocytes. Additionally, the thymus is a target for

CD3, etc.), and changes in the repertoire of produced cytokines [1].

processes of the immune system.

78 Molecular Mechanisms of the Aging Process and Rejuvenation

they function [3].

skewed or offset [4, 6, 7].

With aging, there are several types of changes in the functioning of T-cells including the decrease in the proliferative response after T-cell receptor (TCR)/cluster of differentiation 3 (CD3) activation, expansion memory cells, and simultaneous reduction in the number of naïve T-cells and the accumulation of CD28-T-cells. Though the mechanism of age changes is not fully known, the change in intracellular signaling associated with the TCR/CD3 complex is regarded as the most significant damaging factor [9].

Key sources of the above factors, which regulate the functioning of T-cells, are elements of their microenvironment (niche) in the lymphoid organs. Microenvironmental cells include fibroblasts and antigen-presenting cells (APCs hereinafter), particularly macrophages and dendritic cells (DCs hereinafter), which play a key role in the proliferation and function of Tlymphocytes. It is known that the ability of dendritic cells to stimulate an immune response to the antigens decreases with age. It also reduces the ability of dendritic cells and macrophages to stimulate protective anti-tumor immune response. But information about age-related changes in the co-stimulatory properties of lymphoid niche cells is now somewhat contradic‐ tory as a whole [10, 11].

Thus, the mechanism of changes in the functions of the immune system in aging involves three main components that affect the function: age of hematopoietic stem progenitor cells, age of lymphoid organs microenvironment, and the impact of the overall environment of the old organism on the development of immune responses.

To isolate the effects of these components and to establish mechanisms of age dysfunction of the immune system, we use artificial models of biological systems, composed of elements of different ages, the so-called heterochronic chimeras. We used three main approaches to the study of the mechanisms of age-related changes in the immune system: (1) study of the role of the aging of hematopoietic stem cell disorders in the immune system on the model of lethally irradiated heterochronic chimeras; (2) study of the role of the system environment on the model heterochronic parabiosis, which provides a full exchange with growth, hormonal factors, and blood cells between animals of different ages; and (3) study of disorders of the lymphoid cell niche in reducing immune function with aging.

#### **2. The aging of the immune system and approaches to its rejuvenation**

#### **2.1. Investigation of the effect of hematopoietic stem cells aging on the immune system functions in lethally irradiated heterochronic chimeras**

Animals that underwent lethal irradiation generally show 100% destruction of lymphoid and hematopoietic stem cells in the organism and the complete immune dysfunction causes the death within 10–14 days. Replacement of dead cells on donor hematopoietic stem cells results in the restoration of HSCs pool in the bone marrow and immune system functions. All lymphoid cells in thus chimeras are of donor origin.

Thus, we used this model to identify the age differences of the regenerative properties of HSCs, and to elucidate the ability of stem cells to restore the immune system of old animals.


**Table 1.** Thymus weight and number of lymphocyte subpopulations in the spleen of mice different experimental groups, M ± SE.

**Figure 1.** Proliferative response of splenocytes after mitogen stimulation in vitro.The shaded bars, PHA response (10 μg/ml); dark bars, Con A response (5 μg/ml); the spotted bars, LPS response (30 μg/ml). Symbol of the experimental groups: Y, young intact mice; Yy, young irradiated animals which were recovered of BM cells from young mice; Yo, young irradiated animals which were recovered of BM cells from old mice; O, old intact mice; Oy, old irradiated ani‐ mals which were recovered of BM cells from young mice; Oo, old irradiated animals which were recovered of BM cells from old mice. CBA/Ca female mice were irradiated and recovered of 15 × 106 BM cells at 4 (young) and 22 (old) months of age. At 3 months after the recovering mice were euthanized and immune parameters were analyzed. \*, *P* (*t*) < 0.05 comparing young intact animals; #, *P* (*t*) < 0.05 comparing old intact animals. The mice number in each group is at least 8.

Studies were conducted on female CBA/Ca mice, where the age of the recipients and donors was 4 months (young) and 22 months (old) before the start of the experiment. For 3 h prior to administration of HSCs, mice of recipients were irradiated with X-ray radiation dose of 8.5 Gy (dose rate of 0.72 Gy/min). To eliminate mature T-lymphocytes, donor bone marrow cells were treated with monoclonal antibodies to the Thy1.2 mouse antigen. Bone marrow cells were administered intravenously at a dose of 15 × 106 per mouse. Immunological parameters were tested for 3 months after irradiation and bone marrow transplantation.

Thus, we used this model to identify the age differences of the regenerative properties of HSCs,

splenocytes, % 39.3 ± 2.2 38.3 ± 2.3 32.8\* ± 1.9 23.4\* ± 1.2 22.7\*\* ± 2.3 25.9 ± 2.6

splenocytes, % 37.8 ± 2.2 39.7 ± 2.0 47.5 ± 2.4 59.3\* ± 3.6 57.6\*\* ± 3.1 55.1 ± 2.4

**Figure 1.** Proliferative response of splenocytes after mitogen stimulation in vitro.The shaded bars, PHA response (10 μg/ml); dark bars, Con A response (5 μg/ml); the spotted bars, LPS response (30 μg/ml). Symbol of the experimental groups: Y, young intact mice; Yy, young irradiated animals which were recovered of BM cells from young mice; Yo, young irradiated animals which were recovered of BM cells from old mice; O, old intact mice; Oy, old irradiated ani‐ mals which were recovered of BM cells from young mice; Oo, old irradiated animals which were recovered of BM cells

months of age. At 3 months after the recovering mice were euthanized and immune parameters were analyzed. \*, *P* (*t*) < 0.05 comparing young intact animals; #, *P* (*t*) < 0.05 comparing old intact animals. The mice number in each group is

Studies were conducted on female CBA/Ca mice, where the age of the recipients and donors was 4 months (young) and 22 months (old) before the start of the experiment. For 3 h prior to administration of HSCs, mice of recipients were irradiated with X-ray radiation dose of 8.5 Gy (dose rate of 0.72 Gy/min). To eliminate mature T-lymphocytes, donor bone marrow cells were treated with monoclonal antibodies to the Thy1.2 mouse antigen. Bone marrow cells were

from old mice. CBA/Ca female mice were irradiated and recovered of 15 × 106

**Table 1.** Thymus weight and number of lymphocyte subpopulations in the spleen of mice different experimental

splenocytes, %1 27.5 ± 2.0 27.4 ± 0.7 33.0\*\* ± 2.5 69.7\* ± 5.2 61.2\*,

**Y (***n* **= 8) Yy (***n* **= 10) Yo (***n* **= 14) O (***n* **= 7) Oy (***n* **= 10) Oo (***n* **= 7)**
