**Introduction**

**Chapter 1**

**Provisional chapter**

**Introductory Chapter: Homeostasis**

**Introductory Chapter: Homeostasis**

DOI: 10.5772/intechopen.81130

The human body is composed of several systems and organs, consisting of millions of cells that need relatively stable conditions to function and contribute to the survival of the body as a whole. The maintenance of stable conditions for the cells against the variations of the external environment is an essential function of the body and configures the homeostasis.

In 1870, Claude Bernard described the basic principles of physiological regulation, evidencing the body's need to maintain a stable internal environment. This allows life processes to function optimally during periods of environmental fluctuations and disturbances, and to this end, an array of reflexes and regulatory systems that allow the body to thrive and reproduce despite environmental challenges [1]. Later, Cannon [2] coined the term homeostasis to describe the collective activity of bodily systems that monitor and protect vital life parameters against harmful detours. Many of these processes are unnoticed at the conscious level and depend on behavioral, autonomous, endocrine responses among others to prevent the everyday challenges of the internal environment. Cannon defined homeostasis as "a condition that may vary but remains relatively constant." This balance is guaranteed thanks to the physiological processes that act in a coordinated way in the body and that prevent the changes in the environment interfere in its functioning. Factors such as pH, temperature, plasma osmolality, glucose and calcium are critical for the normal functioning of most organisms and therefore

The importance of these factors is due to its universal biochemical effects. Temperature and pH may alter the spatial conformation of the structure and function of enzymes and functional RNAs; temperature and plasma osmolality are important in the integrity and function of the membranes; glucose is a key currency for moving and storing energy; and calcium plays key roles in muscle physiology and multiple signaling pathways. The variation of these factors constitutes a noise that affects all physiological systems, including the homeostatic systems

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

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.81130

are controlled within narrow limits [3].

Fernanda Lasakosvitsch

Fernanda Lasakosvitsch

**1. Introduction**

#### **Chapter 1 Provisional chapter**

#### **Introductory Chapter: Homeostasis Introductory Chapter: Homeostasis**

#### Fernanda Lasakosvitsch Fernanda Lasakosvitsch

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

http://dx.doi.org/10.5772/intechopen.81130

#### **1. Introduction**

The human body is composed of several systems and organs, consisting of millions of cells that need relatively stable conditions to function and contribute to the survival of the body as a whole. The maintenance of stable conditions for the cells against the variations of the external environment is an essential function of the body and configures the homeostasis.

DOI: 10.5772/intechopen.81130

In 1870, Claude Bernard described the basic principles of physiological regulation, evidencing the body's need to maintain a stable internal environment. This allows life processes to function optimally during periods of environmental fluctuations and disturbances, and to this end, an array of reflexes and regulatory systems that allow the body to thrive and reproduce despite environmental challenges [1]. Later, Cannon [2] coined the term homeostasis to describe the collective activity of bodily systems that monitor and protect vital life parameters against harmful detours. Many of these processes are unnoticed at the conscious level and depend on behavioral, autonomous, endocrine responses among others to prevent the everyday challenges of the internal environment. Cannon defined homeostasis as "a condition that may vary but remains relatively constant." This balance is guaranteed thanks to the physiological processes that act in a coordinated way in the body and that prevent the changes in the environment interfere in its functioning. Factors such as pH, temperature, plasma osmolality, glucose and calcium are critical for the normal functioning of most organisms and therefore are controlled within narrow limits [3].

The importance of these factors is due to its universal biochemical effects. Temperature and pH may alter the spatial conformation of the structure and function of enzymes and functional RNAs; temperature and plasma osmolality are important in the integrity and function of the membranes; glucose is a key currency for moving and storing energy; and calcium plays key roles in muscle physiology and multiple signaling pathways. The variation of these factors constitutes a noise that affects all physiological systems, including the homeostatic systems

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

themselves. Since the noise is global (affects all other physiological components in other homeostatic systems), cells cannot filter it or develop private channels to avoid it; instead, the options are to send redundant signals, minimize the spatial and temporal proximity between the transmitter and the receiver, or minimize the variation in the noise-inducing factors. Of all these events, minimizing noise is a key factor that drives the evolution of homeostasis [4].

**Author details**

**References**

Fernanda Lasakosvitsch1,2\*

[cited 2018 Aug 21]

1929;**9**(3):399-431

\*Address all correspondence to: flcastanho@gmail.com

2 Universidade Federal de Sao Paulo, Brazil

doi/10.1086/216202 [cited 2018 Aug 21]

Dowden, Hutchinson & Ross; 1973. 362 p

1 Funzionali Serviços Médicos Ltda, São Paulo, SP, Brazil

[1] Bernard C, Noble D. Experimental Physiology-Paton Lecture the first systems biologist, and the future of physiology. Experimental Physiology [Internet]. 2007;**93**:16-26. Available from: https://pdfs.semanticscholar.org/9049/44f321fef4a0996b00c36a1ce643f7ddce96.pdf

Introductory Chapter: Homeostasis

5

http://dx.doi.org/10.5772/intechopen.81130

[2] Cannon WB. Organization for physiological homeostasis. Physiological Reviews.

[3] Carlson AJ. The Wisdom of the Body. Walter B. Cannon. American Journal of Sociology [Internet]. 1933 Jan;**38**(4):651-651. Available from: https://www.journals.uchicago.edu/

[4] Ramsay DS, Woods SC. Clarifying the roles of homeostasis and allostasis in physiological regulation. Physiological Reviews [Internet]. 2014 Apr;**121**(2):225-247. Available from:

[5] Langley LL. Homeostasis: Origins of the Concept. 1st ed. In: Langley LL, editor. Michigan:

[6] Morrison SF, Nakamura K. Central neural pathways for thermoregulation. Frontiers in Bioscience [Internet]. 2011;**16**:74-104. Available from: http://www.pubmedcentral.nih.gov/

http://www.ncbi.nlm.nih.gov/pubmed/24730599 [cited 2018 Aug 21]

articlerender.fcgi?artid=3051412&tool=pmcentrez&rendertype=abstract

Until the last decades, homeostasis was considered as a reaction to continuous disturbances in vital systems through negative feedback responses. According to Langley [5], homeostasis is a self-regulating negative feedback system that keeps the internal environment constant. Current views on the subject assume that negative feedback systems that are not activated until a disturbance of the regulated variable occurs may be inefficient and fail in its regulatory role. Negative feedback was the first underlying process used to explain how homeostasis works. This was a reactive strategy through which the perturbation of a regulated variable far from its optimal value was detected and, consequently, provoked corrective responses that served to return the variable back to the pre-disturbance levels. For example, a sudden drop in oxygen content in the blood is detected by sensors that are synaptically linked to areas of the brain that control respiratory rate, and respiration and oxygen in the blood are consequently increased. Although negative feedback is certainly an essential component of regulation, based on past experience, organisms learn to prepare early responses to impending disruption, thereby reducing their impact [4].

A fundamental principle of homeostatic regulation is that the answers work in a coordinated way to defend the body parameters critical to the well-being of an animal; that is, that homeostatic regulation is purposeful. Strategies that may appear to be anticipatory use the initial sensory information in an area of the body as the signal of an event that could potentially disrupt a critical regulated variable, activating corrective effectors and preventing the critical variable from being disturbed. For example, when entering a cold environment, skin temperature sensors are activated before there is any change in core temperature (the variable that is presumably homeostatically regulated); and an effect of increased activity of skin thermometers is to trigger a thermopreflection reflex cycle that induces increased metabolic rate or peripheral vasoconstriction that can stabilize central temperature [6].

If the body cannot maintain the homeostasis of the critical variables, normal function is interrupted and a state, or pathological condition, can develop. Diseases are characterized in two general groups according to their origin: those in which the problem arises from an internal insufficiency or failure of some normal physiological process, and those that originate from some external source. The critical patient develops a condition where the loss of organic homeostasis puts him at immediate risk of death. Therapeutic priorities are focused on correcting these imbalances, such as hemodynamic stabilization, restoration and maintenance of blood volume, stabilization of ventilation conditions and correction of other abnormalities such as electrolyte imbalance, metabolic changes and pain control. Thus, medicine can intervene to restore balance through the activation of self-regulating mechanisms, restoration of the pathways that are altered during the disease process and, finally, to achieve homeostasis in critical patients.

#### **Author details**

themselves. Since the noise is global (affects all other physiological components in other homeostatic systems), cells cannot filter it or develop private channels to avoid it; instead, the options are to send redundant signals, minimize the spatial and temporal proximity between the transmitter and the receiver, or minimize the variation in the noise-inducing factors. Of all these events, minimizing noise is a key factor that drives the evolution of homeostasis [4]. Until the last decades, homeostasis was considered as a reaction to continuous disturbances in vital systems through negative feedback responses. According to Langley [5], homeostasis is a self-regulating negative feedback system that keeps the internal environment constant. Current views on the subject assume that negative feedback systems that are not activated until a disturbance of the regulated variable occurs may be inefficient and fail in its regulatory role. Negative feedback was the first underlying process used to explain how homeostasis works. This was a reactive strategy through which the perturbation of a regulated variable far from its optimal value was detected and, consequently, provoked corrective responses that served to return the variable back to the pre-disturbance levels. For example, a sudden drop in oxygen content in the blood is detected by sensors that are synaptically linked to areas of the brain that control respiratory rate, and respiration and oxygen in the blood are consequently increased. Although negative feedback is certainly an essential component of regulation, based on past experience, organisms learn to prepare early responses to impend-

A fundamental principle of homeostatic regulation is that the answers work in a coordinated way to defend the body parameters critical to the well-being of an animal; that is, that homeostatic regulation is purposeful. Strategies that may appear to be anticipatory use the initial sensory information in an area of the body as the signal of an event that could potentially disrupt a critical regulated variable, activating corrective effectors and preventing the critical variable from being disturbed. For example, when entering a cold environment, skin temperature sensors are activated before there is any change in core temperature (the variable that is presumably homeostatically regulated); and an effect of increased activity of skin thermometers is to trigger a thermopreflection reflex cycle that induces increased metabolic rate

If the body cannot maintain the homeostasis of the critical variables, normal function is interrupted and a state, or pathological condition, can develop. Diseases are characterized in two general groups according to their origin: those in which the problem arises from an internal insufficiency or failure of some normal physiological process, and those that originate from some external source. The critical patient develops a condition where the loss of organic homeostasis puts him at immediate risk of death. Therapeutic priorities are focused on correcting these imbalances, such as hemodynamic stabilization, restoration and maintenance of blood volume, stabilization of ventilation conditions and correction of other abnormalities such as electrolyte imbalance, metabolic changes and pain control. Thus, medicine can intervene to restore balance through the activation of self-regulating mechanisms, restoration of the pathways that are altered during the disease process and, finally, to achieve homeostasis

or peripheral vasoconstriction that can stabilize central temperature [6].

ing disruption, thereby reducing their impact [4].

4 Homeostasis - An Integrated Vision

in critical patients.

Fernanda Lasakosvitsch1,2\*


#### **References**


**Section 2**

**Physiological Mechanisms of Homeostasis**

**Physiological Mechanisms of Homeostasis**

**Chapter 2**

**Provisional chapter**

) rhythm is significantly influenced

rhythm. During the light phase, fasting and ghre-

during the

. By contrast, during

**Circadian Body Temperature Rhythm and the**

**Circadian Body Temperature Rhythm and the** 

DOI: 10.5772/intechopen.76229

by fasting/fasting-related hormones. The effect of circadian mechanism and fasting/fast-

light phase in rodents. For the regulation, the suprachiasmatic nucleus (SCN) and clock genes, such as *Cry* and *Clock*, are necessary. In addition, ghrelin and several hypothalamic nuclei, that is, the medial preoptic area, paraventricular nucleus (PVN), and arcuate

lin affect the hypothalamic areas. The activity of the SCN increases and that of the ARC decreases. The SCN sends inhibitory signals to the PVN, which may result in a lower heat

the dark phase, the activity of the SCN decreases and that of the ARC increases. The inhibitory signal from the SCN is less, and the PVN is activated. Heat production of the iBAT

the circadian and thermoregulation systems. The circadian system modulates thermoregulatory response to hypothermia and/or cold depending on time and feeding condition.

The temperature of the cell(s), tissues, organs, and body is an important factor that determines the biological functions and survival of organisms, from prokaryotes to vertebrates, although the preferred temperature varies. Homeothermic animals can constantly regu-

**Keywords:** fasting, ghrerin, leptin, brown adipose tissue, paraventricular nucleus,

is maintained. There are functional and anatomical connections between

ing-related hormones on thermoregulation was examined. Fasting decreases Tb

production in the interscapular brown adipose tissue (iBAT) and Tb

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

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

). Thermoregulation is the balance between heat loss and heat

**Interaction with Energy State**

**Interaction with Energy State**

Yuki Uchida

**Abstract**

increases and Tb

**1. Introduction**

late body temperature (Tb

Yuki Uchida

Kei Nagashima, Ken Tokizawa, Shuri Marui and

Kei Nagashima, Ken Tokizawa, Shuri Marui and

Additional information is available at the end of the chapter

We have revealed that circadian body temperature (Tb

nucleus (ARC), play a key role in the Tb

arcuate nucleus, suprachiasmatic nucleus

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.76229

#### **Circadian Body Temperature Rhythm and the Interaction with Energy State Circadian Body Temperature Rhythm and the Interaction with Energy State**

DOI: 10.5772/intechopen.76229

Kei Nagashima, Ken Tokizawa, Shuri Marui and Yuki Uchida Kei Nagashima, Ken Tokizawa, Shuri Marui and Yuki Uchida

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

http://dx.doi.org/10.5772/intechopen.76229

#### **Abstract**

We have revealed that circadian body temperature (Tb ) rhythm is significantly influenced by fasting/fasting-related hormones. The effect of circadian mechanism and fasting/fasting-related hormones on thermoregulation was examined. Fasting decreases Tb during the light phase in rodents. For the regulation, the suprachiasmatic nucleus (SCN) and clock genes, such as *Cry* and *Clock*, are necessary. In addition, ghrelin and several hypothalamic nuclei, that is, the medial preoptic area, paraventricular nucleus (PVN), and arcuate nucleus (ARC), play a key role in the Tb rhythm. During the light phase, fasting and ghrelin affect the hypothalamic areas. The activity of the SCN increases and that of the ARC decreases. The SCN sends inhibitory signals to the PVN, which may result in a lower heat production in the interscapular brown adipose tissue (iBAT) and Tb . By contrast, during the dark phase, the activity of the SCN decreases and that of the ARC increases. The inhibitory signal from the SCN is less, and the PVN is activated. Heat production of the iBAT increases and Tb is maintained. There are functional and anatomical connections between the circadian and thermoregulation systems. The circadian system modulates thermoregulatory response to hypothermia and/or cold depending on time and feeding condition.

**Keywords:** fasting, ghrerin, leptin, brown adipose tissue, paraventricular nucleus, arcuate nucleus, suprachiasmatic nucleus

#### **1. Introduction**

The temperature of the cell(s), tissues, organs, and body is an important factor that determines the biological functions and survival of organisms, from prokaryotes to vertebrates, although the preferred temperature varies. Homeothermic animals can constantly regulate body temperature (Tb ). Thermoregulation is the balance between heat loss and heat

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

production in the body. The temperature regulation is different among species, and the range of temperature regulation is fairly narrow compared to the larger temperature ranges in the living environment [1].

Homeothermic animals show a circadian Tb rhythm, although the goal of thermoregulation is to maintain a constant Tb . The Tb is higher in the active phase and lower in the inactive phase in both diurnal and nocturnal animals. The Tb rhythm is also observed under conditions wherein the influence of physical activity (i.e., heat production) is minimized. For example, circadian Tb rhythm is observed even in individuals forced to be on complete bed rest [2]. Results suggested that the circadian Tb rhythm can also be regulated.

Despite the usual small amplitude of the circadian Tb rhythm (e.g., less than 1°C in human beings [3]), the rhythm is still important for preserving energy during the inactive phase. The maintenance of higher Tb is energy-costly because more energy from the total daily intake is used for heat production [4]. Therefore, the circadian Tb rhythm may be important in saving energy in homeothermic animals when energy is not needed.

Environmental temperature significantly affects the thermoregulation system. We assessed the circadian Tb change in rats that were placed in an ambient temperature of 18, 25, or 32°C (unpublished data). Laboratory rats are usually housed at 25°C. However, such cold or heat stress did not alter the circadian Tb rhythm. Results suggested that the Tb rhythm is not simply a result of circadian change in heat loss or production in the body. Rather, it is a result of the coordinated thermoregulatory processes that maintain a specific T<sup>b</sup> at a given time of the day. Moreover, the Tb rhythm is probably generated by the association between the circadian and thermoregulation system. However, the mechanism is not yet known.

As mentioned previously, the Tb rhythm remains unchanged even when environmental temperature is altered. However, our previous studies have revealed that the Tb rhythm is remarkably influenced by fasting/fasting-related hormones. It is hypothesized that a lack of energy affects thermoregulation and the T<sup>b</sup> rhythm. However, several previous studies do not support this hypothesis. In this study, a review of the literature on the mechanism and physiological effects of the circadian T<sup>b</sup> rhythm was carried out.

Compared to that during a normal Tb

**Figure 1.** Circadian changes in Tb

, and how?

**temperature during fasting**

decrease in Tb

Tokizawa et al. [9].

Tb

rhythm, the significant decrease in T<sup>b</sup>

and spontaneous activity during *ad lib* feeding and fasting in settings with normal

Circadian Body Temperature Rhythm and the Interaction with Energy State

http://dx.doi.org/10.5772/intechopen.76229

11

phase (i.e., from 36.0°C during feeding to 34.1°C during fasting) may lessen heat transfer from

temperature (27°C) in mice. The x-axis indicates ZT. The lighting condition is shown at the bottom with the open bar denoting the light phase and the closed bar the dark phase. Data were plotted in 5-min bins. The vertical dashed line shows the time when the 48-h fasting period was started. The modified figure was obtained from the manuscript by

the dark phase may be essential in the maintenance of physical activity. On the basis of previ-

phase a regulated phenomenon by the thermoregulation system? (2) Is the circadian system involved in the phenomenon? and (3) Which factor that is associated with fasting causes the

in homeothermic animals is controlled by heat production and heat loss mechanisms.

fasting is still not verified. Fasting is a strong stimulus that decreases metabolic heat production in rodents and pigeons [5, 6, 10–15]. In addition, fasting decreases thermal conductance

the body to the environment and preserve energy. In contrast, the maintenance of Tb

ous studies, the following questions might be raised: (1) Is the reduction in Tb

**3. Physiological mechanism involved in the decrease in body** 

However, whether such control is maintained and generates the circadian Tb

during the light

during the light

rhythm during

during

## **2. Association between fasting and circadian core body temperature rhythm**

Fasting is a strong stimulus that changes the amplitude of the circadian Tb rhythm in both mammals and birds [5–8]. In a previous study by Tokizawa et al. [9], Tb was obtained with a thermometer placed in the abdominal cavity of mice, and it was continuously and noninvasively monitored with a telemetry. The most significant reduction was during the light (inactive) phase, whereas the reduction in the dark (active) phase was not remarkable (**Figure 1A**). In addition, no difference was observed in the spontaneous activity during both phases (**Figure 1B**). Results suggested that less heat production due to a decreased physical activity was not associated with the mechanism involved in the reduction of Tb during fasting. Moreover, a remarkable change in Tb was observed at a specific time, which may indicate an association with the circadian rhythm, although the phase shift of the rhythm was not observed.

Circadian Body Temperature Rhythm and the Interaction with Energy State http://dx.doi.org/10.5772/intechopen.76229 11

production in the body. The temperature regulation is different among species, and the range of temperature regulation is fairly narrow compared to the larger temperature ranges in the

wherein the influence of physical activity (i.e., heat production) is minimized. For example,

beings [3]), the rhythm is still important for preserving energy during the inactive phase. The

Environmental temperature significantly affects the thermoregulation system. We assessed

(unpublished data). Laboratory rats are usually housed at 25°C. However, such cold or heat

a result of circadian change in heat loss or production in the body. Rather, it is a result of the

remarkably influenced by fasting/fasting-related hormones. It is hypothesized that a lack of

support this hypothesis. In this study, a review of the literature on the mechanism and physi-

rhythm was carried out.

thermometer placed in the abdominal cavity of mice, and it was continuously and noninvasively monitored with a telemetry. The most significant reduction was during the light (inactive) phase, whereas the reduction in the dark (active) phase was not remarkable (**Figure 1A**). In addition, no difference was observed in the spontaneous activity during both phases (**Figure 1B**). Results suggested that less heat production due to a decreased physical activ-

association with the circadian rhythm, although the phase shift of the rhythm was not observed.

**2. Association between fasting and circadian core body temperature** 

Fasting is a strong stimulus that changes the amplitude of the circadian Tb

ity was not associated with the mechanism involved in the reduction of Tb

mammals and birds [5–8]. In a previous study by Tokizawa et al. [9], Tb

rhythm is observed even in individuals forced to be on complete bed rest [2].

rhythm can also be regulated.

change in rats that were placed in an ambient temperature of 18, 25, or 32°C

rhythm is probably generated by the association between the circadian and

rhythm remains unchanged even when environmental

rhythm. However, several previous studies do not

was observed at a specific time, which may indicate an

rhythm. Results suggested that the Tb

is energy-costly because more energy from the total daily intake is

rhythm, although the goal of thermoregulation is

rhythm is also observed under conditions

rhythm (e.g., less than 1°C in human

rhythm may be important in saving

rhythm is not simply

rhythm is

rhythm in both

during fasting.

was obtained with a

at a given time of the day.

is higher in the active phase and lower in the inactive phase

living environment [1].

10 Homeostasis - An Integrated Vision

to maintain a constant Tb

maintenance of higher Tb

circadian Tb

the circadian Tb

Moreover, the Tb

**rhythm**

Homeothermic animals show a circadian Tb

Results suggested that the circadian Tb

stress did not alter the circadian Tb

As mentioned previously, the Tb

ological effects of the circadian T<sup>b</sup>

Moreover, a remarkable change in Tb

energy affects thermoregulation and the T<sup>b</sup>

in both diurnal and nocturnal animals. The Tb

. The Tb

Despite the usual small amplitude of the circadian Tb

used for heat production [4]. Therefore, the circadian Tb

energy in homeothermic animals when energy is not needed.

coordinated thermoregulatory processes that maintain a specific T<sup>b</sup>

thermoregulation system. However, the mechanism is not yet known.

temperature is altered. However, our previous studies have revealed that the Tb

**Figure 1.** Circadian changes in Tb and spontaneous activity during *ad lib* feeding and fasting in settings with normal temperature (27°C) in mice. The x-axis indicates ZT. The lighting condition is shown at the bottom with the open bar denoting the light phase and the closed bar the dark phase. Data were plotted in 5-min bins. The vertical dashed line shows the time when the 48-h fasting period was started. The modified figure was obtained from the manuscript by Tokizawa et al. [9].

Compared to that during a normal Tb rhythm, the significant decrease in T<sup>b</sup> during the light phase (i.e., from 36.0°C during feeding to 34.1°C during fasting) may lessen heat transfer from the body to the environment and preserve energy. In contrast, the maintenance of Tb during the dark phase may be essential in the maintenance of physical activity. On the basis of previous studies, the following questions might be raised: (1) Is the reduction in Tb during the light phase a regulated phenomenon by the thermoregulation system? (2) Is the circadian system involved in the phenomenon? and (3) Which factor that is associated with fasting causes the decrease in Tb , and how?

#### **3. Physiological mechanism involved in the decrease in body temperature during fasting**

Tb in homeothermic animals is controlled by heat production and heat loss mechanisms. However, whether such control is maintained and generates the circadian Tb rhythm during fasting is still not verified. Fasting is a strong stimulus that decreases metabolic heat production in rodents and pigeons [5, 6, 10–15]. In addition, fasting decreases thermal conductance (i.e., opposite of thermal resistance) from the body core to the environment (heat loss [16–18]). If these physiological responses occurred in the same manner during the day of fasting, the reduction of Tb , specifically during the light phase, would not be observed. Therefore, the thermoregulatory responses must be different during the two phases.

Nagashima et al. [19] assessed the Tb , counts of locomotor activity, and oxygen consumption rate (V˙ O<sup>2</sup> ) in rats that were made to fast for 3 days. **Figure 2** illustrates the data before fasting and on the last day of fasting and recovery day. Tb and counts of activity were obtained as the

**Figure 2.** Body core temperature (*A*, Tcore), counts of locomotor activity (*B*), and oxygen consumption rate (*C*, V̇ o2 ) during feeding, day 3 of fasting, and day 4 of recovery. Stippled area, dark phase (1900–0700). Each point has an average of 30 min. The values are the means ± SE in six rats. \* Significant difference from controlled feeding, *P* < 0.05. The figure was obtained from the manuscript by Nagashima et al. [19].

study by Tokizawa et al. [9], and V˙ O2

of Tb

during fasting in both dark and light phases. Although the rhythms of Tb

and lower back. Each point has an average of 30 min. The values are the means ± SE in five rats. \*

pression of heat loss during fasting and the dark. The estimated thermal conductance during the dark phase of the fasting period decreased from that in the fed condition (1.10 and 1.65 ml·min−1•kg body wt−0.75·°C−1, respectively), which supported the hypothesis. On the con-

**Figure 3.** Surface temperatures of the tail (*A*) and trunk (*B*) determined by thermography in a controlled feeding condition, on day 3 of fasting, and day 4 of recovery. Ttail is the average of surface temperatures at one-third of the length of the tail from the root and the tip. Ttrunk is the average of surface temperatures of the head and middle parts of the upper

from controlled feeding conditions, *P* < 0.05. The figure was obtained from the manuscript by Nagashima et al. [19].

nificantly associated during feeding, the amplitude of the V˙ O2

trary, such a change was not observed during the light phase.

increased during the fasting. The result suggested that Tb

was assessed by indirect calorimetry. V˙ O2

Circadian Body Temperature Rhythm and the Interaction with Energy State

http://dx.doi.org/10.5772/intechopen.76229

13

decreased

Significant differences

were sig-

and V˙ O2

rhythm decreased and that

was maintained by the sup-

Circadian Body Temperature Rhythm and the Interaction with Energy State http://dx.doi.org/10.5772/intechopen.76229 13

**Figure 3.** Surface temperatures of the tail (*A*) and trunk (*B*) determined by thermography in a controlled feeding condition, on day 3 of fasting, and day 4 of recovery. Ttail is the average of surface temperatures at one-third of the length of the tail from the root and the tip. Ttrunk is the average of surface temperatures of the head and middle parts of the upper and lower back. Each point has an average of 30 min. The values are the means ± SE in five rats. \* Significant differences from controlled feeding conditions, *P* < 0.05. The figure was obtained from the manuscript by Nagashima et al. [19].

study by Tokizawa et al. [9], and V˙ O2 was assessed by indirect calorimetry. V˙ O2 decreased during fasting in both dark and light phases. Although the rhythms of Tb and V˙ O2 were significantly associated during feeding, the amplitude of the V˙ O2 rhythm decreased and that of Tb increased during the fasting. The result suggested that Tb was maintained by the suppression of heat loss during fasting and the dark. The estimated thermal conductance during the dark phase of the fasting period decreased from that in the fed condition (1.10 and 1.65 ml·min−1•kg body wt−0.75·°C−1, respectively), which supported the hypothesis. On the contrary, such a change was not observed during the light phase.

**Figure 2.** Body core temperature (*A*, Tcore), counts of locomotor activity (*B*), and oxygen consumption rate (*C*, V̇

30 min. The values are the means ± SE in six rats. \*

reduction of Tb

12 Homeostasis - An Integrated Vision

rate (V˙ O<sup>2</sup>

Nagashima et al. [19] assessed the Tb

and on the last day of fasting and recovery day. Tb

obtained from the manuscript by Nagashima et al. [19].

feeding, day 3 of fasting, and day 4 of recovery. Stippled area, dark phase (1900–0700). Each point has an average of

(i.e., opposite of thermal resistance) from the body core to the environment (heat loss [16–18]). If these physiological responses occurred in the same manner during the day of fasting, the

thermoregulatory responses must be different during the two phases.

, specifically during the light phase, would not be observed. Therefore, the

) in rats that were made to fast for 3 days. **Figure 2** illustrates the data before fasting

, counts of locomotor activity, and oxygen consumption

and counts of activity were obtained as the

Significant difference from controlled feeding, *P* < 0.05. The figure was

o2 ) during Animals use several mechanisms to change the efficiency of heat loss. Among the mechanisms, the tail is a crucial site for the regulation of heat loss in rats and mice, with its physiological and anatomic characteristics, that is, a high density of arteriovenous anastomosis [6], the absence of fur, and a remarkable surface-to-volume ratio. Young and Dawson [20] reported that rats could dissipate 25% of basal heat production by changing the blood flow in the tail.

Nagashima et al. [34] assessed the circadian Tb

and peak phase of the Tb

Differences were found in the T<sup>b</sup>

study showed that the circadian Tb

Mesor 37.5 ± 0.1 37.3 ±

Acrophase 18.2 ± 0.3 16.4 ±

0.91†

Significantly different from control, P < 0.05.

Significantly different from fasting period, P< 0.05.

*r* 0.76–

Values are means ± SE. V˙

\*

†

‡

Amplitude 0.5 ± 0.1 0.7 ± 0.1\* 0.4 ±

1 and 2 genes (*Cry*1 and *Cry*2), two of the core clock genes [35–38]. The mice loose the transcription-translation rhythms of the *Cry*1 and *Cry*2 and other clock genes and periodicity in a wheel-running behavior [38], as well as the electrophysiological activity of the SCN cells under constant dark conditions [39]. However, the behavior is suppressed under the light condition, and the daily rhythm is observed under both light and dark conditions. Therefore,

normal wild-type mice that lack *Cry*1/*Cry*2 (*Cry*1−/−/*Cry*2−/−). The mice were placed under three different conditions: (1) constant darkness (DD) with ad lib feeding, (2) 12–12-h light-dark (LD; lights on at 1900) cycle with ad lib feeding, and (3) LD with food restriction. The food restriction protocol aimed to enhance the eating rhythm. Ordinary chow was given at 1900, which is the end of the light period, at 70–80% of the normal daily intake. Both *Cry*1−/−/*Cry*2−/− and wildtype mice finished eating 10–12 h after the appearance of chow in the food-restriction regimen. In the wild-type mice, no significant difference was observed between the mean, amplitude,

days (i.e., greater amplitude of the rhythms during the food-restriction day). The activity rhythm

rhythms in the LD condition, the daily means were lower, and the amplitudes of the rhythms were higher in the food-restriction condition than those under the ad lib feeding condition. The

mechanism, when an external lighting and feeding stimuli that alter heat production are observed.

**Fed Fast Rec Fed Fast Rec Fed Fast Rec**

15.02 ± 0.55\*

1.57 ± 0.27\*

18.2 ± 0.2 16.4 ± 0.3\*

period; Fast, *day* 3 of fasting period; Rec, *day 4* of recovery from fasting. Acrophase is shown in zeitgeber time (ZT, ZT 0 = 0700).

ity became rhythmic in the same peak phases during the LD conditions. As for the Tb

The result showed that the heat production rhythm may be a key component for the Tb

**O<sup>2</sup>**

3.74 ± 0.44

0.51– 0.85†

Significant regression coefficient (r) for fitted cosine curve for daily change of variable, P < 0.05.

37.4 ± 0.1 21.63 ± 0.84

0.1\* , ‡

17.5 ± 0.3\* , ‡

0.56– 0.91†

**Table 1.** Analysis of cosinor rhythmometry for daily changes in core temperature,Vo2

rhythm under the LD condition with ad lib feeding and DD condition.

rhythms between ad lib feeding and food-restriction

, V˙ O<sup>2</sup>

rhythm is observed even in mice that lack the internal circadian

**, ml** ⋅ **min−1** ⋅ **kg body wt−0.75 Activity, Au**

20.11 ± 0.78‡

3.06 ± 0.38\* , ‡

17.6 ± 0.4\* ,

0.36–0.61† 0.45–0.74† 0.38–

 O2, oxygen consumption rate; Au, arbitrary unit; Tcore, core temperature; Fed, *day* 4 of fed control

0.67†

despite the lack of the internal rhythm, lighting rhythm could induce Tb

and V˙ O<sup>2</sup>

did not change throughout the three conditions. In *Cry*1−/−/*Cry*2−/− mice, Tb

ous activity were arrhythmic under the DD condition. However, Tb

**Tcore, °C** <sup>V</sup>˙

0.1\*

0.3\*

0.80– 0.85†

**Table 1** summarizes the cosinor rhythm analysis [34] of Tb

rhythm in mice that lack the cryptochrome

http://dx.doi.org/10.5772/intechopen.76229

Circadian Body Temperature Rhythm and the Interaction with Energy State

rhythm.

in

15

, spontaneous activity, and V˙ O<sup>2</sup>

, V˙ O<sup>2</sup>

, and spontane-

and V˙ O<sup>2</sup>

rhythm.

0.43–0.67†

, and spontaneous activ-

5.0 ± 1.0 3.2 ± 0.3 5.0 ± 0.9

3.4 ± 0.6 2.6 ± 0.3 3.5 ± 0.5‡

‡ 17.7 ± 0.4 17.9 ± 0.4 17.3 ± 0.6

0.39– 0.63†

, and locomotor activity.

To assess the contribution of the tail in thermoregulation, Nagashima et al. [19] estimated the tail surface temperature by thermography (**Figure 3**). Tail temperature during the fasting day was lower than that during the fed control day. However, body trunk temperature during the fasting was lower than that during the fed control only around the light-onset period. Interestingly, different from the fed control, tail temperature in the fasting condition increased after light onset and remained at a higher level during the light phase. The results strongly suggested an attenuation of tail blood flow in the dark phase during the fasting condition and were blunted at the beginning of the light phase. Thus, the tail may help in determining the process of heat loss from the body in the fasting condition, which was the factor regulating the Tb rhythm.

## **4. Involvement of suprachiasmatic nucleus in Tb rhythm during fasting**

The suprachiasmatic nucleus (SCN) in the hypothalamus is thought to be the master clock of the circadian rhythm. In addition, electrical or chemical lesions of the SCN destroy the Tb rhythm in rodents [21–26]. However, these findings may not indicate that the circadian clock regulates the Tb rhythm. Behavioral and/or physiological responses, such as locomotor activity, eating, and the secretion of several hormones, are also inhibited due to the SCN lesion, which may have direct influences on heat production and T<sup>b</sup> [17, 27–29]. Simply put, SCN lesion inhibits such behavioral and/or physiological responses [3, 21, 24, 30], and the Tb rhythm is subsequently destroyed.

Liu et al. [23] assessed the circadian Tb rhythm within 4 days of fasting in rats, of which SCN was electrically lesioned. The rats had arrhythmia of Tb and spontaneous activity, and no difference was observed during the light and dark phases and the non-fasting and fasting periods. Although the experiment did not answer the question of whether the circadian clock directly regulates the Tb rhythm, the result suggested that SCN may be important for the changes in thermoregulation and Tb due to fasting.

#### **5. Importance of molecular circadian mechanism on Tb rhythm during fasting**

Several genes in the central and peripheral tissues have expression rhythms with a periodicity of ~24 h. Among these genes, those with autoregulatory transcription-translation loops with a periodicity of ~24 h may be responsible for the core molecular mechanisms of the circadian clock (i.e., clock genes). These genes are observed in both the central and the peripheral tissues [31–33].

Nagashima et al. [34] assessed the circadian Tb rhythm in mice that lack the cryptochrome 1 and 2 genes (*Cry*1 and *Cry*2), two of the core clock genes [35–38]. The mice loose the transcription-translation rhythms of the *Cry*1 and *Cry*2 and other clock genes and periodicity in a wheel-running behavior [38], as well as the electrophysiological activity of the SCN cells under constant dark conditions [39]. However, the behavior is suppressed under the light condition, and the daily rhythm is observed under both light and dark conditions. Therefore, despite the lack of the internal rhythm, lighting rhythm could induce Tb rhythm.

Animals use several mechanisms to change the efficiency of heat loss. Among the mechanisms, the tail is a crucial site for the regulation of heat loss in rats and mice, with its physiological and anatomic characteristics, that is, a high density of arteriovenous anastomosis [6], the absence of fur, and a remarkable surface-to-volume ratio. Young and Dawson [20] reported that rats

To assess the contribution of the tail in thermoregulation, Nagashima et al. [19] estimated the tail surface temperature by thermography (**Figure 3**). Tail temperature during the fasting day was lower than that during the fed control day. However, body trunk temperature during the fasting was lower than that during the fed control only around the light-onset period. Interestingly, different from the fed control, tail temperature in the fasting condition increased after light onset and remained at a higher level during the light phase. The results strongly suggested an attenuation of tail blood flow in the dark phase during the fasting condition and were blunted at the beginning of the light phase. Thus, the tail may help in determining the process of heat

rhythm.

[17, 27–29]. Simply put,

and spontaneous activity, and no

could dissipate 25% of basal heat production by changing the blood flow in the tail.

loss from the body in the fasting condition, which was the factor regulating the Tb

**4. Involvement of suprachiasmatic nucleus in Tb rhythm during** 

lesion, which may have direct influences on heat production and T<sup>b</sup>

was electrically lesioned. The rats had arrhythmia of Tb

The suprachiasmatic nucleus (SCN) in the hypothalamus is thought to be the master clock of the circadian rhythm. In addition, electrical or chemical lesions of the SCN destroy the

rhythm in rodents [21–26]. However, these findings may not indicate that the circadian

tor activity, eating, and the secretion of several hormones, are also inhibited due to the SCN

SCN lesion inhibits such behavioral and/or physiological responses [3, 21, 24, 30], and the Tb

difference was observed during the light and dark phases and the non-fasting and fasting periods. Although the experiment did not answer the question of whether the circadian clock

Several genes in the central and peripheral tissues have expression rhythms with a periodicity of ~24 h. Among these genes, those with autoregulatory transcription-translation loops with a periodicity of ~24 h may be responsible for the core molecular mechanisms of the circadian clock (i.e., clock genes). These genes are observed in both the central and the peripheral tis-

due to fasting.

**5. Importance of molecular circadian mechanism on Tb rhythm** 

rhythm. Behavioral and/or physiological responses, such as locomo-

rhythm, the result suggested that SCN may be important for the

rhythm within 4 days of fasting in rats, of which SCN

**fasting**

clock regulates the Tb

14 Homeostasis - An Integrated Vision

directly regulates the Tb

**during fasting**

sues [31–33].

rhythm is subsequently destroyed.

Liu et al. [23] assessed the circadian Tb

changes in thermoregulation and Tb

Tb

**Table 1** summarizes the cosinor rhythm analysis [34] of Tb , spontaneous activity, and V˙ O<sup>2</sup> in normal wild-type mice that lack *Cry*1/*Cry*2 (*Cry*1−/−/*Cry*2−/−). The mice were placed under three different conditions: (1) constant darkness (DD) with ad lib feeding, (2) 12–12-h light-dark (LD; lights on at 1900) cycle with ad lib feeding, and (3) LD with food restriction. The food restriction protocol aimed to enhance the eating rhythm. Ordinary chow was given at 1900, which is the end of the light period, at 70–80% of the normal daily intake. Both *Cry*1−/−/*Cry*2−/− and wildtype mice finished eating 10–12 h after the appearance of chow in the food-restriction regimen.

In the wild-type mice, no significant difference was observed between the mean, amplitude, and peak phase of the Tb rhythm under the LD condition with ad lib feeding and DD condition. Differences were found in the T<sup>b</sup> and V˙ O<sup>2</sup> rhythms between ad lib feeding and food-restriction days (i.e., greater amplitude of the rhythms during the food-restriction day). The activity rhythm did not change throughout the three conditions. In *Cry*1−/−/*Cry*2−/− mice, Tb , V˙ O<sup>2</sup> , and spontaneous activity were arrhythmic under the DD condition. However, Tb , V˙ O<sup>2</sup> , and spontaneous activity became rhythmic in the same peak phases during the LD conditions. As for the Tb and V˙ O<sup>2</sup> rhythms in the LD condition, the daily means were lower, and the amplitudes of the rhythms were higher in the food-restriction condition than those under the ad lib feeding condition. The study showed that the circadian Tb rhythm is observed even in mice that lack the internal circadian mechanism, when an external lighting and feeding stimuli that alter heat production are observed. The result showed that the heat production rhythm may be a key component for the Tb rhythm.


Values are means ± SE. V˙  O2, oxygen consumption rate; Au, arbitrary unit; Tcore, core temperature; Fed, *day* 4 of fed control period; Fast, *day* 3 of fasting period; Rec, *day 4* of recovery from fasting. Acrophase is shown in zeitgeber time (ZT, ZT 0 = 0700). \* Significantly different from control, P < 0.05.

† Significant regression coefficient (r) for fitted cosine curve for daily change of variable, P < 0.05. ‡ Significantly different from fasting period, P< 0.05.

**Table 1.** Analysis of cosinor rhythmometry for daily changes in core temperature,Vo2 , and locomotor activity.

and CT or ZT 0–12 in *Cry*1−/−/*Cry*2−/− mice and wild-type mice, respectively. Moreover, the model indicated that the two regressions in the wild-type mice were different (i.e., for the active and inactive phases), and those in the *Cry*1−/−/*Cry*2−/− mice were similar. The results

phase than in the inactive phase regardless of the V˙ O level in wild-type mice. Moreover, the

mice (wild-type: 0.24 and 0.25 in the active and inactive phases, respectively; *Cry*1−/−/*Cry*2−/−:

than the *Cry*1−/−/*Cry*2−/− mice over the same variation of V˙ O in each phase. Thus, the circadian

The mammalian sirtuins (SIRT) modulate the circadian epigenome and provide specificity in transcriptional control [40–43]. It was reported that the circadian clock regulates mitochondrial oxygen consumption rate in the oxidoreductase factor nicotinamide adenine dinucleo-

glucose oxidation, and acetylation of mitochondrial proteins in normal and circadian (Bmal1) mutant mice. They reported that lipid oxidation and mitochondrial protein acetylation exhib-

results suggest a strong interaction between circadian and metabolic rhythms and its destruc-

Tokizawa et al. [9] tested the hypothesis that thermoregulation is modulated by the circadian system (including the SCN and clock genes), depending on the time of day and feeding condition. Moreover, the physiological and neural responses of the mice during exposure to cold (20°C) under ad lib feeding and during 48-h fasting conditions and the dark and light phases were compared. The differences in the responses between wild-type and *Clock*-mutant mice were also examined. *Clock* is also a gene that organizes the core loop of molecular circadian oscillation. The mutation of *Clock* causes the disappearance of oscillation [44]. However, mutant

similar to that of the wild-type mice during both the dark and light phases at normal tem-

at settings with a temperature of 27°C (ZT2.5–3.5; **Figure 5E**). During fasting at low tem-

increased in settings with a temperature of 27°C at ZT13.5–14.5 during the dark phase

rhythm under light-dark conditions because of the masking effect of light.

rhythm in wild-type animals is not a simple byproduct of the heat production rhythm but

in both phases were smaller than those of the *Cry*1−/−/*Cry*2−/−

Circadian Body Temperature Rhythm and the Interaction with Energy State

and oxidative cycles were self-sustained in fasted mice. These

rhythm

17

was kept higher in the active

http://dx.doi.org/10.5772/intechopen.76229


within a narrower range

biosynthesis, lipid and

rhythm during fasting.

of the wild-type mice was

was higher during the light phase

increased during the light phase.

increased during both the light and dark phases (ZT1–4

cycle in the liver;

suggested that the circadian phase can be a significant factor in determining the T<sup>b</sup>

in wild-type mice but not in the *Cry*1−/−/*Cry*2−/− mice. Thus, Tb

a phenomenon regulated by the circadian system.

0.44). Results showed that the wild-type mice can maintain their Tb

)/SIRT3-dependent manner. In addition, NAD+

in fasting and oxidative metabolism. Peek et al. [40] evaluated NAD+

tion during fasting but do not explain for the change in circadian Tb

**controlling the circadian Tb rhythm during fasting**

During ad lib feeding at a low temperature setting (20°C), the T<sup>b</sup>

(**Figure 5F**) but remained unchanged during the light phase (**Figure 5E**).

perature settings (27°C) (**Figure 5A** and **B**). However, V˙ O<sup>2</sup>

and 14.5–16, respectively; **Figure 5A** and **B**). However, Tb

perature settings, the reduction in T<sup>b</sup>

ited circadian oscillations that corresponded with the clock-driven NAD+

**6. Role of the circadian system and associated brain areas in** 

regression slopes of V˙ O and T<sup>b</sup>

Tb

tide (NAD+

mice show a Tb

<sup>V</sup>˙ O<sup>2</sup>

however, rhythmic NAD+

**Figure 4.** Relationship between metabolic heat production (V˙  O<sup>2</sup> ) and Tb in wild-type mice (*A*) and *Cry*1−/−/*Cry*2−/− (*B*) based on three trials. The values are the means (±SE) in five mice for 30 min, and data corresponding to each hour are used for this illustration. Regression lines were applied for the averaged values in each group. The figure was obtained from the manuscript by Nagashima et al. [34].

**Figure 4** shows the results of the regression analysis of the V˙ O<sup>2</sup> and Tb of each group. If heat production was the sole determinant of circadian Tb rhythm, these regressions must be identical regardless of trial, circadian phase, and group. In addition, model equations fitted to all the data in the three trials within the same phase and the group was established. Model equations were also created for all possible associations among the three trials. The analysis showed that the data could be classified into four groups: those at circadian time (CT; a standard of time based on the free-running period of a rhythm). The onset of activity of diurnal organisms defines circadian time 0, CT 0. The onset of activity of nocturnal organisms defines circadian time 12, CT 12, or zeitgeber time (ZT; under standard light–dark cycles, the time of lights on defines zeitgeber time 0 and the time of lights off defines zeitgeber time 12), 12–24 and CT or ZT 0–12 in *Cry*1−/−/*Cry*2−/− mice and wild-type mice, respectively. Moreover, the model indicated that the two regressions in the wild-type mice were different (i.e., for the active and inactive phases), and those in the *Cry*1−/−/*Cry*2−/− mice were similar. The results suggested that the circadian phase can be a significant factor in determining the T<sup>b</sup> rhythm in wild-type mice but not in the *Cry*1−/−/*Cry*2−/− mice. Thus, Tb was kept higher in the active phase than in the inactive phase regardless of the V˙ O level in wild-type mice. Moreover, the regression slopes of V˙ O and T<sup>b</sup> in both phases were smaller than those of the *Cry*1−/−/*Cry*2−/− mice (wild-type: 0.24 and 0.25 in the active and inactive phases, respectively; *Cry*1−/−/*Cry*2−/−: 0.44). Results showed that the wild-type mice can maintain their Tb within a narrower range than the *Cry*1−/−/*Cry*2−/− mice over the same variation of V˙ O in each phase. Thus, the circadian Tb rhythm in wild-type animals is not a simple byproduct of the heat production rhythm but a phenomenon regulated by the circadian system.

The mammalian sirtuins (SIRT) modulate the circadian epigenome and provide specificity in transcriptional control [40–43]. It was reported that the circadian clock regulates mitochondrial oxygen consumption rate in the oxidoreductase factor nicotinamide adenine dinucleotide (NAD+ )/SIRT3-dependent manner. In addition, NAD+ -dependent enzymes are important in fasting and oxidative metabolism. Peek et al. [40] evaluated NAD+ biosynthesis, lipid and glucose oxidation, and acetylation of mitochondrial proteins in normal and circadian (Bmal1) mutant mice. They reported that lipid oxidation and mitochondrial protein acetylation exhibited circadian oscillations that corresponded with the clock-driven NAD+ cycle in the liver; however, rhythmic NAD+ and oxidative cycles were self-sustained in fasted mice. These results suggest a strong interaction between circadian and metabolic rhythms and its destruction during fasting but do not explain for the change in circadian Tb rhythm during fasting.

## **6. Role of the circadian system and associated brain areas in controlling the circadian Tb rhythm during fasting**

**Figure 4.** Relationship between metabolic heat production (V˙

production was the sole determinant of circadian Tb

**Figure 4** shows the results of the regression analysis of the V˙ O<sup>2</sup>

from the manuscript by Nagashima et al. [34].

16 Homeostasis - An Integrated Vision

 O<sup>2</sup> ) and Tb

based on three trials. The values are the means (±SE) in five mice for 30 min, and data corresponding to each hour are used for this illustration. Regression lines were applied for the averaged values in each group. The figure was obtained

tical regardless of trial, circadian phase, and group. In addition, model equations fitted to all the data in the three trials within the same phase and the group was established. Model equations were also created for all possible associations among the three trials. The analysis showed that the data could be classified into four groups: those at circadian time (CT; a standard of time based on the free-running period of a rhythm). The onset of activity of diurnal organisms defines circadian time 0, CT 0. The onset of activity of nocturnal organisms defines circadian time 12, CT 12, or zeitgeber time (ZT; under standard light–dark cycles, the time of lights on defines zeitgeber time 0 and the time of lights off defines zeitgeber time 12), 12–24

in wild-type mice (*A*) and *Cry*1−/−/*Cry*2−/− (*B*)

of each group. If heat

and Tb

rhythm, these regressions must be iden-

Tokizawa et al. [9] tested the hypothesis that thermoregulation is modulated by the circadian system (including the SCN and clock genes), depending on the time of day and feeding condition. Moreover, the physiological and neural responses of the mice during exposure to cold (20°C) under ad lib feeding and during 48-h fasting conditions and the dark and light phases were compared. The differences in the responses between wild-type and *Clock*-mutant mice were also examined. *Clock* is also a gene that organizes the core loop of molecular circadian oscillation. The mutation of *Clock* causes the disappearance of oscillation [44]. However, mutant mice show a Tb rhythm under light-dark conditions because of the masking effect of light.

During ad lib feeding at a low temperature setting (20°C), the T<sup>b</sup> of the wild-type mice was similar to that of the wild-type mice during both the dark and light phases at normal temperature settings (27°C) (**Figure 5A** and **B**). However, V˙ O<sup>2</sup> was higher during the light phase at settings with a temperature of 27°C (ZT2.5–3.5; **Figure 5E**). During fasting at low temperature settings, the reduction in T<sup>b</sup> increased during both the light and dark phases (ZT1–4 and 14.5–16, respectively; **Figure 5A** and **B**). However, Tb increased during the light phase. <sup>V</sup>˙ O<sup>2</sup> increased in settings with a temperature of 27°C at ZT13.5–14.5 during the dark phase (**Figure 5F**) but remained unchanged during the light phase (**Figure 5E**).

neural connections between the SCN exist (the center of the circadian system) and the hypo-

Circadian Body Temperature Rhythm and the Interaction with Energy State

http://dx.doi.org/10.5772/intechopen.76229

19

Liu et al. [23] assessed the cFos (i.e., early gene expression protein) expression in the SCN of rats. cFos can be a marker of neuronal activation in the brain [50]. The number of cFos immunoreactive (cFos-IR) cells changes daily, which is higher during the light phase and smaller during the dark phase. A 4-day fasting did not change the phase difference in the number of cFos-IR cells. However, the number increased during the dark phase and decreased during the light phase. Whether such change in the SCN causes altered Tb rhythm during fasting remains unknown. However, results showed that the SCN receives some information that is associated with fasting and alters Tb rhythm and/

Tokizawa et al. [9] evaluated cFos expressions in the SCN and other hypothalamic areas in wild-type and *Clock*-mutant mice. The mice were exposed to settings with a temperature of 20°C and/or 48-h fasting. Neural associations were also observed between the SCN and the hypothalamic areas involved in thermoregulation. Data are summarized in **Figure 6**. Fasting increased the number of cFos-IR cells in the SCN in both wild-type and *Clock*-mutant mice. The number was smaller in *Clock*-mutant mice than in wild-type mice. cFos-IR cells also increased during fasting in other hypothalamic areas, such as the medial preoptic nucleus (MPO), dorsomedial hypothalamus (DMH), paraventricular nucleus (PVN), and dorsal subparaventricular zone (dSPZ), in wild-type mice. Differences were observed in the number of cFos-IR cells during the dark and light phases (**Figure 6A**, **B**, **D**, **E**). Small increases were also observed in the DMH, ARC, and PVN in *Clock*-mutant mice. However, no phase differences were observed. Neural outputs from the SCN, including the SPZ, reached these hypothalamic areas [51, 52]. Thus, the activation of the SCN during fasting may be linked with the activation

The preoptic area in the hypothalamus is thought to be important in thermoregulation because it has several thermosensitive neurons in the core body and skin temperatures [53]. In addition, stimulatory and inhibitory signals are sent from the area to other brain areas, which regulate the effector organs of thermoregulation, such as vasodilation, shivering, and non-shivering thermogenesis [48, 49]. The sympathetic outflow may originate from the PVN [54–56]. The DMH receives thermal input from the skin and is associated with the control of BAT thermogenesis [57]. Whether all cFos-IR cells are associated with circadian change of Tb and/or thermoregulation is not verified. However, the changes in cFos expression between

Fasting increased the cFos expression in the ARC in mice [58, 59]. The ARC is involved in the regulation of food intake and energy expenditure, which responds to peripheral nutritional signals, such as the levels of leptin and insulin [60]. However, no study on the direct association between ARC and thermoregulation was conducted. The ARC had phase differences in cFos expression in wild-type mice (**Figure 5C**), which increased during fasting. The ARC has neural input from the SCN [61–63]. Therefore, the fasting signals received by the ARC increased during the dark phase and were attenuated during the light phase in wild-type

that was observed during fasting

the phases may, in part, be responsible for the decrease in Tb

mice, and this may be related to the signals from the SCN.

thalamic subregion is involved in the thermoregulation remains unclear.

or the thermoregulatory responses.

of the hypothalamic areas.

and the light phase.

**Figure 5.** Tb and V˙ O2 at low-temperature settings (27 or 20°C) with *ad lib* feeding or fasting in the wild-type and *Clock*mutant mice (*A–H*). The values given are the means ± SEM (*n* = 8). \* *P* < 0.05, 20°C (low-temperature setting) versus 27°C (control setting) in each feeding condition. # *P* < 0.05, versus the dark phase in wild-type mice and both phases in *Clock*mutant mice; § *P* < 0.05, versus dark phase in wild-type mice and light phase in *Clock*-mutant mice. The modified figure was obtained from the manuscript by Tokizawa et al. [9].

During ad lib feeding at low temperature settings, T<sup>b</sup> in *Clock*-mutant mice was also maintained at the 27°C level (**Figure 5C** and **D**). V˙ O<sup>2</sup> increased above the 27°C level during both the dark and light phases (ZT3–4 and 13.5–14; **Figure 5G** and **H**), and no difference was observed between the two phases. During fasting at low temperature conditions, Tb decreased below the 27°C level at ZT3–3.5 during the light phase and ZT14.5–16 during the dark phase (**Figure 5C** and **D**), and no significant difference was observed between the phases. V˙ O<sup>2</sup> was higher than the 27°C level at ZT2 and 13.5–14 (**Figure 5G** and **H**). This increase in V˙O2 was higher during the dark phase than during the light phase.

The study indicated that the physiological response of mice to low temperature conditions is different during ad lib feeding and fasting in wild-type animals and during the dark and light phases. However, such differences were not observed or significantly decreased in *Clock*-mutant mice. The thermoregulatory mechanism of heat production is attenuated or inhibited during fasting and light phases. For such response, the circadian system is important. To support this result, the expression of uncoupling protein 1 (UCP1) mRNA [45–47] in the interscapular brown adipose tissue (iBAT, one of the effector organs for thermoregulatory heat production) was suppressed during the light phase and fasting in wild-type animals. However, cold exposure increased the expression during both dark and light phases in *Clock*-mutant mice.

#### **7. Histological evidence on functional and anatomical connection between the circadian and thermoregulation systems**

On the basis of the physiological findings, the association between the circadian and thermoregulation systems may be significant during fasting. The center of thermoregulation is thought to be in the hypothalamus [48, 49]. However, whether functional and anatomical neural connections between the SCN exist (the center of the circadian system) and the hypothalamic subregion is involved in the thermoregulation remains unclear.

Liu et al. [23] assessed the cFos (i.e., early gene expression protein) expression in the SCN of rats. cFos can be a marker of neuronal activation in the brain [50]. The number of cFos immunoreactive (cFos-IR) cells changes daily, which is higher during the light phase and smaller during the dark phase. A 4-day fasting did not change the phase difference in the number of cFos-IR cells. However, the number increased during the dark phase and decreased during the light phase. Whether such change in the SCN causes altered Tb rhythm during fasting remains unknown. However, results showed that the SCN receives some information that is associated with fasting and alters Tb rhythm and/ or the thermoregulatory responses.

Tokizawa et al. [9] evaluated cFos expressions in the SCN and other hypothalamic areas in wild-type and *Clock*-mutant mice. The mice were exposed to settings with a temperature of 20°C and/or 48-h fasting. Neural associations were also observed between the SCN and the hypothalamic areas involved in thermoregulation. Data are summarized in **Figure 6**. Fasting increased the number of cFos-IR cells in the SCN in both wild-type and *Clock*-mutant mice. The number was smaller in *Clock*-mutant mice than in wild-type mice. cFos-IR cells also increased during fasting in other hypothalamic areas, such as the medial preoptic nucleus (MPO), dorsomedial hypothalamus (DMH), paraventricular nucleus (PVN), and dorsal subparaventricular zone (dSPZ), in wild-type mice. Differences were observed in the number of cFos-IR cells during the dark and light phases (**Figure 6A**, **B**, **D**, **E**). Small increases were also observed in the DMH, ARC, and PVN in *Clock*-mutant mice. However, no phase differences were observed. Neural outputs from the SCN, including the SPZ, reached these hypothalamic areas [51, 52]. Thus, the activation of the SCN during fasting may be linked with the activation of the hypothalamic areas.

During ad lib feeding at low temperature settings, T<sup>b</sup>

mutant mice (*A–H*). The values given are the means ± SEM (*n* = 8). \*

higher during the dark phase than during the light phase.

the dark and light phases (ZT3–4 and 13.5–14; **Figure 5G** and **H**), and no difference was

at low-temperature settings (27 or 20°C) with *ad lib* feeding or fasting in the wild-type and *Clock*-

*P* < 0.05, versus dark phase in wild-type mice and light phase in *Clock*-mutant mice. The modified figure

below the 27°C level at ZT3–3.5 during the light phase and ZT14.5–16 during the dark phase (**Figure 5C** and **D**), and no significant difference was observed between the phases. V˙ O<sup>2</sup>

The study indicated that the physiological response of mice to low temperature conditions is different during ad lib feeding and fasting in wild-type animals and during the dark and light phases. However, such differences were not observed or significantly decreased in *Clock*-mutant mice. The thermoregulatory mechanism of heat production is attenuated or inhibited during fasting and light phases. For such response, the circadian system is important. To support this result, the expression of uncoupling protein 1 (UCP1) mRNA [45–47] in the interscapular brown adipose tissue (iBAT, one of the effector organs for thermoregulatory heat production) was suppressed during the light phase and fasting in wild-type animals. However, cold expo-

higher than the 27°C level at ZT2 and 13.5–14 (**Figure 5G** and **H**). This increase in V˙O2

sure increased the expression during both dark and light phases in *Clock*-mutant mice.

**7. Histological evidence on functional and anatomical connection** 

On the basis of the physiological findings, the association between the circadian and thermoregulation systems may be significant during fasting. The center of thermoregulation is thought to be in the hypothalamus [48, 49]. However, whether functional and anatomical

**between the circadian and thermoregulation systems**

observed between the two phases. During fasting at low temperature conditions, Tb

tained at the 27°C level (**Figure 5C** and **D**). V˙ O<sup>2</sup>

was obtained from the manuscript by Tokizawa et al. [9].

**Figure 5.** Tb

mutant mice; §

and V˙ O2

18 Homeostasis - An Integrated Vision

(control setting) in each feeding condition. #

in *Clock*-mutant mice was also main-

*P* < 0.05, 20°C (low-temperature setting) versus 27°C

*P* < 0.05, versus the dark phase in wild-type mice and both phases in *Clock*-

decreased

was

was

increased above the 27°C level during both

The preoptic area in the hypothalamus is thought to be important in thermoregulation because it has several thermosensitive neurons in the core body and skin temperatures [53]. In addition, stimulatory and inhibitory signals are sent from the area to other brain areas, which regulate the effector organs of thermoregulation, such as vasodilation, shivering, and non-shivering thermogenesis [48, 49]. The sympathetic outflow may originate from the PVN [54–56]. The DMH receives thermal input from the skin and is associated with the control of BAT thermogenesis [57]. Whether all cFos-IR cells are associated with circadian change of Tb and/or thermoregulation is not verified. However, the changes in cFos expression between the phases may, in part, be responsible for the decrease in Tb that was observed during fasting and the light phase.

Fasting increased the cFos expression in the ARC in mice [58, 59]. The ARC is involved in the regulation of food intake and energy expenditure, which responds to peripheral nutritional signals, such as the levels of leptin and insulin [60]. However, no study on the direct association between ARC and thermoregulation was conducted. The ARC had phase differences in cFos expression in wild-type mice (**Figure 5C**), which increased during fasting. The ARC has neural input from the SCN [61–63]. Therefore, the fasting signals received by the ARC increased during the dark phase and were attenuated during the light phase in wild-type mice, and this may be related to the signals from the SCN.

and fasting were also conducted in the same manner as the previous cFos study. The injection of CTb in the PVN resulted in a widely spread labeling in both the dorsomedial and the ventrolateral parts of the SCN of both wild-type and *Clock*-mutant mice. In both mice, 5–10% of CTblabeled neurons in the SCN were also cFos-positive at 27°C during the light phase and ad lib feeding. In the cold exposure during fasting, the ratio of the double-labeled neurons of CTb and cFos increased to 25–30% only in the wild-type mice. Moreover, the double-labeled neurons are GABAergic. When CTb was injected to the MPO, the dorsomedial part of the SCN was labeled. However, the ratio of the double-labeled neurons remained at the same level (15–20%) during ad lib feeding and during fasting and cold exposure. These results suggested that the SCN may send inhibitory signals to the PVN and MPO during the light phase. Fasting and/or cold exposure increased the inhibitory signals only in the PVN. Moreover, in the process, a normal molecular circadian mechanism would be necessary. The sympathetic outflow may originate from the PVN [54–56]. Therefore, such inhibitory signals may attenuate metabolic heat production and/or skin

Circadian Body Temperature Rhythm and the Interaction with Energy State

during fasting.

http://dx.doi.org/10.5772/intechopen.76229

21

during

. Such a response was

vasoconstriction (i.e., cold defense mechanisms), thus decreasing Tb

**8. Signals attenuating thermoregulatory responses during fasting**

ghrelins are the factors that modulate thermoregulatory heat production and decrease Tb

moregulatory heat production during cold exposure and reduced Tb

(light phase) modulation of thermoregulation and Tb

fasting. Moreover, the neurons in the SCN have leptin and ghrelin receptors [68, 70, 71]. In addition, the neural activity of the SCN is modulated in the presence of leptin and ghrelin [72, 73]. Tokizawa et al. [74] reported that the thermoregulatory response to cold was attenuated in *ob/ob* mice (genetically deficient of leptin). However, the response was not different between the light and dark phases. On the contrary, ghrelin injection to normal mice inhibited ther-

observed only during the light phase. Therefore, ghrelin plays a key role in the phase-specific

The administration of ghrelin suppresses sympathetic nerve activity and iBAT temperature in rats [75, 76]. In the study by Tokizawa et al., ghrelin levels after the injection and 48-h fasting did not differ between the two phases. Therefore, a central mechanism that modulates the sensitivity to plasma ghrelin levels must exist, which may affect thermoregulatory responses to cold exposure. Ghrelin induced phase-specific changes in cFos expression in the hypothalamic areas: increased cFos-IR cells in the SCN during the light phase, the ARC during the dark phase, and the PVN during the cold exposure in the dark phase. Ghrelin injection activates NPY neurons in the ARC in both phases, and cFos-IR cell counts are higher in the dark than in the light phase. In the SCN, the ghrelin effect was limited to the light phase, and 25% of the cFos-IR cells were NPY neurons. NPY acts as nonphotic stimuli in the SCN [77]. The activation of the hypothalamic nuclei, that is, the SCN, ARC, and PVN, seems to be involved in the changes in the thermoregulatory metabolic heat production. **Figure 7** shows the summary of the findings.

during fasting.

In Section 6 of the cFos study, the ARC also seems to play a key role in the circadian change of thermoregulation during fasting, which decreases plasma leptin and increases plasma ghrelin [64]. A reduction of leptin results in hypothermia [65]. Gluck et al. [66] showed that ghrelin induces hypothermia. Decreases in both hormones are signals activating neuropeptide Y (NPY) neurons in the ARC [67], which strongly reduces heat production. The receptors for leptin and ghrelin are found in NPY neurons [68, 69]. These experimental results suggest that leptins and/or

**Figure 6.** Counts of cFos-IR cells in the MPO (*A*), dorsomedial hypothalamus (*B*, DMH), ARC (*C*), PVN (*D*), dorsal subparaventricular zone (*E*, dSPZ), and SCN (*F*) under various conditions. Values are the means ± SEM (*n* = 8). \* *P* < 0.05, dark versus light phase; § *P* < 0.05, 20°C (low-temperature setting) versus 27°C (control setting); † *P* < 0.05, fasting versus *ad lib* feeding; # *P* < 0.05, wild-type versus *Clock*-mutant mice. The figure was obtained from the manuscript by Tokizawa et al. [9].

The increase in cFos expression in *Clock*-mutant mice that was attributed to low temperature during fasting was also observed in all the hypothalamic areas besides the SCN. However, no differences were observed between the two phases. In wild-type mice, the number of cFos expression in the SCN increased during the light phase. However, the increases in the MPO and PVN in wild-type mice were lower than those in Clock-mutant mice. Therefore, exposure to cold while fasting increases neural activity in the SCN during the light phase. In addition, normal molecular circadian mechanisms may be necessary for the response. On the basis of the results of cFos expression in the hypothalamic areas, the SCN may send inhibitory signals to the MPO and PVN, which may result in attenuated thermoregulatory responses. Moreover, since the inhibitory signals are stronger in some conditions, such as in fasting, the attenuation of thermoregulation becomes stronger.

On the basis of the speculated neural connection between the SCN and MPO and/or PVN, Tokizawa et al. [9] conducted an experiment, which directly evaluated the associations. A cholera toxin b-subunit was injected (CTb; monosynaptic retrograde neural tracer) to the MPO or PVN during the light phase, and the presence in the SCN was assessed 3 days later. Cold exposure and fasting were also conducted in the same manner as the previous cFos study. The injection of CTb in the PVN resulted in a widely spread labeling in both the dorsomedial and the ventrolateral parts of the SCN of both wild-type and *Clock*-mutant mice. In both mice, 5–10% of CTblabeled neurons in the SCN were also cFos-positive at 27°C during the light phase and ad lib feeding. In the cold exposure during fasting, the ratio of the double-labeled neurons of CTb and cFos increased to 25–30% only in the wild-type mice. Moreover, the double-labeled neurons are GABAergic. When CTb was injected to the MPO, the dorsomedial part of the SCN was labeled. However, the ratio of the double-labeled neurons remained at the same level (15–20%) during ad lib feeding and during fasting and cold exposure. These results suggested that the SCN may send inhibitory signals to the PVN and MPO during the light phase. Fasting and/or cold exposure increased the inhibitory signals only in the PVN. Moreover, in the process, a normal molecular circadian mechanism would be necessary. The sympathetic outflow may originate from the PVN [54–56]. Therefore, such inhibitory signals may attenuate metabolic heat production and/or skin vasoconstriction (i.e., cold defense mechanisms), thus decreasing Tb during fasting.

#### **8. Signals attenuating thermoregulatory responses during fasting**

In Section 6 of the cFos study, the ARC also seems to play a key role in the circadian change of thermoregulation during fasting, which decreases plasma leptin and increases plasma ghrelin [64]. A reduction of leptin results in hypothermia [65]. Gluck et al. [66] showed that ghrelin induces hypothermia. Decreases in both hormones are signals activating neuropeptide Y (NPY) neurons in the ARC [67], which strongly reduces heat production. The receptors for leptin and ghrelin are found in NPY neurons [68, 69]. These experimental results suggest that leptins and/or ghrelins are the factors that modulate thermoregulatory heat production and decrease Tb during fasting. Moreover, the neurons in the SCN have leptin and ghrelin receptors [68, 70, 71]. In addition, the neural activity of the SCN is modulated in the presence of leptin and ghrelin [72, 73].

Tokizawa et al. [74] reported that the thermoregulatory response to cold was attenuated in *ob/ob* mice (genetically deficient of leptin). However, the response was not different between the light and dark phases. On the contrary, ghrelin injection to normal mice inhibited thermoregulatory heat production during cold exposure and reduced Tb . Such a response was observed only during the light phase. Therefore, ghrelin plays a key role in the phase-specific (light phase) modulation of thermoregulation and Tb during fasting.

The increase in cFos expression in *Clock*-mutant mice that was attributed to low temperature during fasting was also observed in all the hypothalamic areas besides the SCN. However, no differences were observed between the two phases. In wild-type mice, the number of cFos expression in the SCN increased during the light phase. However, the increases in the MPO and PVN in wild-type mice were lower than those in Clock-mutant mice. Therefore, exposure to cold while fasting increases neural activity in the SCN during the light phase. In addition, normal molecular circadian mechanisms may be necessary for the response. On the basis of the results of cFos expression in the hypothalamic areas, the SCN may send inhibitory signals to the MPO and PVN, which may result in attenuated thermoregulatory responses. Moreover, since the inhibitory signals are stronger in some conditions, such as in fasting, the attenuation

**Figure 6.** Counts of cFos-IR cells in the MPO (*A*), dorsomedial hypothalamus (*B*, DMH), ARC (*C*), PVN (*D*), dorsal subparaventricular zone (*E*, dSPZ), and SCN (*F*) under various conditions. Values are the means ± SEM (*n* = 8). \*

*P* < 0.05, 20°C (low-temperature setting) versus 27°C (control setting); †

*P* < 0.05, wild-type versus *Clock*-mutant mice. The figure was obtained from the manuscript by Tokizawa

*P* < 0.05,

*P* < 0.05, fasting versus

On the basis of the speculated neural connection between the SCN and MPO and/or PVN, Tokizawa et al. [9] conducted an experiment, which directly evaluated the associations. A cholera toxin b-subunit was injected (CTb; monosynaptic retrograde neural tracer) to the MPO or PVN during the light phase, and the presence in the SCN was assessed 3 days later. Cold exposure

of thermoregulation becomes stronger.

dark versus light phase; §

20 Homeostasis - An Integrated Vision

*ad lib* feeding; #

et al. [9].

The administration of ghrelin suppresses sympathetic nerve activity and iBAT temperature in rats [75, 76]. In the study by Tokizawa et al., ghrelin levels after the injection and 48-h fasting did not differ between the two phases. Therefore, a central mechanism that modulates the sensitivity to plasma ghrelin levels must exist, which may affect thermoregulatory responses to cold exposure. Ghrelin induced phase-specific changes in cFos expression in the hypothalamic areas: increased cFos-IR cells in the SCN during the light phase, the ARC during the dark phase, and the PVN during the cold exposure in the dark phase. Ghrelin injection activates NPY neurons in the ARC in both phases, and cFos-IR cell counts are higher in the dark than in the light phase. In the SCN, the ghrelin effect was limited to the light phase, and 25% of the cFos-IR cells were NPY neurons. NPY acts as nonphotic stimuli in the SCN [77]. The activation of the hypothalamic nuclei, that is, the SCN, ARC, and PVN, seems to be involved in the changes in the thermoregulatory metabolic heat production. **Figure 7** shows the summary of the findings.

**Author details**

Kei Nagashima<sup>1</sup>

**References**

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Tokorozawa, Saitama, Japan

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\*, Ken Tokizawa<sup>2</sup>

\*Address all correspondence to: k-nagashima@waseda.jp

, Shuri Marui1

2 National Institute of Occupational Safety and Health, Kiyose, Tokyo, Japan

1 Body Temperature and Fluid Laboratory, Faculty of Human Sciences, Waseda University,

3 Women's Environmental Science Laboratory, Faculty of Human Life and Environment, Department of Health Sciences, Nara Women's University, Nara-City, Nara, Japan

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and Yuki Uchida3

Circadian Body Temperature Rhythm and the Interaction with Energy State

http://dx.doi.org/10.5772/intechopen.76229

23

**Figure 7.** Summary of the histological findings together with physiological studies. During the light phase, fasting and an increase in plasma ghrelin level affect the hypothalamic areas. The activity of the suprachiasmatic nucleus (SCN) increases and that of the arcuate nucleus (ARC) relatively decreases. The SCN sends inhibitory signals to the paraventricular nucleus (PVN), which may result in a lower metabolic heat production of the interscapular brown adipose tissue (iBAT) and a lower body temperature. On the contrary, during the dark phase, the activity of the SCN decreases and that of the ARC relatively increases. The inhibitory signal from the SCN is less, and the PVN is activated. Metabolic heat production of the iBAT increases and body temperature is maintained.

#### **9. Conclusion**

This is a review article showing our previous series of studies involved in fasting-induced change in Tb rhythm. Interestingly, the change in Tb rhythm is a regulated phenomenon by molecular mechanisms such as *Cry and Clock* and neural mechanisms such as the SCN, MPO, and ARC in the hypothalamus. These studies are important in considering physiological importance and mechanism of the circadian body temperature and metabolic rhythms.

#### **Acknowledgements**

This research was partially supported by the Ministry of Education, Science, Sports, and Culture, Japan; Grant-in-Aid for challenging Exploratory Research, No. 16 K13055; and Ibuka Foundation, Waseda University.

#### **Conflict of interest**

There is no conflict of interest in this review.

## **Author details**

Kei Nagashima<sup>1</sup> \*, Ken Tokizawa<sup>2</sup> , Shuri Marui1 and Yuki Uchida3

\*Address all correspondence to: k-nagashima@waseda.jp

1 Body Temperature and Fluid Laboratory, Faculty of Human Sciences, Waseda University, Tokorozawa, Saitama, Japan

2 National Institute of Occupational Safety and Health, Kiyose, Tokyo, Japan

3 Women's Environmental Science Laboratory, Faculty of Human Life and Environment, Department of Health Sciences, Nara Women's University, Nara-City, Nara, Japan

## **References**

**Figure 7.** Summary of the histological findings together with physiological studies. During the light phase, fasting and an increase in plasma ghrelin level affect the hypothalamic areas. The activity of the suprachiasmatic nucleus (SCN) increases and that of the arcuate nucleus (ARC) relatively decreases. The SCN sends inhibitory signals to the paraventricular nucleus (PVN), which may result in a lower metabolic heat production of the interscapular brown adipose tissue (iBAT) and a lower body temperature. On the contrary, during the dark phase, the activity of the SCN decreases and that of the ARC relatively increases. The inhibitory signal from the SCN is less, and the PVN is activated.

This is a review article showing our previous series of studies involved in fasting-induced

molecular mechanisms such as *Cry and Clock* and neural mechanisms such as the SCN, MPO, and ARC in the hypothalamus. These studies are important in considering physiological importance and mechanism of the circadian body temperature and metabolic rhythms.

This research was partially supported by the Ministry of Education, Science, Sports, and Culture, Japan; Grant-in-Aid for challenging Exploratory Research, No. 16 K13055; and Ibuka

rhythm is a regulated phenomenon by

Metabolic heat production of the iBAT increases and body temperature is maintained.

rhythm. Interestingly, the change in Tb

**9. Conclusion**

22 Homeostasis - An Integrated Vision

**Acknowledgements**

**Conflict of interest**

Foundation, Waseda University.

There is no conflict of interest in this review.

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**Chapter 3**

Provisional chapter

**Sex and Sex Hormones in Tissue Homeostasis**

Women are not small men. Sex-specific differences do not only affect the classical target organs of sexual differentiation and reproduction, but have been found to involve most, if not all the organs and tissues in the body. One of the consequences of this dimorphism is that diseases manifest in a sex- and gender-specific way. Key to maintenance of a healthy state is functioning tissue able to cope with insults. Regulated death of damaged cells and replacement with new cells by proliferation is a prerequisite for maintaining tissue function taking place at different pace in the different organs. The intent of this chapter is to review current evidence for sex-specific differences in tissue homeostasis focusing on the variability of hormone exposure characteristic for the female reproductive life stages.

DOI: 10.5772/intechopen.76177

Keywords: tissue maintenance, sex differences, proliferation, cell death, kidney, menstrual

Living systems are continuously challenged by potentially toxic internal and external processes. The normal metabolic function of the cells produces a plethora of potentially damaging oxidative metabolites inducing damage in DNA, proteins, and lipids. In addition, living cells are exposed to a variety of external factors, which may be internalized as building blocks and/ or energy sources. These vital processes put the organism at risk to be harmed. Coping strategies are necessary to avoid damage. There are several lines of cellular defenses induced via cell stress pathways, including compartmentalization processes, enzymatic modification,

Ultimately, these processes may not be sufficient to prevent major cellular damage. Therefore, every cell is in addition equipped with internal cell death programs, which can be activated in order to prevent a damaged cell to cause harm to the organism [3]. Cell losses are inevitable

> © 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 eproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

Sex and Sex Hormones in Tissue Homeostasis

Judith Lechner and Gerhard Gstraunthaler

Judith Lechner and Gerhard Gstraunthaler

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.76177

Abstract

cycle

1. Introduction

externalization, degradation, and repair [1, 2].


#### **Sex and Sex Hormones in Tissue Homeostasis** Sex and Sex Hormones in Tissue Homeostasis

DOI: 10.5772/intechopen.76177

Judith Lechner and Gerhard Gstraunthaler Judith Lechner and Gerhard Gstraunthaler

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

http://dx.doi.org/10.5772/intechopen.76177

#### Abstract

Regulatory, Integrative and Comparative Physiology. 2006;**291**:R1303-R1309. DOI:

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[74] Tokizawa K, Onoue Y, Uchida Y, Nagashima K. Ghrelin induces time-dependent modulation of thermoregulation in the cold. Chronobiology International. 2012;**29**:736-746.

[75] Mano-Otagiri A, Ohata H, Iwasaki-Sekino A, et al. Ghrelin suppresses noradrenaline release in the brown adipose tissue of rats. The Journal of Endocrinology. 2009;**201**:341-

[76] Yasuda T, Masaki T, Kakuma T, Yoshimatsu H. Centrally administered ghrelin suppresses sympathetic nerve activity in brown adipose tissue of rats. Neuroscience Letters.

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forms in the rat brain. Molecular and Cellular Endocrinology. 1997;**133**:1-7

10.1152/ajpregu.00232.2006

28 Homeostasis - An Integrated Vision

1999;**70**:306-316

10.1002/cne.20823

1111/j.1365-2826.2009.01889.x

JNEUROSCI.3913-06.2007

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2003;**349**:75-78. DOI: 10.1016/S0304-3940(03)00789-4

2005;**65**:513-519. DOI: 10.1016/j.brainresbull.2005.03.004

349. DOI: 10.1677/JOE-08-0374

International Journal of Obesity. 2001;**25**:56-62

by in situ hybridization. FEBS Letters. 1996;**387**:113-116

Women are not small men. Sex-specific differences do not only affect the classical target organs of sexual differentiation and reproduction, but have been found to involve most, if not all the organs and tissues in the body. One of the consequences of this dimorphism is that diseases manifest in a sex- and gender-specific way. Key to maintenance of a healthy state is functioning tissue able to cope with insults. Regulated death of damaged cells and replacement with new cells by proliferation is a prerequisite for maintaining tissue function taking place at different pace in the different organs. The intent of this chapter is to review current evidence for sex-specific differences in tissue homeostasis focusing on the variability of hormone exposure characteristic for the female reproductive life stages.

Keywords: tissue maintenance, sex differences, proliferation, cell death, kidney, menstrual cycle

#### 1. Introduction

Living systems are continuously challenged by potentially toxic internal and external processes. The normal metabolic function of the cells produces a plethora of potentially damaging oxidative metabolites inducing damage in DNA, proteins, and lipids. In addition, living cells are exposed to a variety of external factors, which may be internalized as building blocks and/ or energy sources. These vital processes put the organism at risk to be harmed. Coping strategies are necessary to avoid damage. There are several lines of cellular defenses induced via cell stress pathways, including compartmentalization processes, enzymatic modification, externalization, degradation, and repair [1, 2].

Ultimately, these processes may not be sufficient to prevent major cellular damage. Therefore, every cell is in addition equipped with internal cell death programs, which can be activated in order to prevent a damaged cell to cause harm to the organism [3]. Cell losses are inevitable

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

and take place continuously in our bodies even without a specific trigger. The rate of cell death may be significantly enhanced at times of increased challenges. Cellular losses are necessary in order to prevent detrimental effects like neoplastic transformation [4]. Thus, programmed cell death needs to be carefully balanced, and this ability is a key determinant for the health and survival of the organism.

Ultimately, differences between the sexes derive from chromosomal and hormonal sex differences, which are further influenced by environmental factors. Thus, differences originating from the biological sex are further shaped by gender, which refers to the perceptions of male or

Sex and Sex Hormones in Tissue Homeostasis http://dx.doi.org/10.5772/intechopen.76177 31

Tissue homeostasis is guaranteed, when cells lost in physiological tissue turnover or under stress conditions are replaced by proliferation. Organs with high demanding functions have increased regeneration potential and continually renew their cell populations. This is the case for intestine, skin, and blood, for example. Liver, bone, and blood even have the capacity to fully recover to the original size after loss of tissue [14]. Other organs have lower regeneration potential, like the heart, brain, and kidney [15–17]. Many organs contain stem cell niches hosting adult tissue stem cells that are precursor cells maintained in a relatively undifferentiated state ready to replace lost cells by proliferation followed by differentiation [18, 19].

Sex hormones have classically been implicated in regulation of proliferation of cells of reproductive organs and cancer of reproductive tissue [20–24]. Besides these effects, sex hormones were also found to have pronounced effects on the proliferation of different stem cell populations. Cell proliferation of embryonic stem (ES) cells was found to be enhanced by female gonadal hormones [25]. ES cells are derived from the inner cell mass of the embryoid body. They can selfrenew in vitro and are pluripotent, that is, they can differentiate into all the cell types of the body [26]. Estrogen appears to act via nuclear and cell surface signaling pathways involving Erk1/2 activation, cyclin-dependent kinases and proto-oncogenes like c-myc, c-fos, c-jun, and pRB in ES cells. In addition, store-operated calcium channels were found to play a role in estrogen-

Differentiation of ES cells into dopaminergic neurons was also shown to be affected by estrogen. ERβ promoted differentiation by crosstalk signaling with insulin like growth factor-1 [28]. Motor neuron differentiation from ES cells was found to be enhanced by 17-β estradiol and progesterone through nuclear ERα and progesterone receptor [29]. Dopaminergic precursors derived from ES cells were found to increase proliferation upon treatment with progesterone

Induced pluripotent stem (iPS) cells are similar to ES cells with regard to their ability to differentiate into all cell types, providing a promising tool for in vitro research and regenerative medicine. They are derived from adult mature cells by reprogramming through the introduction of specific transcription factors [31]. Similar to ES cells, sex hormones were shown to affect iPS cells. Neuronal cells derived by differentiation of iPS cells showed increased dendritic branching by treatment with 17-β estradiol [32]. Functional integration of dopaminergic neuronal cells from iPS cells into neuronal circuits was found to be enhanced by estradiol [33]. Testosterone was described to enhance differentiation of iPS cells into insulin-

Sex differences were also described for tissue stem cells in vivo. Adult stem cells are believed to provide a local pool of self-renewing, multipotent cells pivotal in tissue homeostasis and

female identity and depends on sex-based social structures [8].

2.2. Influence of sex and sex hormones on cellular proliferation

mediated cell proliferation through the transcription factor NF-AT [27].

in vitro [30].

producing cells [34].

Cells lost by cell death need to be replaced in order to maintain cell numbers and ultimately tissue function. Controlled regeneration is, thus, required to cope for cell losses due to toxic challenges derived from internal and external sources, be they derived from normal metabolic processes or damaging environmental stimuli. Cell losses and proliferation need to be carefully balanced in order to guarantee proper function. Thus, the process of tissue homeostasis, that is, the capability to send damaged cells into cell death programs, to replace the cells by proliferation, and to regulate the exact balance of these events are crucial processes for preserving a healthy state. Any distortion of the balance between cell death and cell proliferation—be it by overwhelming damaging events beyond the host's range of tolerance and/or primarily ineffective or maladaptive homeostatic mechanisms by the host—is prone to induce malfunctioning of the organs in the body ultimately causing disease and potentially death.

#### 2. Sex and sex hormones in tissue homeostasis

#### 2.1. Origin of sex differences

During development, sex differences originate from genetic and hormonal influences. Master regulators for male sex differentiation, like SRY, are encoded by DNA of the Y chromosome governing the embryonic development of the male phenotype in mammals. Female or male gonadal development gives rise to a sex-specific hormonal environment [5]. Sex hormones induce organizational effects during the life span causing persistent sex-specific changes within the tissues, for example, by epigenetic modifications [6, 7]. Activational effects further introduce sex differences in tissue structure and function depending on the pattern of exposure to gonadal hormones. All the organs in the body are affected throughout life [8]. In this respect, sex differences are based on the different chromosomal equipment that qualifies every cell in the body as male or female. These basic differences are further shaped by sex hormones depending on previous or current, transient or persistent exposure [9]. This hypothesis was phrased by Arnold [10] as follows: "XX and XY cells are different prior to the secretion of gonadal hormones, and gonadal hormones affect XX and XY cells unequally."

Sex hormones act through receptors widely expressed throughout the cells of the body. The classical estrogen (ERα, ERβ), androgen (AR) and progesterone (PR) receptors belong to the nuclear receptor protein family acting through the nucleus as transcription factor or co-factor. In addition to their nuclear actions, they were found to be localized to the cell membrane and mitochondria inducing fast, non-genomic intracellular signaling pathways, for example, by the interaction with growth factor or cytokine receptors. Estrogen-binding cell membrane-localized receptors of the seven transmembrane receptor family were also characterized, for example, G-protein coupled estrogen receptor, GPR30/GPER-1 [11–13].

Ultimately, differences between the sexes derive from chromosomal and hormonal sex differences, which are further influenced by environmental factors. Thus, differences originating from the biological sex are further shaped by gender, which refers to the perceptions of male or female identity and depends on sex-based social structures [8].

#### 2.2. Influence of sex and sex hormones on cellular proliferation

and take place continuously in our bodies even without a specific trigger. The rate of cell death may be significantly enhanced at times of increased challenges. Cellular losses are necessary in order to prevent detrimental effects like neoplastic transformation [4]. Thus, programmed cell death needs to be carefully balanced, and this ability is a key determinant for the health and

Cells lost by cell death need to be replaced in order to maintain cell numbers and ultimately tissue function. Controlled regeneration is, thus, required to cope for cell losses due to toxic challenges derived from internal and external sources, be they derived from normal metabolic processes or damaging environmental stimuli. Cell losses and proliferation need to be carefully balanced in order to guarantee proper function. Thus, the process of tissue homeostasis, that is, the capability to send damaged cells into cell death programs, to replace the cells by proliferation, and to regulate the exact balance of these events are crucial processes for preserving a healthy state. Any distortion of the balance between cell death and cell proliferation—be it by overwhelming damaging events beyond the host's range of tolerance and/or primarily ineffective or maladaptive homeostatic mechanisms by the host—is prone to induce malfunctioning

During development, sex differences originate from genetic and hormonal influences. Master regulators for male sex differentiation, like SRY, are encoded by DNA of the Y chromosome governing the embryonic development of the male phenotype in mammals. Female or male gonadal development gives rise to a sex-specific hormonal environment [5]. Sex hormones induce organizational effects during the life span causing persistent sex-specific changes within the tissues, for example, by epigenetic modifications [6, 7]. Activational effects further introduce sex differences in tissue structure and function depending on the pattern of exposure to gonadal hormones. All the organs in the body are affected throughout life [8]. In this respect, sex differences are based on the different chromosomal equipment that qualifies every cell in the body as male or female. These basic differences are further shaped by sex hormones depending on previous or current, transient or persistent exposure [9]. This hypothesis was phrased by Arnold [10] as follows: "XX and XY cells are different prior to the secretion of

Sex hormones act through receptors widely expressed throughout the cells of the body. The classical estrogen (ERα, ERβ), androgen (AR) and progesterone (PR) receptors belong to the nuclear receptor protein family acting through the nucleus as transcription factor or co-factor. In addition to their nuclear actions, they were found to be localized to the cell membrane and mitochondria inducing fast, non-genomic intracellular signaling pathways, for example, by the interaction with growth factor or cytokine receptors. Estrogen-binding cell membrane-localized receptors of the seven transmembrane receptor family were also characterized, for example,

of the organs in the body ultimately causing disease and potentially death.

gonadal hormones, and gonadal hormones affect XX and XY cells unequally."

G-protein coupled estrogen receptor, GPR30/GPER-1 [11–13].

2. Sex and sex hormones in tissue homeostasis

survival of the organism.

30 Homeostasis - An Integrated Vision

2.1. Origin of sex differences

Tissue homeostasis is guaranteed, when cells lost in physiological tissue turnover or under stress conditions are replaced by proliferation. Organs with high demanding functions have increased regeneration potential and continually renew their cell populations. This is the case for intestine, skin, and blood, for example. Liver, bone, and blood even have the capacity to fully recover to the original size after loss of tissue [14]. Other organs have lower regeneration potential, like the heart, brain, and kidney [15–17]. Many organs contain stem cell niches hosting adult tissue stem cells that are precursor cells maintained in a relatively undifferentiated state ready to replace lost cells by proliferation followed by differentiation [18, 19].

Sex hormones have classically been implicated in regulation of proliferation of cells of reproductive organs and cancer of reproductive tissue [20–24]. Besides these effects, sex hormones were also found to have pronounced effects on the proliferation of different stem cell populations. Cell proliferation of embryonic stem (ES) cells was found to be enhanced by female gonadal hormones [25]. ES cells are derived from the inner cell mass of the embryoid body. They can selfrenew in vitro and are pluripotent, that is, they can differentiate into all the cell types of the body [26]. Estrogen appears to act via nuclear and cell surface signaling pathways involving Erk1/2 activation, cyclin-dependent kinases and proto-oncogenes like c-myc, c-fos, c-jun, and pRB in ES cells. In addition, store-operated calcium channels were found to play a role in estrogenmediated cell proliferation through the transcription factor NF-AT [27].

Differentiation of ES cells into dopaminergic neurons was also shown to be affected by estrogen. ERβ promoted differentiation by crosstalk signaling with insulin like growth factor-1 [28]. Motor neuron differentiation from ES cells was found to be enhanced by 17-β estradiol and progesterone through nuclear ERα and progesterone receptor [29]. Dopaminergic precursors derived from ES cells were found to increase proliferation upon treatment with progesterone in vitro [30].

Induced pluripotent stem (iPS) cells are similar to ES cells with regard to their ability to differentiate into all cell types, providing a promising tool for in vitro research and regenerative medicine. They are derived from adult mature cells by reprogramming through the introduction of specific transcription factors [31]. Similar to ES cells, sex hormones were shown to affect iPS cells. Neuronal cells derived by differentiation of iPS cells showed increased dendritic branching by treatment with 17-β estradiol [32]. Functional integration of dopaminergic neuronal cells from iPS cells into neuronal circuits was found to be enhanced by estradiol [33]. Testosterone was described to enhance differentiation of iPS cells into insulinproducing cells [34].

Sex differences were also described for tissue stem cells in vivo. Adult stem cells are believed to provide a local pool of self-renewing, multipotent cells pivotal in tissue homeostasis and recovery upon damage [35]. Stem cells in many stem cell niches appear to have a higher ability to self-renew, have an increased regeneration potential, and in some cases, show higher proliferative activity in women [36, 37]. Intrinsic sexual dimorphism was described for neural stem cells that hold much promise for potential brain damage repair therapy in the future. Proliferation of neural stem cell was, for example, shown to depend on hormone changes in the adult mouse due to the estrous cycle, pregnancy, reproductive status, and age. Phases of high estrogen exposure like pro-estrus were found to be associated with increased hippocampal adult neurogenesis indicating a role of estrogens [38]. Differential expression of sex steroid receptors and androgen metabolizing enzymes may result in differential outcomes in neural stem cell transplantation [39]. Neural stem cell proliferation was found to be dependent on nuclear ERs, while oligodendroglial differentiation was stimulated by cell membrane-associated ERs [40]. Other researcher also proposed that actions of sex steroids on the brain might be correlated with reduced brain damage. Intact females were found to be less susceptible upon injury than ovariectomized females and males [41]. Similarly, muscle-derived stem cells derived from female mice and transplanted into dystrophic mutant mice showed a better potential to regenerate skeletal muscle than stem cells from males [42].

many cancerous or age-related diseases have been shown to be influenced by the secretome of

Sex and Sex Hormones in Tissue Homeostasis http://dx.doi.org/10.5772/intechopen.76177 33

Alternatively, cells may activate a cell death program as a means to ensure physiological tissue renewal or in response to overwhelming damage. The most common are type I cell death programs or apoptosis, type II or autophagy, type III or necrosis, and mitotic catastrophe [57, 58]. The cell death modalities are characterized by different morphological criteria and are executed by specific intracellular signaling cascades. Specific catabolic enzymes are typically associated with specific forms of cell death, for example, caspases with apoptosis. The pathways are interdependent. The intensity of the damage signal is often decisive for the type of cell death program that is executed or the switch from one modality to the other. In addition, autophagy is not primarily regarded as a cell death mechanism. Autophagy describes a process involving the break-down and recycling of specific subcellular organelles. This process may provide a cell survival strategy by reducing damaged organelles and/or shifting internal resources in order to optimize cell survival. Only if the damaging process exceeds the cellular

Regarding the role of sex in cell fate decisions, several reports have highlighted distinct sexdependent differences. Sex hormones have been shown to influence the propensity of cells to undergo apoptosis. In general, lower concentrations of estrogen were found to be protective, while higher concentrations were found to promote apoptosis. Androgens were found to enhance, but also to suppress apoptosis depending on the cellular context [61, 62]. For example, estrogen and testosterone were described to reduce apoptosis in skeletal muscle cells [63]. Both hormones also appear to prevent apoptosis in neuronal cells adding to their neuroprotective function [64, 65]. An anti-apoptotic action of testosterone was also described in pancreatic β cells from male rats, but not from female rats [66]. Estrogen and estrogenic compounds, however, appeared to enhance apoptosis in pancreatic β cells in elderly mice, while it reduced apoptosis in young animals [67]. Regarding vascular endothelial cells, several studies have shown that estrogens protect from apoptotic cell death [68, 69], while apoptosis increased in coronary artery endothelia from postmenopausal women [70]. Testosterone was found to induce apoptosis in endothelial cells [71–73]. Treatment with testosterone also induced apoptosis or senescence in human dermal papilla cells, a process implied in inherited male alopecia [74, 75]. In addition, androgens were found to promote apoptosis in renal and

Overall, sex hormones appear to influence cell fate decisions depending on the cell context. Hormone independent sex differences are also apparent shaping the cellular response [79]. Thus, female and male cells appear to rely on different coping strategies in response to stressors. For example, vascular smooth muscle cells isolated from aorta of male rats appear to be more inclined to undergo apoptosis in response to UV irradiation, while female cells are more prone to execute the cell senescence program [62, 80]. Female cells showed characteristics of autophagy, which is presumed to help female cells to repair the UV-induced intracellular damages ultimately providing a survival strategy [81]. In addition, female cells were found to better adhere to the growth support, thus avoiding apoptotic cell death initiation by cell detachment, a process called anoikis-resistance. Differences in the intracellular organization

intestinal cell lines and bone marrow-derived macrophages [76–78].

senescence cells [54–56].

defenses, cells die in the process [59, 60].

Hematopoietic stem cells were found to be more abundant and proliferative in female mice in comparison to males dependent on estrogen exposure [43]. 17-β estradiol was found to improve hematopoietic differentiation from human iPS cells and from human umbilical cord blood through ERα signaling suggesting a universal function for estrogen in hematopoietic stem cell differentiation [44, 45].

Estrogens have beneficial effects on bone regeneration [46]. Osteoblasts are stimulated by estrogen to proliferate with distinct roles for ERα and ERβ [47]. In vitro, proliferation of bone marrow mesenchymal stromal cells was found to be enhanced by estrogen [48]. Estrogens enhanced the proliferation and migration of bone marrow-derived endothelial progenitor cells to ischemic regions of the heart facilitating repair and regeneration [49]. Androgens were also described to stimulate the proliferation and angiogenesis/vascular repair capability of circulating endothelial progenitor cells in males, not females [50].

#### 2.3. Sex differences in cell survival

When progenitor cells involved in tissue regeneration enter a cell senescence state, tissue homeostasis may be compromised. The cells are able to permanently halt the cell cycle and persist in a quiescent, but still functional state [51]. This is a possible fate of cells damaged beyond repair. The three major types of senescence are replicative senescence, oncogeneinduced senescence, and DNA damage-induced DNA damage. The DNA damage response pathway appears to be eventually involved in the execution of the program independent of the primary stimulus [52]. Furthermore, senescent cells are able to influence their neighboring cells by secretion of a range of activating signals referred to as senescence-associated secretory phenotype. The signals may favor a pro-inflammatory or—alternatively—an immunosuppressive/pro-fibrotic state. Both phases appear to be important for successful tissue repair and the timing of the shift in the secretome might be crucial [53]. The etiology and progression of many cancerous or age-related diseases have been shown to be influenced by the secretome of senescence cells [54–56].

recovery upon damage [35]. Stem cells in many stem cell niches appear to have a higher ability to self-renew, have an increased regeneration potential, and in some cases, show higher proliferative activity in women [36, 37]. Intrinsic sexual dimorphism was described for neural stem cells that hold much promise for potential brain damage repair therapy in the future. Proliferation of neural stem cell was, for example, shown to depend on hormone changes in the adult mouse due to the estrous cycle, pregnancy, reproductive status, and age. Phases of high estrogen exposure like pro-estrus were found to be associated with increased hippocampal adult neurogenesis indicating a role of estrogens [38]. Differential expression of sex steroid receptors and androgen metabolizing enzymes may result in differential outcomes in neural stem cell transplantation [39]. Neural stem cell proliferation was found to be dependent on nuclear ERs, while oligodendroglial differentiation was stimulated by cell membrane-associated ERs [40]. Other researcher also proposed that actions of sex steroids on the brain might be correlated with reduced brain damage. Intact females were found to be less susceptible upon injury than ovariectomized females and males [41]. Similarly, muscle-derived stem cells derived from female mice and transplanted into dystrophic mutant mice showed a better potential to

Hematopoietic stem cells were found to be more abundant and proliferative in female mice in comparison to males dependent on estrogen exposure [43]. 17-β estradiol was found to improve hematopoietic differentiation from human iPS cells and from human umbilical cord blood through ERα signaling suggesting a universal function for estrogen in hematopoietic

Estrogens have beneficial effects on bone regeneration [46]. Osteoblasts are stimulated by estrogen to proliferate with distinct roles for ERα and ERβ [47]. In vitro, proliferation of bone marrow mesenchymal stromal cells was found to be enhanced by estrogen [48]. Estrogens enhanced the proliferation and migration of bone marrow-derived endothelial progenitor cells to ischemic regions of the heart facilitating repair and regeneration [49]. Androgens were also described to stimulate the proliferation and angiogenesis/vascular repair capability of circulat-

When progenitor cells involved in tissue regeneration enter a cell senescence state, tissue homeostasis may be compromised. The cells are able to permanently halt the cell cycle and persist in a quiescent, but still functional state [51]. This is a possible fate of cells damaged beyond repair. The three major types of senescence are replicative senescence, oncogeneinduced senescence, and DNA damage-induced DNA damage. The DNA damage response pathway appears to be eventually involved in the execution of the program independent of the primary stimulus [52]. Furthermore, senescent cells are able to influence their neighboring cells by secretion of a range of activating signals referred to as senescence-associated secretory phenotype. The signals may favor a pro-inflammatory or—alternatively—an immunosuppressive/pro-fibrotic state. Both phases appear to be important for successful tissue repair and the timing of the shift in the secretome might be crucial [53]. The etiology and progression of

regenerate skeletal muscle than stem cells from males [42].

ing endothelial progenitor cells in males, not females [50].

stem cell differentiation [44, 45].

32 Homeostasis - An Integrated Vision

2.3. Sex differences in cell survival

Alternatively, cells may activate a cell death program as a means to ensure physiological tissue renewal or in response to overwhelming damage. The most common are type I cell death programs or apoptosis, type II or autophagy, type III or necrosis, and mitotic catastrophe [57, 58]. The cell death modalities are characterized by different morphological criteria and are executed by specific intracellular signaling cascades. Specific catabolic enzymes are typically associated with specific forms of cell death, for example, caspases with apoptosis. The pathways are interdependent. The intensity of the damage signal is often decisive for the type of cell death program that is executed or the switch from one modality to the other. In addition, autophagy is not primarily regarded as a cell death mechanism. Autophagy describes a process involving the break-down and recycling of specific subcellular organelles. This process may provide a cell survival strategy by reducing damaged organelles and/or shifting internal resources in order to optimize cell survival. Only if the damaging process exceeds the cellular defenses, cells die in the process [59, 60].

Regarding the role of sex in cell fate decisions, several reports have highlighted distinct sexdependent differences. Sex hormones have been shown to influence the propensity of cells to undergo apoptosis. In general, lower concentrations of estrogen were found to be protective, while higher concentrations were found to promote apoptosis. Androgens were found to enhance, but also to suppress apoptosis depending on the cellular context [61, 62]. For example, estrogen and testosterone were described to reduce apoptosis in skeletal muscle cells [63]. Both hormones also appear to prevent apoptosis in neuronal cells adding to their neuroprotective function [64, 65]. An anti-apoptotic action of testosterone was also described in pancreatic β cells from male rats, but not from female rats [66]. Estrogen and estrogenic compounds, however, appeared to enhance apoptosis in pancreatic β cells in elderly mice, while it reduced apoptosis in young animals [67]. Regarding vascular endothelial cells, several studies have shown that estrogens protect from apoptotic cell death [68, 69], while apoptosis increased in coronary artery endothelia from postmenopausal women [70]. Testosterone was found to induce apoptosis in endothelial cells [71–73]. Treatment with testosterone also induced apoptosis or senescence in human dermal papilla cells, a process implied in inherited male alopecia [74, 75]. In addition, androgens were found to promote apoptosis in renal and intestinal cell lines and bone marrow-derived macrophages [76–78].

Overall, sex hormones appear to influence cell fate decisions depending on the cell context. Hormone independent sex differences are also apparent shaping the cellular response [79]. Thus, female and male cells appear to rely on different coping strategies in response to stressors. For example, vascular smooth muscle cells isolated from aorta of male rats appear to be more inclined to undergo apoptosis in response to UV irradiation, while female cells are more prone to execute the cell senescence program [62, 80]. Female cells showed characteristics of autophagy, which is presumed to help female cells to repair the UV-induced intracellular damages ultimately providing a survival strategy [81]. In addition, female cells were found to better adhere to the growth support, thus avoiding apoptotic cell death initiation by cell detachment, a process called anoikis-resistance. Differences in the intracellular organization of the actin cytoskeleton and increased phosphorylation of focal adhesion kinase were attributed to this higher propensity of female cells to adhere [81]. Apparently, female cells are better equipped to prevent cell death. While autophagic processes were found to protect neuronal cells from cell death due to starving in female rats, male cells were not able to benefit and died more often from autophagic cell death [82]. Organ-specific sex differences were found in constitutive autophagy, a process implicated in physiological tissue turnover. While autophagic marker proteins were increased in the male versus female heart and liver, no such differences were observed in the kidneys [83]. Osteoblasts showed reduced autophagy in aging female mice, while the rate remained constant in males over the life span. This was correlated with higher oxidative stress in female cells, thus potentially enhancing bone loss and playing a role in the pathophysiology of osteoporosis in women [84]. Estrogens alter the redox balance and counteract bone loss [46]. Stem cells involved in generation of osteoblast, namely bone marrow derived mesenchymal stem cells, were found to be influenced by estrogens not only inducing increased proliferation, but also reducing senescence and apoptosis [85].

modulation of mitochondrial function is achieved by hormone effects on the expression of mitochondrial and nuclear genome-encoded mitochondrial proteins [97–101]. Since mitochondria are central in the cellular defense against oxidative stress, mitochondria are especially sensitive to accumulate damage over time. Malfunctioning mitochondria accumulate during aging, a process regarded as a major contributor to the onset of many age-related diseases [102, 103]. Sex differences were observed in this process. Delayed malfunctioning of mitochondria during the aging process might provide females with better strategies to cope with cellular stressors. Maternal transmission of mitochondria appears to provide a more favorable environment in female offspring [104]. Xist, an RNA-coding gene involved in X chromosome inactivation in female cells, appears to be pivotal for mitochondrial maintenance [105]. Mitochondrial biogenesis and degradation by mitophagy are dependent on the transcription factors p53 and FOXO [106, 107]. Sex-specific differences in the activity of these nuclear factors were reported. Males were shown to exhibit relatively greater FOXO activity. Females, on the other hand, had higher p53 activity resulting in sex-specific differences in the ability to maintain

Sex and Sex Hormones in Tissue Homeostasis http://dx.doi.org/10.5772/intechopen.76177 35

healthy mitochondrial functionality during aging [105].

cancer, among others [108–110].

proliferation upon estrogen treatment [126].

2.4. Potential consequences of sex-specific differences in tissue homeostasis

The abovementioned paragraphs have described examples of sex-specific differences regarding processes involved in tissue maintenance, like the control of cell proliferation and cell death. Such effects may ultimately result in differences in the ability of female and male tissues to cope with stressors affecting the ability to repair and restore function or develop disease. Many diseases show different incidence and prevalence rates in men and women derived from sex and gender specific pathophysiological mechanisms. Sex and gender differences have been studied intensively in the neural system, the cardiovascular system, and the development of

Mechanisms underlying differences in kidney diseases between men and women are less well known, despite renal diseases with a high morbidity and mortality risk being a challenging problem for patients, clinicians and society [111, 112]. International registries show that fewer women than men develop kidney failure [113–116]. The underlying causes, however, are widely unknown. The presumed female protective effects appear to be most pronounced in women of reproductive age [117–120]. This finding suggests that female sex hormones might play a key role. Estrogen was proposed to be renoprotective via modulating renal perfusion and effects on the vasculature. Furthermore, a role of estrogen was proposed in the control of the local renal renin-angiotensin system [121–123]. On the other hand, estrogen was implicated in the control of mesangial and tubular cell proliferation and linked to neoplastic transformation of the kidney in hamster kidneys. Low estrogen concentrations were shown to induce proliferation in glomerular mesangial cell, while high concentrations suppressed it [124]. Primary proximal tubular cell explants and subcultured dissociated proximal tubular cells were shown to proliferate, when treated with estrogen at physiologic concentrations [125]. This finding was confirmed in primary rabbit proximal tubular cells, which showed increased

We have previously shown that renal tubular cell-specific proteins appear at higher rates in the urine of healthy women at specific hormonal transition phases of the natural ovulating

In general, increased antioxidative cellular defenses were implicated to provide females with better strategies to cope with oxidative stress and prevent cellular losses [62]. Differences in basal redox state and responses to oxidative imbalance were demonstrated between female and male cells [86]. For example, female cells were shown to produce less hydrogen peroxide and superoxide anion. Anti-oxidative enzymes, such as superoxide dismutase (SOD) and catalase, showed higher basic activity in female versus male cells [87]. Thioredoxin reductases and manganese SOD were increased by estrogen in cardiomyocytes [88, 89]. In vascular smooth muscle cells and circulating monocytes, estrogen was found to stimulate manganese and extracellular SOD expression [90]. Estrogen was, furthermore, shown to modulate the expression of other key molecular defense enzymes differently in XX and XY cells, for example, poly-ADP ribose polymerase (PARP), a DNA damage repair enzyme, or RLIP76, a cellprotective transporter protein [86].

PARP was also found to play a major role in sex differences in stroke. Experiments in mice have shown that ischemic neuronal cell death is dependent on intact neuronal nitric oxide synthase (nNOS)/PARP signaling, while in females a protection is provided by estrogen paradoxically also requiring an intact nNOS/PAPR axis [91]. While male neuronal cells appear to die via a PARP-mediated caspase independent pathway, ischemic cell death pathways appear to be dependent on activation of caspase-dependent cell death pathways in females [9, 92]. Such sex-specific differences may be relevant for the sex-specific difference in stroke prevalence [93, 94].

PARP signaling was also implied in sex differences in cell fate decisions in kidney cells. In a mouse model of immune-mediated nephritis, PARP signaling induced necrosis in male cells and inhibition of PARP shifted the pro-inflammatory necrotic cell death to an antiinflammatory apoptotic pathway. In female cells, by contrast, cell death was independent of PARP and female cells preferentially underwent apoptosis. Estrogen acted in a pro-survival manner in female cells only. In addition to the kidney cells, bone marrow-derived hematopoietic cells showed similar sex differences [95].

Mitochondria play a crucial role in apoptotic cell death programs. Estrogens were described to modulate the propensity for mitochondrial initiation of apoptosis [61, 96]. Estrogen-mediated

modulation of mitochondrial function is achieved by hormone effects on the expression of mitochondrial and nuclear genome-encoded mitochondrial proteins [97–101]. Since mitochondria are central in the cellular defense against oxidative stress, mitochondria are especially sensitive to accumulate damage over time. Malfunctioning mitochondria accumulate during aging, a process regarded as a major contributor to the onset of many age-related diseases [102, 103]. Sex differences were observed in this process. Delayed malfunctioning of mitochondria during the aging process might provide females with better strategies to cope with cellular stressors. Maternal transmission of mitochondria appears to provide a more favorable environment in female offspring [104]. Xist, an RNA-coding gene involved in X chromosome inactivation in female cells, appears to be pivotal for mitochondrial maintenance [105]. Mitochondrial biogenesis and degradation by mitophagy are dependent on the transcription factors p53 and FOXO [106, 107]. Sex-specific differences in the activity of these nuclear factors were reported. Males were shown to exhibit relatively greater FOXO activity. Females, on the other hand, had higher p53 activity resulting in sex-specific differences in the ability to maintain healthy mitochondrial functionality during aging [105].

#### 2.4. Potential consequences of sex-specific differences in tissue homeostasis

of the actin cytoskeleton and increased phosphorylation of focal adhesion kinase were attributed to this higher propensity of female cells to adhere [81]. Apparently, female cells are better equipped to prevent cell death. While autophagic processes were found to protect neuronal cells from cell death due to starving in female rats, male cells were not able to benefit and died more often from autophagic cell death [82]. Organ-specific sex differences were found in constitutive autophagy, a process implicated in physiological tissue turnover. While autophagic marker proteins were increased in the male versus female heart and liver, no such differences were observed in the kidneys [83]. Osteoblasts showed reduced autophagy in aging female mice, while the rate remained constant in males over the life span. This was correlated with higher oxidative stress in female cells, thus potentially enhancing bone loss and playing a role in the pathophysiology of osteoporosis in women [84]. Estrogens alter the redox balance and counteract bone loss [46]. Stem cells involved in generation of osteoblast, namely bone marrow derived mesenchymal stem cells, were found to be influenced by estrogens not only inducing

In general, increased antioxidative cellular defenses were implicated to provide females with better strategies to cope with oxidative stress and prevent cellular losses [62]. Differences in basal redox state and responses to oxidative imbalance were demonstrated between female and male cells [86]. For example, female cells were shown to produce less hydrogen peroxide and superoxide anion. Anti-oxidative enzymes, such as superoxide dismutase (SOD) and catalase, showed higher basic activity in female versus male cells [87]. Thioredoxin reductases and manganese SOD were increased by estrogen in cardiomyocytes [88, 89]. In vascular smooth muscle cells and circulating monocytes, estrogen was found to stimulate manganese and extracellular SOD expression [90]. Estrogen was, furthermore, shown to modulate the expression of other key molecular defense enzymes differently in XX and XY cells, for example, poly-ADP ribose polymerase (PARP), a DNA damage repair enzyme, or RLIP76, a cell-

PARP was also found to play a major role in sex differences in stroke. Experiments in mice have shown that ischemic neuronal cell death is dependent on intact neuronal nitric oxide synthase (nNOS)/PARP signaling, while in females a protection is provided by estrogen paradoxically also requiring an intact nNOS/PAPR axis [91]. While male neuronal cells appear to die via a PARP-mediated caspase independent pathway, ischemic cell death pathways appear to be dependent on activation of caspase-dependent cell death pathways in females [9, 92]. Such sex-specific differences may be relevant for the sex-specific difference in stroke

PARP signaling was also implied in sex differences in cell fate decisions in kidney cells. In a mouse model of immune-mediated nephritis, PARP signaling induced necrosis in male cells and inhibition of PARP shifted the pro-inflammatory necrotic cell death to an antiinflammatory apoptotic pathway. In female cells, by contrast, cell death was independent of PARP and female cells preferentially underwent apoptosis. Estrogen acted in a pro-survival manner in female cells only. In addition to the kidney cells, bone marrow-derived hematopoi-

Mitochondria play a crucial role in apoptotic cell death programs. Estrogens were described to modulate the propensity for mitochondrial initiation of apoptosis [61, 96]. Estrogen-mediated

increased proliferation, but also reducing senescence and apoptosis [85].

protective transporter protein [86].

34 Homeostasis - An Integrated Vision

etic cells showed similar sex differences [95].

prevalence [93, 94].

The abovementioned paragraphs have described examples of sex-specific differences regarding processes involved in tissue maintenance, like the control of cell proliferation and cell death. Such effects may ultimately result in differences in the ability of female and male tissues to cope with stressors affecting the ability to repair and restore function or develop disease. Many diseases show different incidence and prevalence rates in men and women derived from sex and gender specific pathophysiological mechanisms. Sex and gender differences have been studied intensively in the neural system, the cardiovascular system, and the development of cancer, among others [108–110].

Mechanisms underlying differences in kidney diseases between men and women are less well known, despite renal diseases with a high morbidity and mortality risk being a challenging problem for patients, clinicians and society [111, 112]. International registries show that fewer women than men develop kidney failure [113–116]. The underlying causes, however, are widely unknown. The presumed female protective effects appear to be most pronounced in women of reproductive age [117–120]. This finding suggests that female sex hormones might play a key role. Estrogen was proposed to be renoprotective via modulating renal perfusion and effects on the vasculature. Furthermore, a role of estrogen was proposed in the control of the local renal renin-angiotensin system [121–123]. On the other hand, estrogen was implicated in the control of mesangial and tubular cell proliferation and linked to neoplastic transformation of the kidney in hamster kidneys. Low estrogen concentrations were shown to induce proliferation in glomerular mesangial cell, while high concentrations suppressed it [124]. Primary proximal tubular cell explants and subcultured dissociated proximal tubular cells were shown to proliferate, when treated with estrogen at physiologic concentrations [125]. This finding was confirmed in primary rabbit proximal tubular cells, which showed increased proliferation upon estrogen treatment [126].

We have previously shown that renal tubular cell-specific proteins appear at higher rates in the urine of healthy women at specific hormonal transition phases of the natural ovulating menstrual cycle. Urinary samples from healthy probands showed increased rates of urinary excretion of the marker proteins Fructose-1,6-bisphosphatase and Glutathione-S-transferase α, when estrogen levels decreased after a preceding height associated with ovulation and luteal phase [127]. Both enzymes are specifically found in proximal tubular cells, the most populous cell type in the kidney. When proximal tubular cells are damaged, intracellular enzymes are released into the urine, making them clinical markers for kidney injury. In contrast to ovulating women, male probands and postmenopausal women showed consistently low levels of these renal marker proteins over time. Other urinary proteins, for example, albumin, α1 microglobulin, and immunglobulin G, which are markers for functional changes of the glomerular filter and/or tubular protein resorption, showed constant urinary excretion suggesting that the observed increases of proximal tubular marker protein release in ovulating women are not accompanied by major functional distress of the kidneys [127]. This pattern of urinary marker proteins excretion suggests that cyclical changes of female hormones might affect kidney cell health. Tubular enzymes are released into the urine, if proximal tubular cells are sloughed off and/or their plasma membranes become leaky. This could be due to tubular cells being transiently more prone to damage in situ resulting in plasma membrane leakage or to the cells being removed from the tubular epithelium and released into the urinary space, for example, by apoptosis. Both processes lead to increased cell losses. Tissue homeostasis would be maintained, if increased cell removal was accompanied by increased cell proliferation. This could be the case during the high estrogen exposure phases preceding the observed tubular enzyme releases into the urine. The finding that tubular cells are able to proliferate upon estrogen treatment [126] is in line with this hypothesis. Such a periodic interplay between cell proliferation and cell loss brought about by the specific changes in the pattern of sex hormone exposure might result in an increased rate of tissue renewal. If this was the case, then women in their reproductive years would possess an efficient means to easily get rid of potentially injured, dysfunctional or simply older proximal tubular cells by replacing them with fresh new cells. Such a transiently increased repair capacity might provide an efficient means to cyclically renew renal tubular tissue leading to a higher resistance to damage. It is, however, also possible that during the short phases of increased tubular cell death, the kidneys might be especially sensitive to damage. With regard to the potential beneficial action of treatment of renal proximal tubular cells with a proliferation-inducing growth factor, we have previously demonstrated that epidermal growth factor (EGF) treatment was able to accelerate tissue repair after treatment with interferon α (IFNα) in vitro. However, if EGF was present before or during IFNα treatment, epithelial barrier destabilization was intensified [128, 129]. Therefore, the overall effect might be different in other cycle contexts or under hormone therapy, if the vulnerable phases might not be restricted to short periods.

expression levels and activity of detoxifying and repair enzymes, in the propensity to use autophagic processes for repair, in senescence or cell death programs and in the ability to replace cells by proliferation (Figure 1). These effects are apparent in isolated cells and are further shaped by exposure to sex hormones. Sex hormone levels cyclically changing in dependence of the female reproductive hormone cycle might enhance physiological tissue regeneration and provide greater damage repair potential. Overall, female tissues appear to

Figure 1. Sex and sex hormone-induced differences in tissue homeostasis. The figure shows strategies involved in tissue maintenance following cellular stress. Sex differences and sex hormone-dependent effects have been shown in these

Sex and Sex Hormones in Tissue Homeostasis http://dx.doi.org/10.5772/intechopen.76177 37

This work was supported by the Austrian Central Bank Fund project No 13671 to J Lechner.

be more resistant to cellular stress than their male counterparts.

The authors confirm that there are no conflicts of interest.

\*Address all correspondence to: judith.lechner@i-med.ac.at

Division of Physiology, Medical University of Innsbruck, Innsbruck, Austria

Judith Lechner\* and Gerhard Gstraunthaler

Acknowledgements

processes in different organs and tissues.

Conflict of interest

Author details

#### 3. Conclusion

In conclusion, it appears that males and females are equipped with stress coping strategies that may differ between the sexes. Sex differences have been demonstrated in the cellular

Figure 1. Sex and sex hormone-induced differences in tissue homeostasis. The figure shows strategies involved in tissue maintenance following cellular stress. Sex differences and sex hormone-dependent effects have been shown in these processes in different organs and tissues.

expression levels and activity of detoxifying and repair enzymes, in the propensity to use autophagic processes for repair, in senescence or cell death programs and in the ability to replace cells by proliferation (Figure 1). These effects are apparent in isolated cells and are further shaped by exposure to sex hormones. Sex hormone levels cyclically changing in dependence of the female reproductive hormone cycle might enhance physiological tissue regeneration and provide greater damage repair potential. Overall, female tissues appear to be more resistant to cellular stress than their male counterparts.

#### Acknowledgements

menstrual cycle. Urinary samples from healthy probands showed increased rates of urinary excretion of the marker proteins Fructose-1,6-bisphosphatase and Glutathione-S-transferase α, when estrogen levels decreased after a preceding height associated with ovulation and luteal phase [127]. Both enzymes are specifically found in proximal tubular cells, the most populous cell type in the kidney. When proximal tubular cells are damaged, intracellular enzymes are released into the urine, making them clinical markers for kidney injury. In contrast to ovulating women, male probands and postmenopausal women showed consistently low levels of these renal marker proteins over time. Other urinary proteins, for example, albumin, α1 microglobulin, and immunglobulin G, which are markers for functional changes of the glomerular filter and/or tubular protein resorption, showed constant urinary excretion suggesting that the observed increases of proximal tubular marker protein release in ovulating women are not accompanied by major functional distress of the kidneys [127]. This pattern of urinary marker proteins excretion suggests that cyclical changes of female hormones might affect kidney cell health. Tubular enzymes are released into the urine, if proximal tubular cells are sloughed off and/or their plasma membranes become leaky. This could be due to tubular cells being transiently more prone to damage in situ resulting in plasma membrane leakage or to the cells being removed from the tubular epithelium and released into the urinary space, for example, by apoptosis. Both processes lead to increased cell losses. Tissue homeostasis would be maintained, if increased cell removal was accompanied by increased cell proliferation. This could be the case during the high estrogen exposure phases preceding the observed tubular enzyme releases into the urine. The finding that tubular cells are able to proliferate upon estrogen treatment [126] is in line with this hypothesis. Such a periodic interplay between cell proliferation and cell loss brought about by the specific changes in the pattern of sex hormone exposure might result in an increased rate of tissue renewal. If this was the case, then women in their reproductive years would possess an efficient means to easily get rid of potentially injured, dysfunctional or simply older proximal tubular cells by replacing them with fresh new cells. Such a transiently increased repair capacity might provide an efficient means to cyclically renew renal tubular tissue leading to a higher resistance to damage. It is, however, also possible that during the short phases of increased tubular cell death, the kidneys might be especially sensitive to damage. With regard to the potential beneficial action of treatment of renal proximal tubular cells with a proliferation-inducing growth factor, we have previously demonstrated that epidermal growth factor (EGF) treatment was able to accelerate tissue repair after treatment with interferon α (IFNα) in vitro. However, if EGF was present before or during IFNα treatment, epithelial barrier destabilization was intensified [128, 129]. Therefore, the overall effect might be different in other cycle contexts or under hormone therapy, if

the vulnerable phases might not be restricted to short periods.

In conclusion, it appears that males and females are equipped with stress coping strategies that may differ between the sexes. Sex differences have been demonstrated in the cellular

3. Conclusion

36 Homeostasis - An Integrated Vision

This work was supported by the Austrian Central Bank Fund project No 13671 to J Lechner.

#### Conflict of interest

The authors confirm that there are no conflicts of interest.

#### Author details

Judith Lechner\* and Gerhard Gstraunthaler

\*Address all correspondence to: judith.lechner@i-med.ac.at

Division of Physiology, Medical University of Innsbruck, Innsbruck, Austria

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**Section 3**

**Integrated Systems**

**Section 3**

**Integrated Systems**

**Chapter 4**

**Provisional chapter**

**Gateway Reflex: A Neuro-Immune Crosstalk for Organ-**

Homeostasis of the central nervous system (CNS) is strictly regulated by a unique structure of blood vessels, the blood-brain barrier (BBB). Experimental and clinical evidence has revealed that abnormalities in the BBB in chronic inflammatory diseases such as multiple sclerosis (MS). By using an animal model of MS, we identified novel neuro-immune crosstalk to explain how pathogenic immune cells enter the CNS to disrupt its homeostasis, a phenomenon we named the gateway reflex. Regional neural inputs such as gravity, electricity, pain or chronic stress cause specific neural activation to create a gateway of immune cells, particularly pathogenic ones, at specific blood vessels. Moreover, the recently discovered stress-induced gateway reflex uncovered a stress-induced neural link between the brain, gastrointestine, and heart. Thus, the gateway reflex is critical for the homeostasis of various organs, and aberrant activation of neural pathways by the gateway reflex disrupts normal organ homeostasis. The inflammatory reflex is another mechanism for local neuro-immune interactions. It potently exerts a cholinergic antiinflammatory effect on various disease conditions. In this section, we discuss emerging roles for local neuro-immune interactions, with a special focus on the gateway reflex.

**Keywords:** gateway reflex, experimental autoimmune encephalomyelitis, central

A variety of environmental stimulations such as light, temperature, sound, and so on activates specific neurons to trigger biological responses. Gravity is another stimulus on land animals. Without sufficient gravity stimulation, physical functions including bone mass and

nervous system, chemokines, pathogenic CD4+ T cells

**Gateway Reflex: A Neuro-Immune Crosstalk for Organ-**

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

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

DOI: 10.5772/intechopen.77112

**Specific Disease Development**

**Specific Disease Development**

Masaaki Murakami

Masaaki Murakami

**Abstract**

**1. Introduction**

Daisuke Kamimura, Yuki Tanaka, Takuto Ohki and

Daisuke Kamimura, Yuki Tanaka, Takuto Ohki and

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.77112

#### **Gateway Reflex: A Neuro-Immune Crosstalk for Organ-Specific Disease Development Gateway Reflex: A Neuro-Immune Crosstalk for Organ-Specific Disease Development**

DOI: 10.5772/intechopen.77112

Daisuke Kamimura, Yuki Tanaka, Takuto Ohki and Masaaki Murakami Daisuke Kamimura, Yuki Tanaka, Takuto Ohki and Masaaki Murakami

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

http://dx.doi.org/10.5772/intechopen.77112

#### **Abstract**

Homeostasis of the central nervous system (CNS) is strictly regulated by a unique structure of blood vessels, the blood-brain barrier (BBB). Experimental and clinical evidence has revealed that abnormalities in the BBB in chronic inflammatory diseases such as multiple sclerosis (MS). By using an animal model of MS, we identified novel neuro-immune crosstalk to explain how pathogenic immune cells enter the CNS to disrupt its homeostasis, a phenomenon we named the gateway reflex. Regional neural inputs such as gravity, electricity, pain or chronic stress cause specific neural activation to create a gateway of immune cells, particularly pathogenic ones, at specific blood vessels. Moreover, the recently discovered stress-induced gateway reflex uncovered a stress-induced neural link between the brain, gastrointestine, and heart. Thus, the gateway reflex is critical for the homeostasis of various organs, and aberrant activation of neural pathways by the gateway reflex disrupts normal organ homeostasis. The inflammatory reflex is another mechanism for local neuro-immune interactions. It potently exerts a cholinergic antiinflammatory effect on various disease conditions. In this section, we discuss emerging roles for local neuro-immune interactions, with a special focus on the gateway reflex.

**Keywords:** gateway reflex, experimental autoimmune encephalomyelitis, central nervous system, chemokines, pathogenic CD4+ T cells

#### **1. Introduction**

A variety of environmental stimulations such as light, temperature, sound, and so on activates specific neurons to trigger biological responses. Gravity is another stimulus on land animals. Without sufficient gravity stimulation, physical functions including bone mass and

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

muscle strength change, as observed in astronauts who stayed in the International Space Station [1, 2]. In addition to these physical stimulations, various events in daily social interactions associated with psychological alterations including anxiety, mental stresses or positive emotions are also stimulatory factors that trigger specific neural pathways to affect body functions. A well-studied mechanism to cope with these stimulations includes the release of glucocorticoids via the hypothalamus-pituitary-adrenal grand (HPA) axis, which systemically changes various physiological functions including the immune system [3, 4]. Besides this systemic regulation, we and other groups have identified local regulations of the inflammatory status by various environmental cues that cause specific neural activations. Here we summarize examples of specific neuro-immune interactions in organ homeostasis.

EAE symptoms revealed that pathogenic CD4+ T cells mainly accumulated at the L5 spinal cord, around the dorsal vessels in particular [18]. On the other hand, no accumulation was observed in the brain or upper spinal levels at the preclinical time point of EAE. Various chemokines including CCL20 attract type-17 CD4+ T (Th17) cells, which are known to have a key pathogenic role in EAE [21, 22], to accumulate more in the dorsal vessels of L5 than L1 cord. Indeed, CCL20 neutralization or deficiency of its receptor, CCR6, on pathogenic CD4+ T cells

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53

Interestingly, even without EAE induction, chemokine levels were higher in the L5 dorsal vessels than in the L1 cord. These results suggest a distinct property of L5 dorsal vessels both in the presence and absence of pathogenic CD4+ T cells. The L5 spinal level has the largest dorsal root ganglion (DRG) in both human and mice, and it is known that sensory neurons in the L5 DRG distribute to the soleus muscles, the main anti-gravity muscles of the body [23, 24]. These facts led us to hypothesize a link between gravity, L5 vessels and local inflammation. We examined this possibility using a ground experiment employed by the National Aeronautics and Space Administration (NASA) and Japan Aerospace Exploration Agency (JAXA) [25, 26]. The tail suspension method puts mice in a handstand position, such that the hind limbs are released from gravity stimulation. As hypothesized, after the tail suspension, chemokine expressions of the L5 dorsal vessels were reduced and pathogenic CD4+ T cells hardly invaded the L5 cord. Chemokines were instead upregulated at the cervical cords as though another gateway for immune cells was formed by the greater gravity stimulation on the arm muscles imposed by the tail suspension [18]. Consistently, the tail suspension significantly reduced the expression of a neural activation marker, c-Fos, in the L5 DRG, suggesting a correlation between regional neural activation and local inflammation in the CNS. Consistently, stimulation of the soleus muscles by weak electric pulses, which mimic gravity-mediated sensory activation, during tail suspension restored chemokine expression, pathogenic CD4+ T cell accumulation, and c-Fos levels at the L5 dorsal vessels [18]. That study identified L5 dorsal vessels as a blood vessel gateway for immune cells including pathogenic CD4+ T cells to the CNS, and revealed that regional sensory neural activation by gravity affects CNS homeostasis and causes local inflammation at L5 dorsal blood vessels when CNS-autoreactive pathogenic CD4+ T cells are present. This phenomenon, which represents a novel neuro-immune interaction, was termed the "gateway reflex" [27–34]. Since then, we have found various types of gateway reflexes depending on the neural stimulation. Thus, the

abovementioned example is known as the gravity gateway reflex (**Figure 1**).

How the regional neural pathway by gravity stimulation regulates chemokine expressions in L5 blood vessels is an important question. Contribution of the autonomic nervous system was suggested. In fact, neural activation examined by c-Fos expression was higher in sympathetic ganglions at the L5 cord than at the L1 cord. In addition, the blood flow speed of the L5 dorsal vessels but not of other blood vessels including the L1 dorsal vessels, femoral artery or portal vein, slowed after tail suspension. Functionally, the treatment of mice with β-adrenergic receptor antagonists or chemical sympathectomy inhibited chemokine expressions, the accumulation of pathogenic CD4+ T cells at the L5 dorsal vessels, and the clinical scores of EAE [18]. Norepinephrine is one of the major neurotransmitters of sympathetic nerves. Indeed, in vitro experiments suggest that norepinephrine enhances chemokine expressions from

abrogated the cell accumulation in the L5 spinal cord [18].

#### **2. Main text**

#### **2.1. Gravity gateway reflex**

The central nervous system (CNS) is considered an immune privileged site due to structural protections by the blood-brain barrier (BBB) [5]. The BBB is formed by tight cell-cell interactions between blood endothelial cells and tight liner sheets, a structure that lies outside the basement membrane and consists of pericytes, neurons, microglias, and astrocytes. Tight junctions by the interactions of tight-junction molecules including claudins and occludins are critical for the cell-cell interactions in the BBB that sequester cerebrospinal fluid from circulating blood components [6]. However, despite the BBB, there exist a certain number of immune cells in the CNS to prevent brain tumors and viral infections. In pathological conditions of the CNS, excessive immune cells from the blood accumulate from the breached BBB to cause chronic inflammatory diseases including multiple sclerosis (MS). It is also known that brain micro-inflammation is associated with neurodegenerative disorders such as Parkinson's disease and Alzheimer's disease [7]. Inflammatory cytokines including IL-1β, IL-17A, IFNγ and TNFα increase the BBB permeability, while chemokines recruit immune cells from the blood to cause inflammation [8–10]. However, where and how local CNS inflammation is induced in the BBB is poorly understood.

MS is a chronic inflammatory disease of the CNS. It is characterized by impairments in sensory, motor, autonomic, and neurocognitive functions due to autoimmune-mediated demyelination in CNS nerves [11]. Genetic factors strongly contribute to the MS pathogenesis, and genome-wide association studies (GWAS) have revealed that certain alleles of major histocompatibility complex (MHC) class II genes including HLA-DRB1\*15:01 and HLA-DRB1\*13:03 and genes involved in CD4+ T cell activation and homeostasis such as IL-2Rα and IL-7R are genetically associated with MS development [12–15]. These genetic results strongly suggest that autoreactive CD4+ T cells play a vital role in the MS pathogenesis and are supported by animal models of MS including experimental autoimmune encephalomyelitis (EAE) [16–19]. Using an adaptive transfer model of EAE [20], we investigated how and where myelin-autoreactive pathogenic CD4+ T cells initially invade the CNS. Whole-mount sections of adult mice with pathogenic CD4+ T cell transfer sacrificed just before the onset of EAE symptoms revealed that pathogenic CD4+ T cells mainly accumulated at the L5 spinal cord, around the dorsal vessels in particular [18]. On the other hand, no accumulation was observed in the brain or upper spinal levels at the preclinical time point of EAE. Various chemokines including CCL20 attract type-17 CD4+ T (Th17) cells, which are known to have a key pathogenic role in EAE [21, 22], to accumulate more in the dorsal vessels of L5 than L1 cord. Indeed, CCL20 neutralization or deficiency of its receptor, CCR6, on pathogenic CD4+ T cells abrogated the cell accumulation in the L5 spinal cord [18].

muscle strength change, as observed in astronauts who stayed in the International Space Station [1, 2]. In addition to these physical stimulations, various events in daily social interactions associated with psychological alterations including anxiety, mental stresses or positive emotions are also stimulatory factors that trigger specific neural pathways to affect body functions. A well-studied mechanism to cope with these stimulations includes the release of glucocorticoids via the hypothalamus-pituitary-adrenal grand (HPA) axis, which systemically changes various physiological functions including the immune system [3, 4]. Besides this systemic regulation, we and other groups have identified local regulations of the inflammatory status by various environmental cues that cause specific neural activations. Here we

summarize examples of specific neuro-immune interactions in organ homeostasis.

The central nervous system (CNS) is considered an immune privileged site due to structural protections by the blood-brain barrier (BBB) [5]. The BBB is formed by tight cell-cell interactions between blood endothelial cells and tight liner sheets, a structure that lies outside the basement membrane and consists of pericytes, neurons, microglias, and astrocytes. Tight junctions by the interactions of tight-junction molecules including claudins and occludins are critical for the cell-cell interactions in the BBB that sequester cerebrospinal fluid from circulating blood components [6]. However, despite the BBB, there exist a certain number of immune cells in the CNS to prevent brain tumors and viral infections. In pathological conditions of the CNS, excessive immune cells from the blood accumulate from the breached BBB to cause chronic inflammatory diseases including multiple sclerosis (MS). It is also known that brain micro-inflammation is associated with neurodegenerative disorders such as Parkinson's disease and Alzheimer's disease [7]. Inflammatory cytokines including IL-1β, IL-17A, IFNγ and TNFα increase the BBB permeability, while chemokines recruit immune cells from the blood to cause inflammation [8–10]. However, where and how local CNS inflammation is induced

MS is a chronic inflammatory disease of the CNS. It is characterized by impairments in sensory, motor, autonomic, and neurocognitive functions due to autoimmune-mediated demyelination in CNS nerves [11]. Genetic factors strongly contribute to the MS pathogenesis, and genome-wide association studies (GWAS) have revealed that certain alleles of major histocompatibility complex (MHC) class II genes including HLA-DRB1\*15:01 and HLA-DRB1\*13:03 and genes involved in CD4+ T cell activation and homeostasis such as IL-2Rα and IL-7R are genetically associated with MS development [12–15]. These genetic results strongly suggest that autoreactive CD4+ T cells play a vital role in the MS pathogenesis and are supported by animal models of MS including experimental autoimmune encephalomyelitis (EAE) [16–19]. Using an adaptive transfer model of EAE [20], we investigated how and where myelin-autoreactive pathogenic CD4+ T cells initially invade the CNS. Whole-mount sections of adult mice with pathogenic CD4+ T cell transfer sacrificed just before the onset of

**2. Main text**

**2.1. Gravity gateway reflex**

52 Homeostasis - An Integrated Vision

in the BBB is poorly understood.

Interestingly, even without EAE induction, chemokine levels were higher in the L5 dorsal vessels than in the L1 cord. These results suggest a distinct property of L5 dorsal vessels both in the presence and absence of pathogenic CD4+ T cells. The L5 spinal level has the largest dorsal root ganglion (DRG) in both human and mice, and it is known that sensory neurons in the L5 DRG distribute to the soleus muscles, the main anti-gravity muscles of the body [23, 24]. These facts led us to hypothesize a link between gravity, L5 vessels and local inflammation. We examined this possibility using a ground experiment employed by the National Aeronautics and Space Administration (NASA) and Japan Aerospace Exploration Agency (JAXA) [25, 26]. The tail suspension method puts mice in a handstand position, such that the hind limbs are released from gravity stimulation. As hypothesized, after the tail suspension, chemokine expressions of the L5 dorsal vessels were reduced and pathogenic CD4+ T cells hardly invaded the L5 cord. Chemokines were instead upregulated at the cervical cords as though another gateway for immune cells was formed by the greater gravity stimulation on the arm muscles imposed by the tail suspension [18]. Consistently, the tail suspension significantly reduced the expression of a neural activation marker, c-Fos, in the L5 DRG, suggesting a correlation between regional neural activation and local inflammation in the CNS. Consistently, stimulation of the soleus muscles by weak electric pulses, which mimic gravity-mediated sensory activation, during tail suspension restored chemokine expression, pathogenic CD4+ T cell accumulation, and c-Fos levels at the L5 dorsal vessels [18]. That study identified L5 dorsal vessels as a blood vessel gateway for immune cells including pathogenic CD4+ T cells to the CNS, and revealed that regional sensory neural activation by gravity affects CNS homeostasis and causes local inflammation at L5 dorsal blood vessels when CNS-autoreactive pathogenic CD4+ T cells are present. This phenomenon, which represents a novel neuro-immune interaction, was termed the "gateway reflex" [27–34]. Since then, we have found various types of gateway reflexes depending on the neural stimulation. Thus, the abovementioned example is known as the gravity gateway reflex (**Figure 1**).

How the regional neural pathway by gravity stimulation regulates chemokine expressions in L5 blood vessels is an important question. Contribution of the autonomic nervous system was suggested. In fact, neural activation examined by c-Fos expression was higher in sympathetic ganglions at the L5 cord than at the L1 cord. In addition, the blood flow speed of the L5 dorsal vessels but not of other blood vessels including the L1 dorsal vessels, femoral artery or portal vein, slowed after tail suspension. Functionally, the treatment of mice with β-adrenergic receptor antagonists or chemical sympathectomy inhibited chemokine expressions, the accumulation of pathogenic CD4+ T cells at the L5 dorsal vessels, and the clinical scores of EAE [18]. Norepinephrine is one of the major neurotransmitters of sympathetic nerves. Indeed, in vitro experiments suggest that norepinephrine enhances chemokine expressions from

**Figure 1.** Gravity gateway reflex. Stimulation of the soleus muscles by gravity (1) induces the activation of specific sensory nerves (2). The cell bodies of these sensory neurons are located at the dorsal root ganglion (DRG) of the fifth lumbar (L5) spinal cord (3). Neural activation via the L5 DRG neurons travels to the L5 sympathetic ganglion (4) and induces the activation of sympathetic nerves (5), which results in norepinephrine (NE) secretion (6) at the L5 dorsal vessels. NE enhances the inflammation amplifier in the L5 dorsal vessels, causing an upregulation of chemokines and recruiting pathogenic CD4+ T cells from the vessels (7).

endothelial cell lines with activation of the inflammation amplifier [18]. The inflammation amplifier is a molecular mechanism that operates in nonimmune cells including endothelial cells to produce a large amount of pro-inflammatory mediators including chemokines, cytokines and growth factors upon the concomitant activation of two transcription factors, NF-κB and STAT3 [35–37]. The co-activation of NF-κB and STAT3 by cytokines such as IL-17, IL-6, and TNFα augments NF-κB activity and upregulates NF-κB-target genes including chemokines, which can be further enhanced by neurotransmitters including norepinephrine and ATP (**Figure 2**) [18, 38]. The contribution of nonimmune cells to chronic inflammation via the inflammation amplifier has been found significant in the development of various disease models and evidence of inflammation amplifier activation has been observed in patients with MS, rheumatoid arthritis, atherosclerosis, and chronic rejection after lung transplantation [39–45]. Thus, the gateway reflex represents novel local neuro-immune communication involving the activation of specific sensory and sympathetic neurons for the formation of blood vessel gateways (**Figure 1**). Because chemokine levels are constantly higher at the L5 dorsal vessels under normal conditions without EAE induction, the gateway reflex could have a physiological role as well. Indeed, it is reported in mice that after a learning task, CD4+ T cells, but not CD8+ T cells, accumulated in the CNS, and mice devoid of CD4+ T cells showed impaired learning performance and neurogenesis in the hippocampus [46–48]. The gravity gateway reflex may be utilized by these cells to enter the CNS to maintain and/or control the homeostasis and function of the CNS.

**2.2. Electric gateway reflex**

NE, norepinephrine; Th, helper T cells.

**2.3. Pain gateway reflex**

The gateway reflex is not specific to the soleus-L5 axis because electrical stimulation of regional sensory neurons in different muscles can induce the formation of gateways in the dorsal vessels of different spinal cord levels. For instance, electric stimulation of the quadriceps, which are controlled by the L3 DRG neurons, induces chemokine expressions at the L3 dorsal vessels. Similarly, electric stimulations of the triceps upregulate chemokines at the dorsal vessels of the cervical to thoracic spinal cords (**Figure 3**) [18]. These results suggest that the electric gateway reflex can be artificially controlled, raising a possibility for a therapeutic

**Figure 2.** Inflammation amplifier. Co-activation of the transcription factors NF-κB and STAT3 in nonimmune cells such as endothelial cells and fibroblasts induces a synergistic effect on the production of inflammation factors such as chemokines, growth factors, and IL-6. Various factors activating NF-κB and STAT3 including IL-17, TNFα, and IL-6 can drive the amplifier. Secreted IL-6 is thought to act on nonimmune cells to form a positive feedback loop for this synergistic effect. Massive production of chemokines and growth factors by the inflammation amplifier play an essential role in the pathogenesis of many inflammatory diseases. DC, dendritic cells; GF, growth factors; NK, natural killer cells;

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We have examined whether other sensory stimulations can also generate the gateway reflex. We focused on pain because it is a tonic sensory stimulation [49, 50] and associated with various diseases that significantly compromise quality of life [51]. Some studies suggest that

application of the gateway reflex to various CNS diseases like MS and brain tumors.

**Figure 2.** Inflammation amplifier. Co-activation of the transcription factors NF-κB and STAT3 in nonimmune cells such as endothelial cells and fibroblasts induces a synergistic effect on the production of inflammation factors such as chemokines, growth factors, and IL-6. Various factors activating NF-κB and STAT3 including IL-17, TNFα, and IL-6 can drive the amplifier. Secreted IL-6 is thought to act on nonimmune cells to form a positive feedback loop for this synergistic effect. Massive production of chemokines and growth factors by the inflammation amplifier play an essential role in the pathogenesis of many inflammatory diseases. DC, dendritic cells; GF, growth factors; NK, natural killer cells; NE, norepinephrine; Th, helper T cells.

#### **2.2. Electric gateway reflex**

endothelial cell lines with activation of the inflammation amplifier [18]. The inflammation amplifier is a molecular mechanism that operates in nonimmune cells including endothelial cells to produce a large amount of pro-inflammatory mediators including chemokines, cytokines and growth factors upon the concomitant activation of two transcription factors, NF-κB and STAT3 [35–37]. The co-activation of NF-κB and STAT3 by cytokines such as IL-17, IL-6, and TNFα augments NF-κB activity and upregulates NF-κB-target genes including chemokines, which can be further enhanced by neurotransmitters including norepinephrine and ATP (**Figure 2**) [18, 38]. The contribution of nonimmune cells to chronic inflammation via the inflammation amplifier has been found significant in the development of various disease models and evidence of inflammation amplifier activation has been observed in patients with MS, rheumatoid arthritis, atherosclerosis, and chronic rejection after lung transplantation [39–45]. Thus, the gateway reflex represents novel local neuro-immune communication involving the activation of specific sensory and sympathetic neurons for the formation of blood vessel gateways (**Figure 1**). Because chemokine levels are constantly higher at the L5 dorsal vessels under normal conditions without EAE induction, the gateway reflex could have a physiological role as well. Indeed, it is reported in mice that after a learning task, CD4+ T cells, but not CD8+ T cells, accumulated in the CNS, and mice devoid of CD4+ T cells showed impaired learning performance and neurogenesis in the hippocampus [46–48]. The gravity gateway reflex may be utilized by these cells to enter the CNS to maintain and/or

**Figure 1.** Gravity gateway reflex. Stimulation of the soleus muscles by gravity (1) induces the activation of specific sensory nerves (2). The cell bodies of these sensory neurons are located at the dorsal root ganglion (DRG) of the fifth lumbar (L5) spinal cord (3). Neural activation via the L5 DRG neurons travels to the L5 sympathetic ganglion (4) and induces the activation of sympathetic nerves (5), which results in norepinephrine (NE) secretion (6) at the L5 dorsal vessels. NE enhances the inflammation amplifier in the L5 dorsal vessels, causing an upregulation of chemokines and

control the homeostasis and function of the CNS.

recruiting pathogenic CD4+ T cells from the vessels (7).

54 Homeostasis - An Integrated Vision

The gateway reflex is not specific to the soleus-L5 axis because electrical stimulation of regional sensory neurons in different muscles can induce the formation of gateways in the dorsal vessels of different spinal cord levels. For instance, electric stimulation of the quadriceps, which are controlled by the L3 DRG neurons, induces chemokine expressions at the L3 dorsal vessels. Similarly, electric stimulations of the triceps upregulate chemokines at the dorsal vessels of the cervical to thoracic spinal cords (**Figure 3**) [18]. These results suggest that the electric gateway reflex can be artificially controlled, raising a possibility for a therapeutic application of the gateway reflex to various CNS diseases like MS and brain tumors.

#### **2.3. Pain gateway reflex**

We have examined whether other sensory stimulations can also generate the gateway reflex. We focused on pain because it is a tonic sensory stimulation [49, 50] and associated with various diseases that significantly compromise quality of life [51]. Some studies suggest that

monocytes that expressed high levels of MHC class II [18]. These MHC class II high monocytes distributed around the meningeal region of the L5 cord in EAE-recovered mice. After pain induction, however, these cells accumulated at the ventral vessels of the L5 cord bilaterally. Experimental evidence that (1) norepinephrine signaling could be detected around the L5 ventral vessels, (2) MHC class II high monocytes expressed chemokine receptor CX3CR1, and (3) MHC class II high monocytes secreted chemokine CX3CL1, a ligand for CX3CR1, after norepinephrine stimulation suggested an auto/paracrine loop is responsible for the accumulation of MHC class II high monocytes around the L5 ventral vessels via norepinephrine regulation of the CX3CL1-CX3CR1 axis. Because freshly isolated MHC class II high monocytes from EAE-recovered mice have autoantigen (MOG)-presenting capacity without additional peptide loading, it can be suggested that MHC class II high monocytes accumulated around the L5 ventral vessels would activate pathogenic CD4+ T cells through autoantigen presentation, followed by regional inflammation and disease relapse. Suppression of norepinephrine signaling by a β1 blocker or sympathetic nerve ablation by chemical sympathectomy inhibited the pain-mediated accumulation of MHC class II high monocytes around the L5 ventral vessels [19]. These results identified the L5 ventral vessels as the gateway for pain-induced relapse in the EAE model (**Figure 4**). This pain gateway reflex is the third example of the gate-

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Chronic stresses deteriorate illness, an effect attributed to the proverbs "Illness starts in the mind" and "Care killed the cat." Chronic stress conditions often cause gastric and intestinal diseases via the brain-gut axis. Although these diseases are well known and often experienced, the underlying molecular mechanisms remain to be elucidated. Stresses generate neural activations involving multiple brain areas such as the paraventricular nucleus (PVN), dorsomedial nucleus of hypothalamus (DMH), dorsal motor nucleus of the vagal nerve (DMX), and vagal nerve pathway [58]. We therefore hypothesized that chronic stresses might induce a specific gateway reflex. Indeed, experiments confirmed the stress gateway reflex. We serendipitously found that micro-inflammation induced by the stress gateway reflex activates an otherwise resting neural circuit to enhance a stress response that causes fatal gastrointestinal and heart failure in mice [38]. Therefore, the stress gateway reflex may explain the

The gravity gateway reflex directs donor pathogenic CD4+ T cells to the dorsal vessels of the L5 spinal cord under normal conditions (**Figure 1**). However, under chronic stress conditions, the cells instead invade specific blood vessels of the boundary area of the third ventricle (3V), thalamus, and part of hippocampus [dentate gyrus, (DG)] to establish micro-inflammation. Therefore, chronic stresses can alter the location of the immune cell gateway from the L5 dorsal vessels to specific vessels in the brain. Immunohistochemistry of c-Fos revealed that the resulting micro-inflammation led to enhanced neural activations in the PVN and DMH, which are stress sensing areas of the hypothalamus. Neural tracing experiments identified direct connections via noradrenergic neurons between the PVN and the specific vessels of the boundary area of 3V, thalamus and DG. Moreover, these experiments showed direct

way reflex following the aforementioned gravity and electric examples.

mechanisms for the abovementioned proverbs, as described below.

**2.4. Stress gateway reflex**

**Figure 3.** Electric gateway reflex. Electric stimulation to the triceps induces chemokine upregulation at the dorsal vessels of the fifth cervical (C5) to fifth thoracic (T5) spinal cord through sensory-sympathetic neural activation, which leads to activation of the inflammation amplifier at the C5-T5 dorsal vessels. In a similar fashion, electric stimulation of the quadriceps and soleus muscles induces a gateway at the L3 and L5 dorsal vessels, respectively.

pain occurrence is positively correlated with disease severity in MS [52–54], and mechanical allodynia and thermal hyperalgesia are induced in mice during EAE [55, 56]. In the adoptive transfer EAE model we used, recipient mice that received myelin-specific pathogenic CD4+ T cells developed transient paralysis, but then recovered and hardly relapsed even 100 days later. To examine how pain affects EAE symptoms, we induced pain sensation in mice by partial ligation of the middle branch of the trigeminal nerves, which are composed of sensory nerves alone [57]. Pain induction with pathogenic CD4+ T cell transfer significantly worsened the paralysis. By contrast, pain medicines improved EAE development [19]. Thus, pain is not simply an alert of the disease or injury status, but it also has a pathogenic role, triggering EAE relapse. Since a large part of MS patients show relapse and remission and pain is more frequently claimed by MS patients with higher disease scores [52, 53], we examined the effects of pain induction in mice that had recovered from EAE (EAE-recovered mice). As expected, EAE-recovered mice showed a clear sign of relapse after the partial ligation of the trigeminal nerve or injection of pain-inducing chemicals such as substance P and capsaicin [19]. As described above, under normal conditions, the blood vessel gateway for immune cells is the L5 dorsal vessels due to the effect of gravity [18]. To identify specific blood vessels that act as a gateway for immune cells that cause the pain-induced relapse, immunohistological examination of the CNS from EAE-recovered mice was performed. Although the motility of EAE-recovered mice was not significantly different from normal healthy mice, the meningeal region of the L5 cord of EAE-recovered mice contained a high number of periphery-derived monocytes that expressed high levels of MHC class II [18]. These MHC class II high monocytes distributed around the meningeal region of the L5 cord in EAE-recovered mice. After pain induction, however, these cells accumulated at the ventral vessels of the L5 cord bilaterally. Experimental evidence that (1) norepinephrine signaling could be detected around the L5 ventral vessels, (2) MHC class II high monocytes expressed chemokine receptor CX3CR1, and (3) MHC class II high monocytes secreted chemokine CX3CL1, a ligand for CX3CR1, after norepinephrine stimulation suggested an auto/paracrine loop is responsible for the accumulation of MHC class II high monocytes around the L5 ventral vessels via norepinephrine regulation of the CX3CL1-CX3CR1 axis. Because freshly isolated MHC class II high monocytes from EAE-recovered mice have autoantigen (MOG)-presenting capacity without additional peptide loading, it can be suggested that MHC class II high monocytes accumulated around the L5 ventral vessels would activate pathogenic CD4+ T cells through autoantigen presentation, followed by regional inflammation and disease relapse. Suppression of norepinephrine signaling by a β1 blocker or sympathetic nerve ablation by chemical sympathectomy inhibited the pain-mediated accumulation of MHC class II high monocytes around the L5 ventral vessels [19]. These results identified the L5 ventral vessels as the gateway for pain-induced relapse in the EAE model (**Figure 4**). This pain gateway reflex is the third example of the gateway reflex following the aforementioned gravity and electric examples.

#### **2.4. Stress gateway reflex**

pain occurrence is positively correlated with disease severity in MS [52–54], and mechanical allodynia and thermal hyperalgesia are induced in mice during EAE [55, 56]. In the adoptive transfer EAE model we used, recipient mice that received myelin-specific pathogenic CD4+ T cells developed transient paralysis, but then recovered and hardly relapsed even 100 days later. To examine how pain affects EAE symptoms, we induced pain sensation in mice by partial ligation of the middle branch of the trigeminal nerves, which are composed of sensory nerves alone [57]. Pain induction with pathogenic CD4+ T cell transfer significantly worsened the paralysis. By contrast, pain medicines improved EAE development [19]. Thus, pain is not simply an alert of the disease or injury status, but it also has a pathogenic role, triggering EAE relapse. Since a large part of MS patients show relapse and remission and pain is more frequently claimed by MS patients with higher disease scores [52, 53], we examined the effects of pain induction in mice that had recovered from EAE (EAE-recovered mice). As expected, EAE-recovered mice showed a clear sign of relapse after the partial ligation of the trigeminal nerve or injection of pain-inducing chemicals such as substance P and capsaicin [19]. As described above, under normal conditions, the blood vessel gateway for immune cells is the L5 dorsal vessels due to the effect of gravity [18]. To identify specific blood vessels that act as a gateway for immune cells that cause the pain-induced relapse, immunohistological examination of the CNS from EAE-recovered mice was performed. Although the motility of EAE-recovered mice was not significantly different from normal healthy mice, the meningeal region of the L5 cord of EAE-recovered mice contained a high number of periphery-derived

quadriceps and soleus muscles induces a gateway at the L3 and L5 dorsal vessels, respectively.

56 Homeostasis - An Integrated Vision

**Figure 3.** Electric gateway reflex. Electric stimulation to the triceps induces chemokine upregulation at the dorsal vessels of the fifth cervical (C5) to fifth thoracic (T5) spinal cord through sensory-sympathetic neural activation, which leads to activation of the inflammation amplifier at the C5-T5 dorsal vessels. In a similar fashion, electric stimulation of the

Chronic stresses deteriorate illness, an effect attributed to the proverbs "Illness starts in the mind" and "Care killed the cat." Chronic stress conditions often cause gastric and intestinal diseases via the brain-gut axis. Although these diseases are well known and often experienced, the underlying molecular mechanisms remain to be elucidated. Stresses generate neural activations involving multiple brain areas such as the paraventricular nucleus (PVN), dorsomedial nucleus of hypothalamus (DMH), dorsal motor nucleus of the vagal nerve (DMX), and vagal nerve pathway [58]. We therefore hypothesized that chronic stresses might induce a specific gateway reflex. Indeed, experiments confirmed the stress gateway reflex. We serendipitously found that micro-inflammation induced by the stress gateway reflex activates an otherwise resting neural circuit to enhance a stress response that causes fatal gastrointestinal and heart failure in mice [38]. Therefore, the stress gateway reflex may explain the mechanisms for the abovementioned proverbs, as described below.

The gravity gateway reflex directs donor pathogenic CD4+ T cells to the dorsal vessels of the L5 spinal cord under normal conditions (**Figure 1**). However, under chronic stress conditions, the cells instead invade specific blood vessels of the boundary area of the third ventricle (3V), thalamus, and part of hippocampus [dentate gyrus, (DG)] to establish micro-inflammation. Therefore, chronic stresses can alter the location of the immune cell gateway from the L5 dorsal vessels to specific vessels in the brain. Immunohistochemistry of c-Fos revealed that the resulting micro-inflammation led to enhanced neural activations in the PVN and DMH, which are stress sensing areas of the hypothalamus. Neural tracing experiments identified direct connections via noradrenergic neurons between the PVN and the specific vessels of the boundary area of 3V, thalamus and DG. Moreover, these experiments showed direct

**Figure 4.** Pain gateway reflex. Pain induces nociceptive sensory nerve stimulation (1, 2), leading to activation of the anterior cingulate cortex (ACC), a pain-processing area of the brain (3). Specific sympathetic nerves are then activated (4) to induce norepinephrine (NE) release around the ventral vessels of the spinal cords (5). NE around the L5 ventral vessels induces the production of chemokine CX3CL1 from MHC class II high (MHC class IIHI) monocytes, further recruiting these cells in an auto/paracrine manner (6). MHC class II high monocytes are able to present myelin autoantigens to activate pathogenic CD4+ T cells, leading to disease relapse (7).

connections via nonnoradrenergic neurons between the specific vessels and the DMH. Since the PVN is a key orchestrator of stress signals, its activation is expected to affect specific blood vessels via a new noradrenergic neural pathway. Indeed, the expression of chemokines such as CCL5 was upregulated at specific vessels in mice with chronic stress alone. CNS-reactive pathogenic CD4+ T cells in the blood circulation of the stressed mice sensed the upregulation of CCL5, caused micro-inflammation with peripheral derived MHC class II monocytes at specific vessels of the boundary area of the 3V, thalamus, and DG [38]. ATP is released during an inflammatory response, but it also acts as a neurotransmitter [59, 60]. The injection of an ATP receptor antagonist at specific blood vessels of the boundary area of the 3V, thalamus and DG suppressed both neural activation in the DMH and the mortality rate of EAE mice with chronic stress. Importantly, the direct injection of ATP or cytokines at these specific vessels, which mimicked the micro-inflammation, induced fatal gastrointestinal dysfunction in stressed mice without EAE induction. These results suggest that brain micro-inflammation at specific vessels of the boundary area of the 3V, thalamus, and DG activates a resting neural pathway through ATP production, thus strongly enhancing the stress response to cause fatal gastrointestinal damage via the DMX and vagal nerve activation. These results uncovered the stress gateway reflex acting as a direct link between micro-inflammation at a particular site in the brain and gastrointestinal homeostasis (**Figure 5**) [38].

Several reports have shown the concurrence of inflammatory bowel diseases and MS [61–65]. Moreover, brain micro-inflammations were observed in patients with neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease [66, 67], epilepsy [68], and psychological disorders [69]. We therefore suggest that brain micro-inflammations could regulate the homeostasis of organ functions including the brain itself by acting as a switch to stimulate resting neural pathways, which could account for the comorbidities observed in many

) in blood circulation by the upper GI tract failure (6) explains at least in part heart

Gateway Reflex: A Neuro-Immune Crosstalk for Organ-Specific Disease Development

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59

**Figure 5.** Stress gateway reflex. Chronic stress induces activation of the paraventricular nucleus (PVN) (1). We identified neural connections from the PVN to the specific vessels of the boundary area of the third ventricle, thalamus and dentate gyrus, which induces micro-inflammation around the specific vessels (2). The resulting micro-inflammation induces activation of a neural pathway that connects to the dorsomedial nucleus of hypothalamus (DMH) (3) and dorsal motor nucleus of the vagal nerve (DMX) (4), resulting in severe upper gastrointestinal (GI) tract failure via efferent vagal nerves

The Tracey laboratory has demonstrated using a mouse model of sepsis that the activation of vagal nerves, which mainly consist of parasympathetic nerves, suppresses an inflammatory response [70]. They revealed that lipopolysaccharide induces norepinephrine release in the spleen via vagal and splenic nerves. The norepinephrine stimulates a novel subset of CD4+ T cells that express β1/2 adrenergic receptor and produce acetylcholine. The resulting acetylcholine then acts on activated macrophages expressing α7 nicotinic receptor to suppress the

diseases.

**2.5. Cholinergic anti-inflammatory pathway**

(5). The increase of potassium ions (K<sup>+</sup>

failure associated with cardiac myocyte necrosis (7).

Gateway Reflex: A Neuro-Immune Crosstalk for Organ-Specific Disease Development http://dx.doi.org/10.5772/intechopen.77112 59

**Figure 5.** Stress gateway reflex. Chronic stress induces activation of the paraventricular nucleus (PVN) (1). We identified neural connections from the PVN to the specific vessels of the boundary area of the third ventricle, thalamus and dentate gyrus, which induces micro-inflammation around the specific vessels (2). The resulting micro-inflammation induces activation of a neural pathway that connects to the dorsomedial nucleus of hypothalamus (DMH) (3) and dorsal motor nucleus of the vagal nerve (DMX) (4), resulting in severe upper gastrointestinal (GI) tract failure via efferent vagal nerves (5). The increase of potassium ions (K<sup>+</sup> ) in blood circulation by the upper GI tract failure (6) explains at least in part heart failure associated with cardiac myocyte necrosis (7).

Several reports have shown the concurrence of inflammatory bowel diseases and MS [61–65]. Moreover, brain micro-inflammations were observed in patients with neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease [66, 67], epilepsy [68], and psychological disorders [69]. We therefore suggest that brain micro-inflammations could regulate the homeostasis of organ functions including the brain itself by acting as a switch to stimulate resting neural pathways, which could account for the comorbidities observed in many diseases.

#### **2.5. Cholinergic anti-inflammatory pathway**

connections via nonnoradrenergic neurons between the specific vessels and the DMH. Since the PVN is a key orchestrator of stress signals, its activation is expected to affect specific blood vessels via a new noradrenergic neural pathway. Indeed, the expression of chemokines such as CCL5 was upregulated at specific vessels in mice with chronic stress alone. CNS-reactive pathogenic CD4+ T cells in the blood circulation of the stressed mice sensed the upregulation of CCL5, caused micro-inflammation with peripheral derived MHC class II monocytes at specific vessels of the boundary area of the 3V, thalamus, and DG [38]. ATP is released during an inflammatory response, but it also acts as a neurotransmitter [59, 60]. The injection of an ATP receptor antagonist at specific blood vessels of the boundary area of the 3V, thalamus and DG suppressed both neural activation in the DMH and the mortality rate of EAE mice with chronic stress. Importantly, the direct injection of ATP or cytokines at these specific vessels, which mimicked the micro-inflammation, induced fatal gastrointestinal dysfunction in stressed mice without EAE induction. These results suggest that brain micro-inflammation at specific vessels of the boundary area of the 3V, thalamus, and DG activates a resting neural pathway through ATP production, thus strongly enhancing the stress response to cause fatal gastrointestinal damage via the DMX and vagal nerve activation. These results uncovered the stress gateway reflex acting as a direct link between micro-inflammation at a particular site in

**Figure 4.** Pain gateway reflex. Pain induces nociceptive sensory nerve stimulation (1, 2), leading to activation of the anterior cingulate cortex (ACC), a pain-processing area of the brain (3). Specific sympathetic nerves are then activated (4) to induce norepinephrine (NE) release around the ventral vessels of the spinal cords (5). NE around the L5 ventral vessels induces the production of chemokine CX3CL1 from MHC class II high (MHC class IIHI) monocytes, further recruiting these cells in an auto/paracrine manner (6). MHC class II high monocytes are able to present myelin autoantigens to

the brain and gastrointestinal homeostasis (**Figure 5**) [38].

activate pathogenic CD4+ T cells, leading to disease relapse (7).

58 Homeostasis - An Integrated Vision

The Tracey laboratory has demonstrated using a mouse model of sepsis that the activation of vagal nerves, which mainly consist of parasympathetic nerves, suppresses an inflammatory response [70]. They revealed that lipopolysaccharide induces norepinephrine release in the spleen via vagal and splenic nerves. The norepinephrine stimulates a novel subset of CD4+ T cells that express β1/2 adrenergic receptor and produce acetylcholine. The resulting acetylcholine then acts on activated macrophages expressing α7 nicotinic receptor to suppress the

**2.6. Future directions**

**Acknowledgements**

**Conflict of interest**

**Author details**

Accumulating evidence has demonstrated the significant effects of regional neuro-immune interactions on organ homeostasis, particularly during inflammation and diseases. The gateway reflex and inflammatory reflex can be induced by various stimulations to activate these interactions. Stimulation of the vagal nerves by an implantable device has already shown an anti-inflammatory effect in humans [84, 85]. The stimulation of the neurons responsible for the gateway reflex and inflammatory reflex at the body surface, as is the case with acupuncture, could lead to therapies that are less invasive and less costly, although elucidation of the precise neural circuits activated by the reflexes is required. Neural mapping is still challenging, but recent techniques and tools including the tissue clearing method CUBIC [86–89], optogenetics and chemogenetics [90, 91], and transgenic mice reporting neural circuitry and activations [92–94] will greatly contribute. Because neural circuits run throughout the body and because nonimmune cells as well as immune cells [18, 19, 71, 95–97] are able to secrete and respond to neurotransmitters, specific regional neuro-immune interactions such as the gateway reflex and inflammatory reflex bear

Gateway Reflex: A Neuro-Immune Crosstalk for Organ-Specific Disease Development

http://dx.doi.org/10.5772/intechopen.77112

61

We appreciate the outstanding experimental contributions provided by Dr. Y. Arima and the excellent technical assistances by Ms. Ezawa and Ms. Nakayama for our work described here. We also thank Ms. Fukumoto for her excellent secretarial assistance, and Dr. P. Karagiannis (CiRA, Kyoto University, Kyoto, Japan) for carefully reading the manuscript. Our work described here was supported by KAKENHI (D. K. and M. M.), Takeda Science Foundation (M. M.), Institute for Fermentation Osaka (M. M.), Mitsubishi Foundation (M. M.), Mochida Memorial Foundation for Medical and Pharmaceutical Research (D. K.), Suzuken Memorial

potential as a significant therapeutic strategy to recover organ homeostasis.

Foundation (D. K.), and Tokyo Medical Research Foundation (M. M.).

Daisuke Kamimura, Yuki Tanaka, Takuto Ohki and Masaaki Murakami\*

Molecular Psychoimmunology, Institute for Genetic Medicine, Graduate School of Medicine,

\*Address all correspondence to: murakami@igm.hokudai.ac.jp

The authors have no conflict of interest for the article.

Hokkaido University, Sapporo, Hokkaido, Japan

**Figure 6.** Inflammatory reflex. Afferent (1) and efferent vagal nerve (2) activation by infection, and so on induces neural activation in the celiac ganglion (3), followed by the production of norepinephrine (NE) by the splenic nerves (4). NE stimulates the release of acetylcholine (ACh) from a subset of CD4+ T cells expressing β1/2 adrenaline receptor (AR) (5). Then, ACh (6) acts on macrophages expressing α7 nicotinic acetylcholine receptor (α7nAChR) to suppress the expression of pro-inflammatory factors such as HMGB1 and TNFα. It is suggested that C1 neurons in the brain mediate the cholinergic anti-inflammatory effect.

expression of pro-inflammatory factors such as TNFα and HMGB1 (**Figure 6**) [71–73]. The stimulation of macrophages with nicotine inhibits NF-κB activation, but not MAP kinase activation in response to endotoxin [72]. This cholinergic anti-inflammatory pathway is called the "inflammatory reflex" [27–29, 31, 32, 34, 74–76]. In addition to infection, this pathway exerts anti-inflammatory effects in various disease models including renal ischemia–reperfusion injury, acute kidney injury, pressure overload-induced cardiac hypertrophy and neointimal hyperplasia [77–81]. The direct stimulation of C1 neurons in the medullary reticular formation induced this anti-inflammatory effect in mice [79], suggesting the involvement of C1 neurons between afferent and efferent vagal nerves. Activation of the cholinergic anti-inflammatory pathway can be induced by acupuncture and ultrasound, thus inhibiting the pathology of animal models [82, 83]. These findings establish a scientific basis for acupuncture and physical therapy. In addition, activation of the inflammatory reflex through vagal nerve stimulation by an implantable device has been tested in humans for chronic inflammatory diseases including rheumatoid arthritis with promising results [84, 85].

#### **2.6. Future directions**

Accumulating evidence has demonstrated the significant effects of regional neuro-immune interactions on organ homeostasis, particularly during inflammation and diseases. The gateway reflex and inflammatory reflex can be induced by various stimulations to activate these interactions. Stimulation of the vagal nerves by an implantable device has already shown an anti-inflammatory effect in humans [84, 85]. The stimulation of the neurons responsible for the gateway reflex and inflammatory reflex at the body surface, as is the case with acupuncture, could lead to therapies that are less invasive and less costly, although elucidation of the precise neural circuits activated by the reflexes is required. Neural mapping is still challenging, but recent techniques and tools including the tissue clearing method CUBIC [86–89], optogenetics and chemogenetics [90, 91], and transgenic mice reporting neural circuitry and activations [92–94] will greatly contribute. Because neural circuits run throughout the body and because nonimmune cells as well as immune cells [18, 19, 71, 95–97] are able to secrete and respond to neurotransmitters, specific regional neuro-immune interactions such as the gateway reflex and inflammatory reflex bear potential as a significant therapeutic strategy to recover organ homeostasis.

#### **Acknowledgements**

We appreciate the outstanding experimental contributions provided by Dr. Y. Arima and the excellent technical assistances by Ms. Ezawa and Ms. Nakayama for our work described here. We also thank Ms. Fukumoto for her excellent secretarial assistance, and Dr. P. Karagiannis (CiRA, Kyoto University, Kyoto, Japan) for carefully reading the manuscript. Our work described here was supported by KAKENHI (D. K. and M. M.), Takeda Science Foundation (M. M.), Institute for Fermentation Osaka (M. M.), Mitsubishi Foundation (M. M.), Mochida Memorial Foundation for Medical and Pharmaceutical Research (D. K.), Suzuken Memorial Foundation (D. K.), and Tokyo Medical Research Foundation (M. M.).

#### **Conflict of interest**

expression of pro-inflammatory factors such as TNFα and HMGB1 (**Figure 6**) [71–73]. The stimulation of macrophages with nicotine inhibits NF-κB activation, but not MAP kinase activation in response to endotoxin [72]. This cholinergic anti-inflammatory pathway is called the "inflammatory reflex" [27–29, 31, 32, 34, 74–76]. In addition to infection, this pathway exerts anti-inflammatory effects in various disease models including renal ischemia–reperfusion injury, acute kidney injury, pressure overload-induced cardiac hypertrophy and neointimal hyperplasia [77–81]. The direct stimulation of C1 neurons in the medullary reticular formation induced this anti-inflammatory effect in mice [79], suggesting the involvement of C1 neurons between afferent and efferent vagal nerves. Activation of the cholinergic anti-inflammatory pathway can be induced by acupuncture and ultrasound, thus inhibiting the pathology of animal models [82, 83]. These findings establish a scientific basis for acupuncture and physical therapy. In addition, activation of the inflammatory reflex through vagal nerve stimulation by an implantable device has been tested in humans for chronic inflammatory diseases including

**Figure 6.** Inflammatory reflex. Afferent (1) and efferent vagal nerve (2) activation by infection, and so on induces neural activation in the celiac ganglion (3), followed by the production of norepinephrine (NE) by the splenic nerves (4). NE stimulates the release of acetylcholine (ACh) from a subset of CD4+ T cells expressing β1/2 adrenaline receptor (AR) (5). Then, ACh (6) acts on macrophages expressing α7 nicotinic acetylcholine receptor (α7nAChR) to suppress the expression of pro-inflammatory factors such as HMGB1 and TNFα. It is suggested that C1 neurons in the brain mediate

rheumatoid arthritis with promising results [84, 85].

the cholinergic anti-inflammatory effect.

60 Homeostasis - An Integrated Vision

The authors have no conflict of interest for the article.

#### **Author details**

Daisuke Kamimura, Yuki Tanaka, Takuto Ohki and Masaaki Murakami\*

\*Address all correspondence to: murakami@igm.hokudai.ac.jp

Molecular Psychoimmunology, Institute for Genetic Medicine, Graduate School of Medicine, Hokkaido University, Sapporo, Hokkaido, Japan

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68 Homeostasis - An Integrated Vision

0109-1


**Chapter 5**

**Provisional chapter**

**Platelets: From Formation to Function**

**Platelets: From Formation to Function**

DOI: 10.5772/intechopen.80924

Platelets are small, anucleate cells that travel as resting discoid fragments in the circulation. Their average circulating life span is 8–9 days, and their formation is an elegant and finely orchestrated series of cellular processes known as *megakaryocytopoiesis* and t*hrombopoiesis*. This involves the commitment of haematopoietic stem cells, proliferation, terminal differentiation of megakaryocytic progenitors and maturation of megakaryocytes to produce functional platelets. This complex process occurs in specialised endosteal and vascular niches in the bone marrow where megakaryocytes form proplatelet projections, releasing platelets into the circulation. Upon contact with an injured blood vessel, they prevent blood loss through processes of adhesion, activation and aggregation. Platelets play a central role in cardiovascular disease (CVD), both in the development of atherosclerosis and as the cellular mediator in the development of thrombosis. Platelets have diverse roles not limited to thrombosis/haemostasis, also being involved in many vascular inflammatory conditions. Depending on the physiological context, platelet functions may be protective or contribute to adverse thrombotic and inflammatory outcomes. In this chapter, we will discuss platelets in context of their formation and function. Because of their multifaceted role in maintaining physiological homeostasis, current and development of platelet function testing platforms will be

**Keywords:** platelets, megakaryocytes, megakaryocytopoiesis and thrombopoiesis, thrombosis and haemostasis, cardiovascular disease, platelet function testing

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

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

Sinead Sheridan, Michael Harrison, Niall Moyna, Gerardene Meade-Murphy, Nastassia Navasiolava,

Sinead Sheridan, Michael Harrison, Niall Moyna, Gerardene Meade-Murphy, Nastassia Navasiolava,

Marc-Antoine Custaud and Ronan P. Murphy

Marc-Antoine Custaud and Ronan P. Murphy

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

Laura Twomey, Robert G. Wallace, Philip M. Cummins, Bernard Degryse,

Laura Twomey, Robert G. Wallace, Philip M. Cummins, Bernard Degryse,

http://dx.doi.org/10.5772/intechopen.80924

**Abstract**

discussed.

#### **Platelets: From Formation to Function Platelets: From Formation to Function**

DOI: 10.5772/intechopen.80924

Laura Twomey, Robert G. Wallace, Philip M. Cummins, Bernard Degryse, Sinead Sheridan, Michael Harrison, Niall Moyna, Gerardene Meade-Murphy, Nastassia Navasiolava, Marc-Antoine Custaud and Ronan P. Murphy Laura Twomey, Robert G. Wallace, Philip M. Cummins, Bernard Degryse, Sinead Sheridan, Michael Harrison, Niall Moyna, Gerardene Meade-Murphy, Nastassia Navasiolava, Marc-Antoine Custaud and Ronan P. Murphy

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

http://dx.doi.org/10.5772/intechopen.80924

#### **Abstract**

Platelets are small, anucleate cells that travel as resting discoid fragments in the circulation. Their average circulating life span is 8–9 days, and their formation is an elegant and finely orchestrated series of cellular processes known as *megakaryocytopoiesis* and t*hrombopoiesis*. This involves the commitment of haematopoietic stem cells, proliferation, terminal differentiation of megakaryocytic progenitors and maturation of megakaryocytes to produce functional platelets. This complex process occurs in specialised endosteal and vascular niches in the bone marrow where megakaryocytes form proplatelet projections, releasing platelets into the circulation. Upon contact with an injured blood vessel, they prevent blood loss through processes of adhesion, activation and aggregation. Platelets play a central role in cardiovascular disease (CVD), both in the development of atherosclerosis and as the cellular mediator in the development of thrombosis. Platelets have diverse roles not limited to thrombosis/haemostasis, also being involved in many vascular inflammatory conditions. Depending on the physiological context, platelet functions may be protective or contribute to adverse thrombotic and inflammatory outcomes. In this chapter, we will discuss platelets in context of their formation and function. Because of their multifaceted role in maintaining physiological homeostasis, current and development of platelet function testing platforms will be discussed.

**Keywords:** platelets, megakaryocytes, megakaryocytopoiesis and thrombopoiesis, thrombosis and haemostasis, cardiovascular disease, platelet function testing

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

#### **1. Introduction**

Efforts in coping with the socio-economic and health burden of CVD require further understanding of its aetiology and the risk factors behind it in order to develop cost-effective preventive strategies (primordial, primary, secondary and tertiary) to prevent and manage it. CVD risk factors can be classed as modifiable or non-modifiable. Modifiable risk factors included smoking, diabetes, unhealthy diet, cholesterol, physical inactivity (sedentary lifestyle and low cardiorespiratory fitness), overweight and obesity. Risk factors for CVD track from childhood into adulthood and are strong predictors of subclinical disease in early adulthood [1]. Up to 80% of CVD may be prevented if modifiable risk factors are evaded [2].

cell lines. In addition, the principle hypothesis and paradigm discussed will be that the modifiable lifestyle factors of physical activity and inactivity can impact on the processes of both *megakaryocytopoiesis and thrombopoiesis*, *via* epigenetic mechanisms. We propose that physical activity/ inactivity can modulate and program platelet phenotype and therefore function. Through recent studies, including our own (manuscripts in preparation), it is becoming increasingly evident that lifestyle factors such as physical (in)-activity and high BMI do impact on platelet function. Thus, various research studies have collectively demonstrated that platelets are indeed reflective of physiological and lifestyle changes, making them sensitive biomarkers of human health. Platelets represent a tangible link to physiological and pathological changes within the body. Future investigations will undoubtedly contribute to a greater mechanistic understanding of the

Gulio Bizzozero first described platelets as 'spherules piastrine' (little plates) as small cell fragments that clumped together at an injured blood vessel site. He also showed that these blood elements did not have a nucleus [6]. Circulating anucleate platelets are now described as dynamic specialised cells, formed in an elaborate style from their precursor cell, the mega-

constituting the second most abundant cell type in blood after red blood cells. The average adult produces 1011 platelets per day to preserve this count. Platelets travel as resting (quiescent) discoid fragments in the circulation, while an elaborate internal cytoskeleton allows

The size of a mature platelet is approximately 2–4 μm, making them the smallest cells in circu-

facilitates in their role as 'guardians of the vasculature', as under laminar flow environments, platelets are pushed to the periphery by larger white and red blood cells. Consequently, they remain in close proximity to the blood vessel wall where they can quickly respond to any vascular damage [8]. This enables platelets to perform their main physiological function to

Platelets are formed and released into the bloodstream from megakaryocytes (MKs), which reside in the bone marrow [10, 11]. Their production is arguably the most elegant and distinct developmental process in eukaryotes [12]. While accounting for only 0.01% of nucleated bone marrow cells, MKs are also the largest cells, measuring between 50 and 100 μm [13]. Both MK and platelet production, termed megakaryocytopoiesis and thrombopoiesis, are regulated by multiple cytokines, with thrombopoietin (TPO), a hormone produced by the liver and kidneys, being the key regulator. In response to TPO, HSCs differentiate into MKs by differential expression of various transcription factors. This maturation is characterised by a growth in MK size and DNA ploidy levels (endomitosis), enabling the accumulation of RNA, protein and organelles in the MK for packaging into platelets. MKs then migrate to the sinusoidal

per microliter of blood,

Platelets: From Formation to Function http://dx.doi.org/10.5772/intechopen.80924 73

. Their small size

relationship between cardiovascular health, lifestyle factors and platelet biology.

karyocyte. Normal platelet counts ranges between 150 and 450 × 10<sup>3</sup>

shape changes to occur upon contact with an injured blood vessel.

lation, while their average thickness is 0.5 μm [7] and their volume about 7 μm3

prevent blood loss in primary haemostasis by the formation of a 'platelet plug' [9].

**2. Platelets**

**2.1. Platelet production**

The main functions of blood are to supply oxygen and nutrients to tissues and cells, removal of waste, and regulation of pH and body temperature [3]. An average adult has approximately five litres of blood, accounting for about 7% of their body weight. Blood is composed of approximately 55% plasma; a pale yellow fluid mainly consisting of water, proteins, sugars and fat particles, and 45% blood cells. Blood cells include erythrocytes, leukocytes and platelets. Each of these cells are derived from a haematopoietic stem cell (HSC), which reside in the bone marrow and sit at the peak of a developmental hierarchy, with unique ability to self-renew and give rise to cells of all of the blood lineages [4]. In adults, nearly a trillion new blood cells are produced daily to sustain steady state in circulation [5]. In the classic model of haematopoiesis (the production of blood cells), an important bifurcation occurs between the lymphoid and myeloid branches, which then further divide into a number of progenitor cells (**Figure 1**). While the main function of red blood cells (RBCs) and white blood cells (WBCs) is oxygen transport and defence, respectively, this chapter will focus on platelets, the final product of one of the myeloid

**Figure 1.** Pictorial representation of megakaryocytopoiesis and thrombopoiesis, and the potential for exposome and epigenome modulation of platelet phenotype and function during this dynamic cellular differentiation process.

cell lines. In addition, the principle hypothesis and paradigm discussed will be that the modifiable lifestyle factors of physical activity and inactivity can impact on the processes of both *megakaryocytopoiesis and thrombopoiesis*, *via* epigenetic mechanisms. We propose that physical activity/ inactivity can modulate and program platelet phenotype and therefore function. Through recent studies, including our own (manuscripts in preparation), it is becoming increasingly evident that lifestyle factors such as physical (in)-activity and high BMI do impact on platelet function. Thus, various research studies have collectively demonstrated that platelets are indeed reflective of physiological and lifestyle changes, making them sensitive biomarkers of human health. Platelets represent a tangible link to physiological and pathological changes within the body. Future investigations will undoubtedly contribute to a greater mechanistic understanding of the relationship between cardiovascular health, lifestyle factors and platelet biology.

#### **2. Platelets**

**1. Introduction**

72 Homeostasis - An Integrated Vision

Efforts in coping with the socio-economic and health burden of CVD require further understanding of its aetiology and the risk factors behind it in order to develop cost-effective preventive strategies (primordial, primary, secondary and tertiary) to prevent and manage it. CVD risk factors can be classed as modifiable or non-modifiable. Modifiable risk factors included smoking, diabetes, unhealthy diet, cholesterol, physical inactivity (sedentary lifestyle and low cardiorespiratory fitness), overweight and obesity. Risk factors for CVD track from childhood into adulthood and are strong predictors of subclinical disease in early adulthood [1]. Up to

The main functions of blood are to supply oxygen and nutrients to tissues and cells, removal of waste, and regulation of pH and body temperature [3]. An average adult has approximately five litres of blood, accounting for about 7% of their body weight. Blood is composed of approximately 55% plasma; a pale yellow fluid mainly consisting of water, proteins, sugars and fat particles, and 45% blood cells. Blood cells include erythrocytes, leukocytes and platelets. Each of these cells are derived from a haematopoietic stem cell (HSC), which reside in the bone marrow and sit at the peak of a developmental hierarchy, with unique ability to self-renew and give rise to cells of all of the blood lineages [4]. In adults, nearly a trillion new blood cells are produced daily to sustain steady state in circulation [5]. In the classic model of haematopoiesis (the production of blood cells), an important bifurcation occurs between the lymphoid and myeloid branches, which then further divide into a number of progenitor cells (**Figure 1**). While the main function of red blood cells (RBCs) and white blood cells (WBCs) is oxygen transport and defence, respectively, this chapter will focus on platelets, the final product of one of the myeloid

**Figure 1.** Pictorial representation of megakaryocytopoiesis and thrombopoiesis, and the potential for exposome and epigenome modulation of platelet phenotype and function during this dynamic cellular differentiation process.

80% of CVD may be prevented if modifiable risk factors are evaded [2].

#### **2.1. Platelet production**

Gulio Bizzozero first described platelets as 'spherules piastrine' (little plates) as small cell fragments that clumped together at an injured blood vessel site. He also showed that these blood elements did not have a nucleus [6]. Circulating anucleate platelets are now described as dynamic specialised cells, formed in an elaborate style from their precursor cell, the megakaryocyte. Normal platelet counts ranges between 150 and 450 × 10<sup>3</sup> per microliter of blood, constituting the second most abundant cell type in blood after red blood cells. The average adult produces 1011 platelets per day to preserve this count. Platelets travel as resting (quiescent) discoid fragments in the circulation, while an elaborate internal cytoskeleton allows shape changes to occur upon contact with an injured blood vessel.

The size of a mature platelet is approximately 2–4 μm, making them the smallest cells in circulation, while their average thickness is 0.5 μm [7] and their volume about 7 μm3 . Their small size facilitates in their role as 'guardians of the vasculature', as under laminar flow environments, platelets are pushed to the periphery by larger white and red blood cells. Consequently, they remain in close proximity to the blood vessel wall where they can quickly respond to any vascular damage [8]. This enables platelets to perform their main physiological function to prevent blood loss in primary haemostasis by the formation of a 'platelet plug' [9].

Platelets are formed and released into the bloodstream from megakaryocytes (MKs), which reside in the bone marrow [10, 11]. Their production is arguably the most elegant and distinct developmental process in eukaryotes [12]. While accounting for only 0.01% of nucleated bone marrow cells, MKs are also the largest cells, measuring between 50 and 100 μm [13]. Both MK and platelet production, termed megakaryocytopoiesis and thrombopoiesis, are regulated by multiple cytokines, with thrombopoietin (TPO), a hormone produced by the liver and kidneys, being the key regulator. In response to TPO, HSCs differentiate into MKs by differential expression of various transcription factors. This maturation is characterised by a growth in MK size and DNA ploidy levels (endomitosis), enabling the accumulation of RNA, protein and organelles in the MK for packaging into platelets. MKs then migrate to the sinusoidal blood vessels in the vascular niche where numerous long processes called pro-platelets are formed. MKs can extend as many as 20 pro-platelets which branch repeatedly over time. Platelets form at the tips of pro-platelets, receiving organelles, genetic material and granule contents that are transported from the MK cell body. The final point of platelet production occurs in circulation whereby anucleate fragments of pro-platelets bud into pre-platelets [14] and barbell-shaped platelets [15] that are subsequently converted into single platelets in a microtubule-driven process [16] aided by the shear forces within the bloodstream [11]. Platelets then have an average lifespan of 8–10 days, after which they are cleared via phagocytic cells such as macrophages in the spleen. Apoptosis (programmed cell death) is also well recognised in the anucleate platelet [17]. The constant number of platelets in circulation is a consequence of a homeostatic balance between their production and destruction/clearance.

active [26]. The traditional role of mitochondria in the platelet is to the supply of energy in the form of ATP for primary platelet functions. However, novel functions for mitochondria continue to emerge. Dual activation of platelets with collagen and thrombin results in a subtype of platelets known as collagen and thrombin activated (COAT) platelets. COAT platelets display striking alterations in function and structure to typical "activated platelets" by exhibiting a myriad of features such as phosphatidylserine exposure due to cytoskeletal reorganisation, high microparticle release, and increased levels of fibrinogen on the platelet surface [27]. Mitochondrial membrane potential (Δ*Ψ*m) is reduced in (COAT) platelets and decreases in parallel with elevated mitochondrial ROS levels that are necessary for facilitating platelet PS exposure upon activation [28]. Mitochondria are involved in the process of platelet apoptosis [29] and can be released from platelets as potential inflammatory mediators [30].

Platelets express a wide variety of receptors on their membrane, which are fundamental to platelet function and downstream signalling [9]. Major receptors include integrins, leucinerich repeat receptors (Glycoprotein GPIb/IX/V, Toll-like receptors), C-type lectin receptors (P-Selectin, CLEC-2), tyrosine kinase receptors (Ephrins and Eph kinases), proteins belonging to the immunoglobulin superfamily (GPVI, FcγRIIA) and other receptors shared with vascu-

Integrins are type I transmembrane cell adhesion receptors [31] consisting of a short intracellular and larger extracellular domain. All integrins contain an α subunit and a β subunit, capable of bi-directional signalling. During signal transduction, they transmit information concerning the chemical and mechanical status of the ECM to the cell [32, 33]. Platelets express five integrin receptors—each with affinity for specific ligands; αIIbβ3 (fibrinogen), α2β1(collagen), α5β1(fibronectin), αVβ3 (vitronectin) and α6β1 (laminin), all of which share related signal transduction processes [34]. On the cytoplasmic face of the plasma membrane, integrins regulate cytoskeletal dynamics and signalling complexes. On the extracellular side, integrins bind with high affinity to either ECM ligands or counter receptors on adjacent cell surfaces [35]. αIIbβ3 is the most abundant platelet integrin, with copy numbers of ~50,000 per platelet, and is present in both alpha granules and the platelet surface. Platelets express a number of G-protein coupled receptors (GPCRs) which constitute a large family of receptors that can identify molecules outside the cell and initiate signal transduction pathways and ultimately cell function. The main GPCRs present on platelets include thrombin receptors

called protease-activated receptors (PARs) (PAR1 and PAR4), ADP receptors (P2Y<sup>1</sup>

Haemostasis can be subdivided into primary haemostasis, secondary haemostasis and fibrinolysis (**Figure 2**) [38]. Platelets prevent blood loss in primary haemostasis, the physiological

receptors are present on the platelet [36], thromboxane recep-

, P2Y12,), of

Platelets: From Formation to Function http://dx.doi.org/10.5772/intechopen.80924 75

*2.2.2. Surface receptors*

which approximately 150 P2Y<sup>1</sup>

**2.3. Platelet function**

tors (TPα and TPβ) and glycoprotein receptors [37].

*2.3.1. Platelet function in primary haemostasis*

lar cells (TNF receptor type, CD63, CD36, PSGL-1).

#### **2.2. Platelet structure**

#### *2.2.1. Internal*

Platelets are unique in their structural composition and, while anucleate, contain a large variety of cellular organelles, granules and mitochondria. Granules are generally secretory vesicles that release their contents either to the platelet surface or to extracellular fluid by endocytosis. Over 300 proteins from platelet granules have been identified in the platelet releasate following activation [18, 19]. Three types of platelet granules have been identified: α-granules, dense granules, and lysosomal granules all of which derive their cargo from MKs. α-granules are the largest and most numerous (50–80 per platelet) encompassing roughly 10% of the platelet volume [20]. They harbour a vast assortment of proteins important for primary haemostasis including integrins (αIIbβ3) immunoglobulin family receptors (e.g. GPVI, PECAM), leucine-rich repeat family receptors (e.g., GPIb-IX-V complex), tetraspanins (e.g., CD9) and other adhesive proteins such as von Willebrand Factor (vWF), fibrinogen, and coagulation factors (Factors V, XI) that participate in secondary haemostasis. While it was previously assumed that platelet α-granules were homogenous populations, [21] suggested that platelets have distinct subpopulations of alpha granules which differentially release their cargo in a context-dependent manner. Dense granules are smaller in size and number (3–8 per platelet) storing high concentrations of non-protein molecules that potentiate platelet activation such as adenosine diphosphate (ADP), adenosine triphosphate (ATP), calcium, histamine, polyphosphate and serotonin [22, 23]. Lysosomal granules are sparse and harbour enzymes such as acid hydrolases and proteases). They function in the digestion of cytosolic components. Secretion of the lysosomal content has key extracellular functions including receptor cleavage, fibrinolysis and degradation of extracellular matrix (ECM) [24]. A recent report has described a possible new type of secretory granule termed a T-granule, after their tubular morphology [11]. These novel electron-dense granules have been proposed to function in toll-like receptor (TLR) organisation and signalling. Platelet granule deficiencies or defects such as the Grey Platelet Syndrome (α-granule deficiency) or Hermansky-Pudlak Syndrome (dense granule deficiency) can cause mild to severe bleeding disorders [25].

Platelets contain functional mitochondria, which despite being few in number, have higher rates of ATP turnover than resting mammalian muscle, suggesting they are very metabolically active [26]. The traditional role of mitochondria in the platelet is to the supply of energy in the form of ATP for primary platelet functions. However, novel functions for mitochondria continue to emerge. Dual activation of platelets with collagen and thrombin results in a subtype of platelets known as collagen and thrombin activated (COAT) platelets. COAT platelets display striking alterations in function and structure to typical "activated platelets" by exhibiting a myriad of features such as phosphatidylserine exposure due to cytoskeletal reorganisation, high microparticle release, and increased levels of fibrinogen on the platelet surface [27]. Mitochondrial membrane potential (Δ*Ψ*m) is reduced in (COAT) platelets and decreases in parallel with elevated mitochondrial ROS levels that are necessary for facilitating platelet PS exposure upon activation [28]. Mitochondria are involved in the process of platelet apoptosis [29] and can be released from platelets as potential inflammatory mediators [30].

#### *2.2.2. Surface receptors*

blood vessels in the vascular niche where numerous long processes called pro-platelets are formed. MKs can extend as many as 20 pro-platelets which branch repeatedly over time. Platelets form at the tips of pro-platelets, receiving organelles, genetic material and granule contents that are transported from the MK cell body. The final point of platelet production occurs in circulation whereby anucleate fragments of pro-platelets bud into pre-platelets [14] and barbell-shaped platelets [15] that are subsequently converted into single platelets in a microtubule-driven process [16] aided by the shear forces within the bloodstream [11]. Platelets then have an average lifespan of 8–10 days, after which they are cleared via phagocytic cells such as macrophages in the spleen. Apoptosis (programmed cell death) is also well recognised in the anucleate platelet [17]. The constant number of platelets in circulation is a consequence of a homeostatic balance between their production and destruction/clearance.

Platelets are unique in their structural composition and, while anucleate, contain a large variety of cellular organelles, granules and mitochondria. Granules are generally secretory vesicles that release their contents either to the platelet surface or to extracellular fluid by endocytosis. Over 300 proteins from platelet granules have been identified in the platelet releasate following activation [18, 19]. Three types of platelet granules have been identified: α-granules, dense granules, and lysosomal granules all of which derive their cargo from MKs. α-granules are the largest and most numerous (50–80 per platelet) encompassing roughly 10% of the platelet volume [20]. They harbour a vast assortment of proteins important for primary haemostasis including integrins (αIIbβ3) immunoglobulin family receptors (e.g. GPVI, PECAM), leucine-rich repeat family receptors (e.g., GPIb-IX-V complex), tetraspanins (e.g., CD9) and other adhesive proteins such as von Willebrand Factor (vWF), fibrinogen, and coagulation factors (Factors V, XI) that participate in secondary haemostasis. While it was previously assumed that platelet α-granules were homogenous populations, [21] suggested that platelets have distinct subpopulations of alpha granules which differentially release their cargo in a context-dependent manner. Dense granules are smaller in size and number (3–8 per platelet) storing high concentrations of non-protein molecules that potentiate platelet activation such as adenosine diphosphate (ADP), adenosine triphosphate (ATP), calcium, histamine, polyphosphate and serotonin [22, 23]. Lysosomal granules are sparse and harbour enzymes such as acid hydrolases and proteases). They function in the digestion of cytosolic components. Secretion of the lysosomal content has key extracellular functions including receptor cleavage, fibrinolysis and degradation of extracellular matrix (ECM) [24]. A recent report has described a possible new type of secretory granule termed a T-granule, after their tubular morphology [11]. These novel electron-dense granules have been proposed to function in toll-like receptor (TLR) organisation and signalling. Platelet granule deficiencies or defects such as the Grey Platelet Syndrome (α-granule deficiency) or Hermansky-Pudlak Syndrome (dense granule deficiency) can cause mild to severe bleeding disorders [25].

Platelets contain functional mitochondria, which despite being few in number, have higher rates of ATP turnover than resting mammalian muscle, suggesting they are very metabolically

**2.2. Platelet structure**

74 Homeostasis - An Integrated Vision

*2.2.1. Internal*

Platelets express a wide variety of receptors on their membrane, which are fundamental to platelet function and downstream signalling [9]. Major receptors include integrins, leucinerich repeat receptors (Glycoprotein GPIb/IX/V, Toll-like receptors), C-type lectin receptors (P-Selectin, CLEC-2), tyrosine kinase receptors (Ephrins and Eph kinases), proteins belonging to the immunoglobulin superfamily (GPVI, FcγRIIA) and other receptors shared with vascular cells (TNF receptor type, CD63, CD36, PSGL-1).

Integrins are type I transmembrane cell adhesion receptors [31] consisting of a short intracellular and larger extracellular domain. All integrins contain an α subunit and a β subunit, capable of bi-directional signalling. During signal transduction, they transmit information concerning the chemical and mechanical status of the ECM to the cell [32, 33]. Platelets express five integrin receptors—each with affinity for specific ligands; αIIbβ3 (fibrinogen), α2β1(collagen), α5β1(fibronectin), αVβ3 (vitronectin) and α6β1 (laminin), all of which share related signal transduction processes [34]. On the cytoplasmic face of the plasma membrane, integrins regulate cytoskeletal dynamics and signalling complexes. On the extracellular side, integrins bind with high affinity to either ECM ligands or counter receptors on adjacent cell surfaces [35]. αIIbβ3 is the most abundant platelet integrin, with copy numbers of ~50,000 per platelet, and is present in both alpha granules and the platelet surface. Platelets express a number of G-protein coupled receptors (GPCRs) which constitute a large family of receptors that can identify molecules outside the cell and initiate signal transduction pathways and ultimately cell function. The main GPCRs present on platelets include thrombin receptors called protease-activated receptors (PARs) (PAR1 and PAR4), ADP receptors (P2Y<sup>1</sup> , P2Y12,), of which approximately 150 P2Y<sup>1</sup> receptors are present on the platelet [36], thromboxane receptors (TPα and TPβ) and glycoprotein receptors [37].

#### **2.3. Platelet function**

#### *2.3.1. Platelet function in primary haemostasis*

Haemostasis can be subdivided into primary haemostasis, secondary haemostasis and fibrinolysis (**Figure 2**) [38]. Platelets prevent blood loss in primary haemostasis, the physiological process which halts bleeding at an injured blood vessel, while maintaining normal blood flow elsewhere in circulation, by the formation of a 'platelet plug' [11]. Secondary haemostasis refers to the deposition of insoluble fibrin that is generated by the coagulation cascade. Finally, fibrinolysis results in the breakdown of blood clots during wound healing involving the interplay of a number of enzymes [38]. A healthy endothelium provides a non-adhesive surface for platelets. However, in areas of vascular injury, the sub-endothelium is exposed and platelets may adhere quickly to different extracellular matrix components, and then form a platelet plug. This process is achieved through three distinct processes—platelet adhesion,

Platelets: From Formation to Function http://dx.doi.org/10.5772/intechopen.80924 77

Platelet adhesion entails a collaborative effort of various platelet receptors, fundamentally leading to platelet activation and aggregation. ECM elements that platelets adhere to include proteins such as collagen, vWF, fibronectin, laminin and fibrinogen among others [40, 41]. Among these subendothelial substrates, the thrombogenic fibrillar collagens type I and III are the most powerful intermediaries of platelet adhesion due to their robust activating potential and affinity for vWF [42]. Following vascular damage, initial platelet 'tethering' is mediated by the interaction between the A1 domain of vWF deposited in the subendothelial matrix of the damaged vessel wall, and the GPIbα in the platelet receptor GPIb-IX-V. This interaction is particularly important at high shear rates supporting platelet translocation (i.e. decelerating platelets and keeping them in close contact with the endothelium) over the subendothelium, but not stable adhesion [43]. This interaction allows engagement of other platelet receptors. vWF/GPIb-IX interface also induces platelet activation signalling events, resulting in integrin activation [44]. Following platelet tethering, platelet collagen receptors, GPVI and α2β1 interact with exposed collagen and promote platelet adhesion and activation. GPVI is non-covalently coupled to the Fc Receptor chain (FcRγ) [45, 46] and has been acknowledged as the major signalling receptor for collagen. FcRγ has an immunoreceptor tyrosine-based activation motif (ITAM) on its cytoplasmic sphere. After collagen binding to GPVI, the ITAM motif on the GPVI/FcRγ complex is phosphorylated, resulting in activation of the Syk kinase pathways that phosphorylate downstream targets, ultimately resulting in increased cytosolic Ca2+ and subsequent platelet shape change, granule secretion and integrin activation. GPVI has a low affinity for collagen, rendering it unable to mediate stable adhesion alone. The α2β1 integrin then maintains stable adhesion to collagen. α2β1 stimulates downstream steps indirectly by reinforcing GPVI-collagen interactions [41, 47] and by direct signalling leading to activation of αIIbβ3. Platelet Ca2+ signalling is markedly dissimilar between GPVI and α2β1 suggesting that the alliance regarding GPVI and α2β1 supports optimal platelet adhesion. The final step of platelet adhesion occurs via binding of platelets to other ECM components such as fibronectin, laminin and immobilised vWF. Platelets bind to fibronectin via the α5β1 receptor and αIIbβ3, whilst adhesion to laminin is mediated by their α6β1 receptor. Stable binding of platelets elicits activation pathways involving tyrosine kinases and signal transduction GPCR receptor signalling, cumulatively resulting in elevated cytosolic Ca2+ levels,

Once platelet adhesion has occurred at the site of vessel wall damage, platelet activation needs to be maintained for haemostasis to continue. Essential for the amplification of platelet

platelet activation and secretion, and platelet aggregation [39].

cytoskeletal reorganisation and integrin activation.

*2.3.3. Platelet activation and secretion*

*2.3.2. Platelet adhesion*

**Figure 2.** (A) Diagram showing key platelet membrane receptors involved in platelet function in haemostasis and thrombosis. (B) Distinct phases of platelet function in primary haemostasis: Initial platelet tethering and adhesion at a site of vascular damage, firm adhesion and shape change, activation, granule secretion and further recruitment of platelets leading to platelet aggregation. (C) Secondary haemostasis: Formation, deposition and cross-linking of insoluble fibrin, generated by the coagulation cascade, to stabilise the primary platelet plug. (D) Image of platelet aggregates (fluorescently-labelled, green) on collagen fibrils following *in vitro* blood flow at arterial shear (1800 s−<sup>1</sup> ) using a parallelplate blood perfusion chamber (*unpublished data*—*Gerardene Meade-Murphy*).

Finally, fibrinolysis results in the breakdown of blood clots during wound healing involving the interplay of a number of enzymes [38]. A healthy endothelium provides a non-adhesive surface for platelets. However, in areas of vascular injury, the sub-endothelium is exposed and platelets may adhere quickly to different extracellular matrix components, and then form a platelet plug. This process is achieved through three distinct processes—platelet adhesion, platelet activation and secretion, and platelet aggregation [39].

#### *2.3.2. Platelet adhesion*

Platelet adhesion entails a collaborative effort of various platelet receptors, fundamentally leading to platelet activation and aggregation. ECM elements that platelets adhere to include proteins such as collagen, vWF, fibronectin, laminin and fibrinogen among others [40, 41]. Among these subendothelial substrates, the thrombogenic fibrillar collagens type I and III are the most powerful intermediaries of platelet adhesion due to their robust activating potential and affinity for vWF [42]. Following vascular damage, initial platelet 'tethering' is mediated by the interaction between the A1 domain of vWF deposited in the subendothelial matrix of the damaged vessel wall, and the GPIbα in the platelet receptor GPIb-IX-V. This interaction is particularly important at high shear rates supporting platelet translocation (i.e. decelerating platelets and keeping them in close contact with the endothelium) over the subendothelium, but not stable adhesion [43]. This interaction allows engagement of other platelet receptors. vWF/GPIb-IX interface also induces platelet activation signalling events, resulting in integrin activation [44].

Following platelet tethering, platelet collagen receptors, GPVI and α2β1 interact with exposed collagen and promote platelet adhesion and activation. GPVI is non-covalently coupled to the Fc Receptor chain (FcRγ) [45, 46] and has been acknowledged as the major signalling receptor for collagen. FcRγ has an immunoreceptor tyrosine-based activation motif (ITAM) on its cytoplasmic sphere. After collagen binding to GPVI, the ITAM motif on the GPVI/FcRγ complex is phosphorylated, resulting in activation of the Syk kinase pathways that phosphorylate downstream targets, ultimately resulting in increased cytosolic Ca2+ and subsequent platelet shape change, granule secretion and integrin activation. GPVI has a low affinity for collagen, rendering it unable to mediate stable adhesion alone. The α2β1 integrin then maintains stable adhesion to collagen. α2β1 stimulates downstream steps indirectly by reinforcing GPVI-collagen interactions [41, 47] and by direct signalling leading to activation of αIIbβ3. Platelet Ca2+ signalling is markedly dissimilar between GPVI and α2β1 suggesting that the alliance regarding GPVI and α2β1 supports optimal platelet adhesion.

The final step of platelet adhesion occurs via binding of platelets to other ECM components such as fibronectin, laminin and immobilised vWF. Platelets bind to fibronectin via the α5β1 receptor and αIIbβ3, whilst adhesion to laminin is mediated by their α6β1 receptor. Stable binding of platelets elicits activation pathways involving tyrosine kinases and signal transduction GPCR receptor signalling, cumulatively resulting in elevated cytosolic Ca2+ levels, cytoskeletal reorganisation and integrin activation.

#### *2.3.3. Platelet activation and secretion*

**Figure 2.** (A) Diagram showing key platelet membrane receptors involved in platelet function in haemostasis and thrombosis. (B) Distinct phases of platelet function in primary haemostasis: Initial platelet tethering and adhesion at a site of vascular damage, firm adhesion and shape change, activation, granule secretion and further recruitment of platelets leading to platelet aggregation. (C) Secondary haemostasis: Formation, deposition and cross-linking of insoluble fibrin, generated by the coagulation cascade, to stabilise the primary platelet plug. (D) Image of platelet aggregates

process which halts bleeding at an injured blood vessel, while maintaining normal blood flow elsewhere in circulation, by the formation of a 'platelet plug' [11]. Secondary haemostasis refers to the deposition of insoluble fibrin that is generated by the coagulation cascade.

76 Homeostasis - An Integrated Vision

) using a parallel-

(fluorescently-labelled, green) on collagen fibrils following *in vitro* blood flow at arterial shear (1800 s−<sup>1</sup>

plate blood perfusion chamber (*unpublished data*—*Gerardene Meade-Murphy*).

Once platelet adhesion has occurred at the site of vessel wall damage, platelet activation needs to be maintained for haemostasis to continue. Essential for the amplification of platelet activation is the production and release of soluble agonists at the site of damage [26], which act in an autocrine and paracrine manner to amplify platelet activation and recruit further circulating platelets. These agonists consist of TxA<sup>2</sup> , ADP, epinephrine and thrombin. ADP is secreted from platelet dense granules and binds to its relevant receptors, P2Y12 and P2Y<sup>1</sup> on the platelet surface [48]. ADP is also released from red blood cells at the site of vascular damage [37]. Binding of ADP initiates a full complement of activation events such as elevation of intracellular platelet Ca2+, TxA<sup>2</sup> synthesis, protein phosphorylation, shape change, granule release, and most importantly, activation of αIIbβ3 [49]. P2Y12 is also the target of a class of antiplatelet drugs called thienopyridines (ticlopidine, clopidogrel, prasugrel), widely used in the prevention of vascular events in patients with CVD.

low affinity resting state to a high affinity activated state in order to bind extracellular ligands such as fibrinogen and vWF. Irreversible activation of αIIbβ3 is a prerequisite for the development of irreversible platelet aggregates. Due to the symmetrical nature of fibrinogen, platelets can be 'bridged' and platelet aggregates are formed [40]. While αIIbβ3 is the major player in platelet aggregation, couplings between other platelet receptors and their ligands could be incorporated in aggregation. Some of these comprise CD40 ligand [55] interaction with αIIbβ3, the vWF-GPIb complex [56], and an involvement of fibronectin in stabilising platelet aggregation [57, 58]. Cadherin-6 was recently acknowledged as a new counter-receptor for

Bi-directional 'inside out' and 'outside in' signals are transferred by both integrin subunits, mediating receptor conformation and platelet function [44]. After platelet stimulation with ADP, the signal from the ADP receptors is conducted to the intracellular domain of the cytoplasmic tail of αIIbβ3 and subsequently transmitted through a series of events to the extracellular domain (inside-out) causing a conformational change in the extracellular domain that binds to its ligand. Inside-out signalling necessitates binding of talin and kindlins to the cytoplasmic domain of β3. After ligand binding, a signal is sent to the cell (outside-in) to control platelet function such as filopodia and lamellipodia extension to dull platelet spreading [34, 44]. Furthermore, αIIbβ3 outside-in signals can also act as a break to curb excessive platelet

A significant factor influencing platelet aggregation is the distinct shear environment experienced within the vascular system. Platelets are subjected to fluctuating haemodynamic conditions *in vivo* such as shear stress and shear rate. The latter refers to the rate of increase of blood flow velocity, whereas the former denotes the force per unit area on the vessel wall [61]. Shear

shear rates (up to 40,000) have been described [61, 62]. Increasing shear rate activates platelets

a two-step sequential process occurs. The first depends on the adhesive properties of GP1bα and αIIbβ3 and is facilitated by the formation of reversible platelet aggregates. The second relies on the generation of platelet agonists and involves the irreversible activation of αIIbβ3 to form stable aggregates [63]. Accordingly, both fibrinogen and vWF, and receptors GPIbα and αIIbβ3, have distinctive but complementary roles in platelet aggregation subject to the

The role of platelets in haemostasis is reliant on the equilibrium between activatory and inhibitory signals [64]. Inhibitory signals from the vasculature prevent platelet activation in healthy vessels. Activatory signals present at an injured blood vessel initiate platelet activation, and are managed by endogenous negative signalling regulators. Negative regulators

) to diseased or pathological arteries, where extremely high

), platelet aggregation is primarily facilitated by αIIbβ3-

to 500 s−<sup>1</sup>

(but between 1000 and 10,000),

Platelets: From Formation to Function http://dx.doi.org/10.5772/intechopen.80924 79

) to

rates experienced by platelets range from slow flow in veins (shear rate 10 s−<sup>1</sup>

fibrinogen interactions. At shear rates typically over 5000 s−<sup>1</sup>

αIIbβ3, involved in platelet aggregation [59].

activation by activated SHIP-1 [60].

small arteries (approx. 2000 s−<sup>1</sup>

haemodynamic environment.

*2.3.6. Signal transduction during platelet function*

itself. At low shear rates (<1000 s−<sup>1</sup>

*2.3.5. Bio rheological factors in platelet aggregation*

TxA<sup>2</sup> is a potent platelet agonist synthesised from arachidonic acid through the COX pathway and TxA<sup>2</sup> synthase enzymes. It subsequently binds to TPα and TPβ receptors that differ in their cytoplasmic tails, causing vasoconstriction, shape change, protein phosphorylation, secretion and platelet aggregation [50, 51]. Indeed, high levels of TxA<sup>2</sup> have been implicated in CVD, whilst inhibition of TxA<sup>2</sup> synthesis through aspirin-mediated COX inhibition is a major anti-platelet target.

The agonist thrombin rapidly accumulates at sites of vascular damage and has major functions in promoting and stabilising thrombus formation. Platelets release factors that support the activation of prothrombin, which after a complex series of sequential events in the coagulation cascade, results in the generation of thrombin [52]. The increase in cytosolic Ca2+ after platelet activation results in platelet phosphatidylserine (PS) exposure on the activated platelet membrane providing a procoagulant surface for thrombin to interact with its PAR1 and PAR4 (G protein-coupled) receptors.

Uniquely, thrombin activates its PAR-receptors by cleaving an N-terminal part at a consensus site. Cleavage exposes a new binding site that acts as a ligand to activate the receptor. Thrombin is the most powerful platelet activator, initiating an entire complement of platelet responses (shape change, granule secretion, TxA<sup>2</sup> synthesis, aggregation etc.) [53]. Thrombin can activate platelets at extremely low concentrations and within seconds, increases cytosolic level of Ca2+, eliciting downstream signalling events. Unlike ADP, TxA<sup>2</sup> or thrombin, the catecholamine epinephrine is a weak agonist unable to cause shape change alone. However, it works collectively with the other agonists increasing their potential to activate. Epinephrine's mode of action is to inhibit cAMP formation by the platelet α<sup>2</sup> A-adrenergic receptor [54].

#### *2.3.4. Platelet aggregation*

The ultimate step in primary haemostasis is platelet aggregation, caused by crosslinking of αIIbβ3 on adjacent platelets by fibrinogen (**Figure 2**). While platelet aggregation is a complex process involving different receptors (αIIbβ3 and GPIbα) and ligands (fibrinogen, fibronectin, and vWF), the main process involves the integrin αIIbβ3. On resting platelets, integrin αIIbβ3 has a low affinity for its ligands fibrinogen and vWF, which dramatically increases upon platelet activation. Binding of the main agonists to their respective receptors induce intracellular signals that disrupt the complex between the cytoplasmic tails of αIIbβ3. This ultimately leads to a conformational change in its extracellular globular head domains from a low affinity resting state to a high affinity activated state in order to bind extracellular ligands such as fibrinogen and vWF. Irreversible activation of αIIbβ3 is a prerequisite for the development of irreversible platelet aggregates. Due to the symmetrical nature of fibrinogen, platelets can be 'bridged' and platelet aggregates are formed [40]. While αIIbβ3 is the major player in platelet aggregation, couplings between other platelet receptors and their ligands could be incorporated in aggregation. Some of these comprise CD40 ligand [55] interaction with αIIbβ3, the vWF-GPIb complex [56], and an involvement of fibronectin in stabilising platelet aggregation [57, 58]. Cadherin-6 was recently acknowledged as a new counter-receptor for αIIbβ3, involved in platelet aggregation [59].

Bi-directional 'inside out' and 'outside in' signals are transferred by both integrin subunits, mediating receptor conformation and platelet function [44]. After platelet stimulation with ADP, the signal from the ADP receptors is conducted to the intracellular domain of the cytoplasmic tail of αIIbβ3 and subsequently transmitted through a series of events to the extracellular domain (inside-out) causing a conformational change in the extracellular domain that binds to its ligand. Inside-out signalling necessitates binding of talin and kindlins to the cytoplasmic domain of β3. After ligand binding, a signal is sent to the cell (outside-in) to control platelet function such as filopodia and lamellipodia extension to dull platelet spreading [34, 44]. Furthermore, αIIbβ3 outside-in signals can also act as a break to curb excessive platelet activation by activated SHIP-1 [60].

#### *2.3.5. Bio rheological factors in platelet aggregation*

activation is the production and release of soluble agonists at the site of damage [26], which act in an autocrine and paracrine manner to amplify platelet activation and recruit further

is secreted from platelet dense granules and binds to its relevant receptors, P2Y12 and P2Y<sup>1</sup> on the platelet surface [48]. ADP is also released from red blood cells at the site of vascular damage [37]. Binding of ADP initiates a full complement of activation events such as elevation

release, and most importantly, activation of αIIbβ3 [49]. P2Y12 is also the target of a class of antiplatelet drugs called thienopyridines (ticlopidine, clopidogrel, prasugrel), widely used in

in their cytoplasmic tails, causing vasoconstriction, shape change, protein phosphorylation,

The agonist thrombin rapidly accumulates at sites of vascular damage and has major functions in promoting and stabilising thrombus formation. Platelets release factors that support the activation of prothrombin, which after a complex series of sequential events in the coagulation cascade, results in the generation of thrombin [52]. The increase in cytosolic Ca2+ after platelet activation results in platelet phosphatidylserine (PS) exposure on the activated platelet membrane providing a procoagulant surface for thrombin to interact with its PAR1

Uniquely, thrombin activates its PAR-receptors by cleaving an N-terminal part at a consensus site. Cleavage exposes a new binding site that acts as a ligand to activate the receptor. Thrombin is the most powerful platelet activator, initiating an entire complement of platelet

can activate platelets at extremely low concentrations and within seconds, increases cytosolic

echolamine epinephrine is a weak agonist unable to cause shape change alone. However, it works collectively with the other agonists increasing their potential to activate. Epinephrine's

The ultimate step in primary haemostasis is platelet aggregation, caused by crosslinking of αIIbβ3 on adjacent platelets by fibrinogen (**Figure 2**). While platelet aggregation is a complex process involving different receptors (αIIbβ3 and GPIbα) and ligands (fibrinogen, fibronectin, and vWF), the main process involves the integrin αIIbβ3. On resting platelets, integrin αIIbβ3 has a low affinity for its ligands fibrinogen and vWF, which dramatically increases upon platelet activation. Binding of the main agonists to their respective receptors induce intracellular signals that disrupt the complex between the cytoplasmic tails of αIIbβ3. This ultimately leads to a conformational change in its extracellular globular head domains from a

is a potent platelet agonist synthesised from arachidonic acid through the COX path-

synthase enzymes. It subsequently binds to TPα and TPβ receptors that differ

, ADP, epinephrine and thrombin. ADP

have been implicated

or thrombin, the cat-

synthesis, protein phosphorylation, shape change, granule

synthesis through aspirin-mediated COX inhibition is a

synthesis, aggregation etc.) [53]. Thrombin

A-adrenergic receptor [54].

circulating platelets. These agonists consist of TxA<sup>2</sup>

the prevention of vascular events in patients with CVD.

secretion and platelet aggregation [50, 51]. Indeed, high levels of TxA<sup>2</sup>

level of Ca2+, eliciting downstream signalling events. Unlike ADP, TxA<sup>2</sup>

mode of action is to inhibit cAMP formation by the platelet α<sup>2</sup>

of intracellular platelet Ca2+, TxA<sup>2</sup>

78 Homeostasis - An Integrated Vision

in CVD, whilst inhibition of TxA<sup>2</sup>

and PAR4 (G protein-coupled) receptors.

responses (shape change, granule secretion, TxA<sup>2</sup>

major anti-platelet target.

*2.3.4. Platelet aggregation*

TxA<sup>2</sup>

way and TxA<sup>2</sup>

A significant factor influencing platelet aggregation is the distinct shear environment experienced within the vascular system. Platelets are subjected to fluctuating haemodynamic conditions *in vivo* such as shear stress and shear rate. The latter refers to the rate of increase of blood flow velocity, whereas the former denotes the force per unit area on the vessel wall [61]. Shear rates experienced by platelets range from slow flow in veins (shear rate 10 s−<sup>1</sup> to 500 s−<sup>1</sup> ) to small arteries (approx. 2000 s−<sup>1</sup> ) to diseased or pathological arteries, where extremely high shear rates (up to 40,000) have been described [61, 62]. Increasing shear rate activates platelets itself. At low shear rates (<1000 s−<sup>1</sup> ), platelet aggregation is primarily facilitated by αIIbβ3 fibrinogen interactions. At shear rates typically over 5000 s−<sup>1</sup> (but between 1000 and 10,000), a two-step sequential process occurs. The first depends on the adhesive properties of GP1bα and αIIbβ3 and is facilitated by the formation of reversible platelet aggregates. The second relies on the generation of platelet agonists and involves the irreversible activation of αIIbβ3 to form stable aggregates [63]. Accordingly, both fibrinogen and vWF, and receptors GPIbα and αIIbβ3, have distinctive but complementary roles in platelet aggregation subject to the haemodynamic environment.

#### *2.3.6. Signal transduction during platelet function*

The role of platelets in haemostasis is reliant on the equilibrium between activatory and inhibitory signals [64]. Inhibitory signals from the vasculature prevent platelet activation in healthy vessels. Activatory signals present at an injured blood vessel initiate platelet activation, and are managed by endogenous negative signalling regulators. Negative regulators and pathways include the ITIM containing receptors that are postulated to reduce activation of PLC, PI3K and integrin αIIbβ3 [64]. The Wnt-β-catenin pathway has recently gained attention as negative regulator of platelet function [65, 66]. Wnt3a is one of these glycoproteins, which is secreted from activated platelets. It has been suggested to activate the canonical Wnt-β-catenin pathway as constituents of this pathway have been identified in platelets. The regulation of small GTPases such as Rap1, Rac1, RhoA and Cdc42 has been suggested as players in this inhibition of platelet function [65]. These pathways are less well characterised than the classical activation pathways [67]. The primary platelet inhibiting signals produced by healthy ECs are nitric oxide and prostacyclin. Both NO and prostacyclin relax blood vessels and prevent platelet activation [68]. NO is synthesised from several cells including platelets, ECs and RBCs and plays vital roles in maintaining platelets in a resting state [69–71]. Prostacyclin (PGI<sup>2</sup> ) is a physiological anti-aggregating agent produced constitutively by ECs as a result of arachidonic acid metabolism by cyclooxygenase (COX) enzymes.

Platelets have significant roles in the pathogenesis of metastasis [77]. Tumour cells can aggregate platelets *in vitro* and it has been proposed that platelet adhesion to metastatic cells can act as a 'cloak' around circulating tumour cells therefore acting as a shield for immune clearance. This phenomenon of platelet cloaking has resulted in pro-survival, pro-angiogenic and epithelial mesenchymal transition (EMT) in cancer cells [78]. Platelets also release growth factors such as VEGF and PDGF that can expedite tumour growth [79]. Platelets are deeply implicated in wound healing and bone health, and indeed the use of platelet rich plasma (PRP) therapy (i.e. rich in growth factors and bioactive substances) is effective in osteoarthritis

The different functions of platelets may be reliably detected with a wide spectrum of tests (**Table 1**). These can be utilised to identify inherited or acquired platelet dysfunction, monitor antiplatelet therapy, manage various aspects of platelet banking and transfusion, and to aid in the understanding of platelet physiology in basic research. PFTs are centred around principles of platelet function such as platelet adhesion and aggregation, platelet function under shear conditions, and measurement of the platelet releasate [81]. Platelet function testing began with the evaluation of the bleeding time (the time taken for platelets to occlude an *in vivo* wound), using the Duke procedure [82], before the development of light transmission aggregometry (LTA) revolutionised the study of platelet function. Considered the historical gold standard, LTA is a relatively easy technique that involves stirring a suspension of platelet rich plasma in a cuvette in the presence of a platelet agonist (such as ADP or collagen). The cuvette is placed between a light source and photocell. Agonist addition causes *in vitro* platelet aggregation

Investigation of platelet function in the environmental *milieu* of whole blood under conditions that take into account most of the physiological parameters that influence platelet adhesion and aggregation (red blood cells, white blood cells, plasma) is important. The PFA-100, The Impact-R Cone and Plate analyser and the global thrombosis test are examples of such assays. The PFA-100 assesses platelet aggregation under high shear where platelets are activated

collagen and ADP), resulting in closure of an aperture [84]. The Impact-R Cone and Plate analyser is a point-of-care (POC) device which measures global platelet function by testing platelet adhesion and aggregation in whole blood, under arterial shear conditions [85, 86]. In this assay, platelet adhesion is dependent on plasma proteins vWF, fibrinogen and RBCs and WBCs. The addition of platelet agonists such as arachidonic acid (AA) and ADP in the system has enabled the evaluation of dual anti-platelet therapy [87–89]. The system is effective in the assessment of platelet function disorders in adults, [90] children [91] and new-borns [92].

Platelet indices are useful as inexpensive non-invasive biomarkers for assessing platelet activation [93]. Platelet indices are straightforwardly measured by semi-automated counters in complete blood counts (CBC) and usually include four factors; platelet count (PLT), mean

) and agonists (e.g.

Platelets: From Formation to Function http://dx.doi.org/10.5772/intechopen.80924 81

[10] and in muscle damage such as rotator cuff tendinopathy [80].

and changes in light absorbance, which is detected by the photocell [83].

in whole blood by an amalgamation of high shear stress (5000–6000 s−<sup>1</sup>

**2.6. Platelet indices**

**2.5. Assessing function: platelet function tests (PFT)**

Following endothelial damage, endogenous inhibitory signals are overcome and platelets react rapidly to limit blood loss. The activating stimuli, such as collagen, vWF, ADP, TxA<sup>2</sup> and thrombin, which induce platelet adhesion, activation and aggregation ultimately regulate a central set of signalling mediators that support activation. Three principle mediator families of platelet activation are phospholipase C (PLC), protein kinase C (PKC) and phosphatidylinositide-3-kinase (PI3K), and underlie two crucial events in platelet activation—secretion of amplifying mediators and activation of integrin αIIbβ3.

#### **2.4. Platelet function beyond haemostasis**

Platelets are recognised as pivotal players in numerous other processes ranging from inflammation and atherosclerosis, fighting microbial infection and tumour growth and metastasis. Platelets are equipped to influence inflammation, the innate immune response and infection, by acting as sentinels in pathogen detection. They express a collection of pattern recognition receptors called toll-like receptors (TLRs) that identify molecular motifs called pathogen associated molecular patterns (PAMPs) and initiate immune responses [72]. Platelets express functional TLR 1–9, whilst TLR2 stimulation in platelets by bacteria through the activation of the PI3K signalling pathway induces a pro-inflammatory response [20]. Platelet TLR9 activation has been associated with thrombosis and oxidative stress, and is found within the T-granules in platelets [11].

Platelets interact with leukocytes, monocytes and granulocytes through different receptorligand interactions (P-Selectin and PSGL-1 interaction) enabling intercellular communication [73]. The capacity of platelets to store and release copious inflammatory cytokines and chemokines is intimately associated to their role in inflammation. Platelets release microparticles linked to inflammatory pathways and which are associated with inflammatory diseases such as rheumatoid arthritis [74]. Platelets play key roles in infection and immune response to bacterial and viral infections. They are the first cell type to arrive at areas of vascular infection [75]. Thrombocytopenia (low platelet count) is a well-established manifestation of sepsis and studies suggests platelets play a functional role in the pathogenesis of sepsis and multi-organ failure [76].

Platelets have significant roles in the pathogenesis of metastasis [77]. Tumour cells can aggregate platelets *in vitro* and it has been proposed that platelet adhesion to metastatic cells can act as a 'cloak' around circulating tumour cells therefore acting as a shield for immune clearance. This phenomenon of platelet cloaking has resulted in pro-survival, pro-angiogenic and epithelial mesenchymal transition (EMT) in cancer cells [78]. Platelets also release growth factors such as VEGF and PDGF that can expedite tumour growth [79]. Platelets are deeply implicated in wound healing and bone health, and indeed the use of platelet rich plasma (PRP) therapy (i.e. rich in growth factors and bioactive substances) is effective in osteoarthritis [10] and in muscle damage such as rotator cuff tendinopathy [80].

#### **2.5. Assessing function: platelet function tests (PFT)**

and pathways include the ITIM containing receptors that are postulated to reduce activation of PLC, PI3K and integrin αIIbβ3 [64]. The Wnt-β-catenin pathway has recently gained attention as negative regulator of platelet function [65, 66]. Wnt3a is one of these glycoproteins, which is secreted from activated platelets. It has been suggested to activate the canonical Wnt-β-catenin pathway as constituents of this pathway have been identified in platelets. The regulation of small GTPases such as Rap1, Rac1, RhoA and Cdc42 has been suggested as players in this inhibition of platelet function [65]. These pathways are less well characterised than the classical activation pathways [67]. The primary platelet inhibiting signals produced by healthy ECs are nitric oxide and prostacyclin. Both NO and prostacyclin relax blood vessels and prevent platelet activation [68]. NO is synthesised from several cells including platelets, ECs and RBCs and plays vital roles in maintaining platelets in a resting state [69–71].

Following endothelial damage, endogenous inhibitory signals are overcome and platelets react rapidly to limit blood loss. The activating stimuli, such as collagen, vWF, ADP, TxA<sup>2</sup> and thrombin, which induce platelet adhesion, activation and aggregation ultimately regulate a central set of signalling mediators that support activation. Three principle mediator families of platelet activation are phospholipase C (PLC), protein kinase C (PKC) and phosphatidylinositide-3-kinase (PI3K), and underlie two crucial events in platelet activation—secretion

Platelets are recognised as pivotal players in numerous other processes ranging from inflammation and atherosclerosis, fighting microbial infection and tumour growth and metastasis. Platelets are equipped to influence inflammation, the innate immune response and infection, by acting as sentinels in pathogen detection. They express a collection of pattern recognition receptors called toll-like receptors (TLRs) that identify molecular motifs called pathogen associated molecular patterns (PAMPs) and initiate immune responses [72]. Platelets express functional TLR 1–9, whilst TLR2 stimulation in platelets by bacteria through the activation of the PI3K signalling pathway induces a pro-inflammatory response [20]. Platelet TLR9 activation has been associated with thrombosis and oxidative stress, and is found within the

Platelets interact with leukocytes, monocytes and granulocytes through different receptorligand interactions (P-Selectin and PSGL-1 interaction) enabling intercellular communication [73]. The capacity of platelets to store and release copious inflammatory cytokines and chemokines is intimately associated to their role in inflammation. Platelets release microparticles linked to inflammatory pathways and which are associated with inflammatory diseases such as rheumatoid arthritis [74]. Platelets play key roles in infection and immune response to bacterial and viral infections. They are the first cell type to arrive at areas of vascular infection [75]. Thrombocytopenia (low platelet count) is a well-established manifestation of sepsis and studies suggests platelets play a functional role in the pathogenesis of sepsis and multi-organ

as a result of arachidonic acid metabolism by cyclooxygenase (COX) enzymes.

of amplifying mediators and activation of integrin αIIbβ3.

**2.4. Platelet function beyond haemostasis**

T-granules in platelets [11].

failure [76].

) is a physiological anti-aggregating agent produced constitutively by ECs

Prostacyclin (PGI<sup>2</sup>

80 Homeostasis - An Integrated Vision

The different functions of platelets may be reliably detected with a wide spectrum of tests (**Table 1**). These can be utilised to identify inherited or acquired platelet dysfunction, monitor antiplatelet therapy, manage various aspects of platelet banking and transfusion, and to aid in the understanding of platelet physiology in basic research. PFTs are centred around principles of platelet function such as platelet adhesion and aggregation, platelet function under shear conditions, and measurement of the platelet releasate [81]. Platelet function testing began with the evaluation of the bleeding time (the time taken for platelets to occlude an *in vivo* wound), using the Duke procedure [82], before the development of light transmission aggregometry (LTA) revolutionised the study of platelet function. Considered the historical gold standard, LTA is a relatively easy technique that involves stirring a suspension of platelet rich plasma in a cuvette in the presence of a platelet agonist (such as ADP or collagen). The cuvette is placed between a light source and photocell. Agonist addition causes *in vitro* platelet aggregation and changes in light absorbance, which is detected by the photocell [83].

Investigation of platelet function in the environmental *milieu* of whole blood under conditions that take into account most of the physiological parameters that influence platelet adhesion and aggregation (red blood cells, white blood cells, plasma) is important. The PFA-100, The Impact-R Cone and Plate analyser and the global thrombosis test are examples of such assays. The PFA-100 assesses platelet aggregation under high shear where platelets are activated in whole blood by an amalgamation of high shear stress (5000–6000 s−<sup>1</sup> ) and agonists (e.g. collagen and ADP), resulting in closure of an aperture [84]. The Impact-R Cone and Plate analyser is a point-of-care (POC) device which measures global platelet function by testing platelet adhesion and aggregation in whole blood, under arterial shear conditions [85, 86]. In this assay, platelet adhesion is dependent on plasma proteins vWF, fibrinogen and RBCs and WBCs. The addition of platelet agonists such as arachidonic acid (AA) and ADP in the system has enabled the evaluation of dual anti-platelet therapy [87–89]. The system is effective in the assessment of platelet function disorders in adults, [90] children [91] and new-borns [92].

#### **2.6. Platelet indices**

Platelet indices are useful as inexpensive non-invasive biomarkers for assessing platelet activation [93]. Platelet indices are straightforwardly measured by semi-automated counters in complete blood counts (CBC) and usually include four factors; platelet count (PLT), mean


[94, 96]. The volume of circulating platelets is heterogeneous with subsequent functional differences. Some authors suggest that larger platelets are metabolically more active than smaller platelets, that they have faster rates of aggregation and release higher quantities of pro-thrombotic

including T2DM [98], CVD and atherosclerosis [93] (Berger et al., 2010), and in this regard they have been suggested as markers of subclinical platelet activation. PLCR and PCT may serve as sensitive biomarkers of platelet health [99]. PLCR indicates the percentage of large platelets present in blood [93]. PLCR is significantly higher in subjects with dyslipidaemia compared to healthy subjects [100] and higher in children with T2DM compared to healthy children. Moreover, Rechcinski et al., have hypothesised that PLCR has the potential to be a prognostic biomarker [101]. Importantly, thrombogenicity of large platelets may put individuals at higher risk of acute cardiovascular events. PCT is the volume of blood occupied by platelets as a percentage, similar to the erythrocyte measurement of haematocrit (HCT). PCT reflects total platelet mass and is

has been proposed as a novel predictor of cardiovascular risk and higher PCT is associated with the risk of re-infarction and long-term mortality in CVD patients [102]. However, the clinical significance, reference values and efficacy of some of these parameters are still under exploration. In peripheral blood, there is ample interplay between RBCs, WBCs and platelets [103] and altered levels of blood cells and their morphology have been associated with CVD [104]. Platelet adhesion and aggregate size, is influenced by platelet indices, RBC and WBC [86]. RBCs encourage platelets towards the vessel wall [105], which can affect platelet adhesion and aggregation. In this context, it is important to investigate the associations between the various indices of each blood cell to interpret the multicellular contribution to both thrombogenesis and CVD risk.

Novel techniques continue to emerge and develop the knowledge surrounding the platelet function regulation. The modern "omics" revolution enables simultaneous quantification of hundreds of molecules (e.g. protein or mRNA) from a single sample and their signatures may be reflective of platelet function changes. The amalgamation of transcriptomic and proteomic data and subsequent bioinformatic analysis will lead to a more complete characterisation of platelet function in response to environmental stimuli [106, 107]. Epigenetics and its ancillary elements, including platelet secreted microvesicles (MVs), and microRNA (miRNA), and regulation of the platelet mitochondrial genome are new avenues of investigation and testing

This work was supported through the Irish Higher Education Authority Programme for Research in Third Level Institutes (HEA-PRTLI Cycle 4: T3 Targeted Therapeutics & Theranostics), the Health Research Board of Ireland (HRB-RP/2005/184, HRA/2009/122, HRA/2009/122/R),

and ADP [97]. MPV and PDW levels can be altered in several diseases

Platelets: From Formation to Function http://dx.doi.org/10.5772/intechopen.80924 83

, providing comprehensive information about platelet activity. PCT

elements such as TxA<sup>2</sup>

calculated as PLT × MPV/107

**3. Conclusion**

in platelet research [108–110].

**Acknowledgements**

Typical platelet function tests range from assessment of their primary haemostatic function including measurement of granule secretion using lumi-aggregometry to whole blood shear based assays, which measure platelet function under flow conditions. Platelet function can be tested in washed platelets, whole blood or platelet rich plasma.

**Table 1.** Platelet function tests.

platelet volume (MPV), platelet distribution width (PDW), plateletcrit (PCT), and depending on the analyser, platelet large cell ratio (PLCR). PLT is a universal indicator of haemostasis in a clinical setting and is utilised as a sensitive biomarker for a range of diseases. High PLT, even within the physiological range of 150–450 μl, is associated with a greater risk of thrombosis and CVD suggesting that enhanced PLT encourage platelet hyperactivity and a pro-inflammatory state [94]. However, the consequence of high platelet numbers that are still within physiologic ranges remains unclear [95].

The indices MPV, PDW and PLCR are quantitative measures of the variability in platelet size. MPV reflects the average platelet size while PDW reflects the volume variability in platelet size [94, 96]. The volume of circulating platelets is heterogeneous with subsequent functional differences. Some authors suggest that larger platelets are metabolically more active than smaller platelets, that they have faster rates of aggregation and release higher quantities of pro-thrombotic elements such as TxA<sup>2</sup> and ADP [97]. MPV and PDW levels can be altered in several diseases including T2DM [98], CVD and atherosclerosis [93] (Berger et al., 2010), and in this regard they have been suggested as markers of subclinical platelet activation. PLCR and PCT may serve as sensitive biomarkers of platelet health [99]. PLCR indicates the percentage of large platelets present in blood [93]. PLCR is significantly higher in subjects with dyslipidaemia compared to healthy subjects [100] and higher in children with T2DM compared to healthy children. Moreover, Rechcinski et al., have hypothesised that PLCR has the potential to be a prognostic biomarker [101]. Importantly, thrombogenicity of large platelets may put individuals at higher risk of acute cardiovascular events. PCT is the volume of blood occupied by platelets as a percentage, similar to the erythrocyte measurement of haematocrit (HCT). PCT reflects total platelet mass and is calculated as PLT × MPV/107 , providing comprehensive information about platelet activity. PCT has been proposed as a novel predictor of cardiovascular risk and higher PCT is associated with the risk of re-infarction and long-term mortality in CVD patients [102]. However, the clinical significance, reference values and efficacy of some of these parameters are still under exploration.

In peripheral blood, there is ample interplay between RBCs, WBCs and platelets [103] and altered levels of blood cells and their morphology have been associated with CVD [104]. Platelet adhesion and aggregate size, is influenced by platelet indices, RBC and WBC [86]. RBCs encourage platelets towards the vessel wall [105], which can affect platelet adhesion and aggregation. In this context, it is important to investigate the associations between the various indices of each blood cell to interpret the multicellular contribution to both thrombogenesis and CVD risk.

### **3. Conclusion**

platelet volume (MPV), platelet distribution width (PDW), plateletcrit (PCT), and depending on the analyser, platelet large cell ratio (PLCR). PLT is a universal indicator of haemostasis in a clinical setting and is utilised as a sensitive biomarker for a range of diseases. High PLT, even within the physiological range of 150–450 μl, is associated with a greater risk of thrombosis and CVD suggesting that enhanced PLT encourage platelet hyperactivity and a pro-inflammatory state [94]. However, the consequence of high platelet numbers that are still

Typical platelet function tests range from assessment of their primary haemostatic function including measurement of granule secretion using lumi-aggregometry to whole blood shear based assays, which measure platelet function under

Citrated PRP Measurement of light transmission in response to agonist-induced platelet activation

> to agonist-induced platelet aggregation. Whole blood specimen diluted 1:1 with physiologic saline, prior to

whole blood in response to agonists/inhibitors

Typically measured by ligand binding ELISA

Measurement of platelet glycoproteins, and activation markers; platelet leukocyte aggregates, platelet

Impedance Aggregation Citrated WB Monitors changes in electrical impedance in relation

VerifyNow Citrated WB Turbidimetric optical detection of platelet aggregation in

Plateletworks Citrated WB Platelet counting pre- and post-activation

Urine, serum, citrated plasma

Lumi-aggregometry Citrated WB Combination of LTA/WBA with nucleotide release

PFA-100/200 Citrated WB High-shear platelet adhesion and aggregation during

Impact R cone and plate analyser Citrated WB Shear-induced platelet adhesion-aggregation upon

Global thrombosis test Native WB High-shear platelet plug formation—measurement of

VASP phosphorylation Citrated WB Flow cytometry or ELISA measurement of VASP

washed platelets

flow conditions. Platelet function can be tested in washed platelets, whole blood or platelet rich plasma.

testing

immunoassays

specific surface

phosphorylation

microvesicles

formation of a platelet plug

time cessation of WB flow

The indices MPV, PDW and PLCR are quantitative measures of the variability in platelet size. MPV reflects the average platelet size while PDW reflects the volume variability in platelet size

within physiologic ranges remains unclear [95].

Flow cytometry Citrated WB, PRP,

**Method Sample Principle**

*Platelet aggregation assays*

82 Homeostasis - An Integrated Vision

(LTA)

Light transmission aggregometry

*Assays measuring release reactions*

Soluble platelet release markers (Txb2, PF4, BTF, scD40L)

*Platelet activation based assays*

**Table 1.** Platelet function tests.

*Shear-based assays*

Novel techniques continue to emerge and develop the knowledge surrounding the platelet function regulation. The modern "omics" revolution enables simultaneous quantification of hundreds of molecules (e.g. protein or mRNA) from a single sample and their signatures may be reflective of platelet function changes. The amalgamation of transcriptomic and proteomic data and subsequent bioinformatic analysis will lead to a more complete characterisation of platelet function in response to environmental stimuli [106, 107]. Epigenetics and its ancillary elements, including platelet secreted microvesicles (MVs), and microRNA (miRNA), and regulation of the platelet mitochondrial genome are new avenues of investigation and testing in platelet research [108–110].

#### **Acknowledgements**

This work was supported through the Irish Higher Education Authority Programme for Research in Third Level Institutes (HEA-PRTLI Cycle 4: T3 Targeted Therapeutics & Theranostics), the Health Research Board of Ireland (HRB-RP/2005/184, HRA/2009/122, HRA/2009/122/R), Science Foundation Ireland (SFI-04/BR/B0577, 11/TIDA/B1927) to Dr. R.P. Murphy, and the International Visitor Award, Dublin City University, Dublin, Ireland to Drs. B. Degryse and R.P. Murphy. R.P. Murphy is a management committee member of the EU H2020 COST action-CM1406, EpiChemBio. The ground based protocol and studies of microgravity (3-Day Dry Immersion) were undertaken in MEDES, The Institute for Space Medicine and Physiology, *Toulouse* University Hospital, France and was supported and funded by the Centre National d'Etudes Spatiales (*CNES*—National Centre for Space Studies) to M-A.C., R.P.M., N.N., L.T.

[2] McNeal CJ, Dajani T, Wilson D, Cassidy-Bushrow AE, Dickerson JB, Ory M. Hypercholesterolemia in youth: Opportunities and obstacles to prevent premature atheroscle-

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### **Conflict of interest**

We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.

#### **Author details**

Laura Twomey<sup>1</sup> , Robert G. Wallace<sup>1</sup> , Philip M. Cummins<sup>2</sup> , Bernard Degryse1 , Sinead Sheridan<sup>3</sup> , Michael Harrison<sup>4</sup> , Niall Moyna<sup>1</sup> , Gerardene Meade-Murphy<sup>5</sup> , Nastassia Navasiolava<sup>6</sup> , Marc-Antoine Custaud<sup>6</sup> and Ronan P. Murphy<sup>1</sup> \*

\*Address all correspondence to: ronan.murphy@dcu.ie

1 School of Health and Human Performance, Dublin City University, Dublin, Ireland

2 School of Biotechnology, Dublin City University, Dublin, Ireland

3 School of Public Health, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong

4 School of Health Sciences—Health Sport and Exercise Science, Waterford Institute of Technology, Ireland

5 Department of Pharmacology and Therapeutics, University College Cork, Cork, Ireland

6 Centre de Recherche Clinique du CHU d'Angers, Angers, France

#### **References**

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[2] McNeal CJ, Dajani T, Wilson D, Cassidy-Bushrow AE, Dickerson JB, Ory M. Hypercholesterolemia in youth: Opportunities and obstacles to prevent premature atherosclerotic cardiovascular disease. Current Atherosclerosis Reports. 2010;**12**(1):20-28

Science Foundation Ireland (SFI-04/BR/B0577, 11/TIDA/B1927) to Dr. R.P. Murphy, and the International Visitor Award, Dublin City University, Dublin, Ireland to Drs. B. Degryse and R.P. Murphy. R.P. Murphy is a management committee member of the EU H2020 COST action-CM1406, EpiChemBio. The ground based protocol and studies of microgravity (3-Day Dry Immersion) were undertaken in MEDES, The Institute for Space Medicine and Physiology, *Toulouse* University Hospital, France and was supported and funded by the Centre National d'Etudes Spatiales (*CNES*—National Centre for Space Studies) to M-A.C., R.P.M., N.N., L.T.

We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have

, Philip M. Cummins<sup>2</sup>

, Niall Moyna<sup>1</sup>

1 School of Health and Human Performance, Dublin City University, Dublin, Ireland

3 School of Public Health, Li Ka Shing Faculty of Medicine, The University of Hong Kong,

4 School of Health Sciences—Health Sport and Exercise Science, Waterford Institute of

5 Department of Pharmacology and Therapeutics, University College Cork, Cork, Ireland

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, Bernard Degryse1

, Gerardene Meade-Murphy<sup>5</sup>

and Ronan P. Murphy<sup>1</sup>

,

\*

,

**Conflict of interest**

84 Homeostasis - An Integrated Vision

influenced its outcome.

, Robert G. Wallace<sup>1</sup>

, Michael Harrison<sup>4</sup>

\*Address all correspondence to: ronan.murphy@dcu.ie

**Author details**

Laura Twomey<sup>1</sup>

Sinead Sheridan<sup>3</sup>

Hong Kong

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**Chapter 6**

**Provisional chapter**

**Platelets: Functional Biomarkers of Epigenetic Drift**

Cardiovascular disease (CVD) risk factors can be classed as modifiable or non-modifiable. Physical inactivity and obesity represent major behavioural risk factors for the initiation, development and progression of CVD. Platelet dysfunction is pivotal to the aetiology of CVD, a chronic vascular inflammatory condition, which is characterised by a lag time between onset and clinical manifestation. This indicates the role of epigenetic drift, defined by stochastic patterns of gene expression not dependent on dynamic changes in coding DNA. The epigenome, a collection of chemical marks on DNA and histones, is established during embryogenesis and modified by age and lifestyle. Biogenesis and effector function of non-coding RNA, such as microRNA, play a regulatory role in gene expression and thus the epigenetic mechanism. In this chapter, we will focus on the effect of the modifiable risk factors of physical activity/inactivity and overweight/obesity on platelet function, via epigenetic changes in both *megakaryocytopoiesis* and *thrombopoiesis*. We will also discuss the role of acute exercise on platelet function and the impact of cardiorespiratory fitness (CRF) on platelet responses to acute exercise. This chapter will highlight the potential role of platelets as circulating functional biomarkers of epigenetic drift to implement, optimise

**Keywords:** platelets, epigenetics, microRNA, lifestyle, physical activity, physical

**Platelets: Functional Biomarkers of Epigenetic Drift**

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

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

DOI: 10.5772/intechopen.83447

Laura Twomey, Robert G. Wallace, Marco Mangone,

Gerardene Meade-Murphy, Nastassia Navasiolava,

and monitor CVD preventive management strategies.

inactivity, cardiovascular disease, preventive medicine

Sinead Sheridan, Michael Harrison, Niall Moyna, Gerardene Meade-Murphy, Nastassia Navasiolava,

Marc-Antoine Custaud and Ronan P. Murphy

Marc-Antoine Custaud and Ronan P. Murphy

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

Bernard Degryse, Sinead Sheridan, Michael Harrison, Niall Moyna,

Laura Twomey, Robert G. Wallace, Marco Mangone, Bernard Degryse,

http://dx.doi.org/10.5772/intechopen.83447

**Abstract**


#### **Platelets: Functional Biomarkers of Epigenetic Drift Platelets: Functional Biomarkers of Epigenetic Drift**

DOI: 10.5772/intechopen.83447

Laura Twomey, Robert G. Wallace, Marco Mangone, Bernard Degryse, Sinead Sheridan, Michael Harrison, Niall Moyna, Gerardene Meade-Murphy, Nastassia Navasiolava, Marc-Antoine Custaud and Ronan P. Murphy Laura Twomey, Robert G. Wallace, Marco Mangone, Bernard Degryse, Sinead Sheridan, Michael Harrison, Niall Moyna, Gerardene Meade-Murphy, Nastassia Navasiolava, Marc-Antoine Custaud and Ronan P. Murphy

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

http://dx.doi.org/10.5772/intechopen.83447

#### **Abstract**

[101] Rechcinski T, Jasinska A, Forys J, Krzeminska-Pakula M, Wierzbowska-Drabik K, Plewka M, et al. Prognostic value of platelet indices after acute myocardial infarction treated with primary percutaneous coronary intervention. Cardiology Journal.

[102] Gul M, Uyarel H, Akgul O, Akkaya E, Surgit O, Cakmak HA, et al. Long-term prognostic significance of admission plateletcrit values in patients with non-ST elevation

[103] Ho CH. White blood cell and platelet counts could affect whole blood viscosity. Journal

[104] Cetin M, Bakirci EM, Baysal E, Tasolar H, Balli M, Cakici M, et al. Increased platelet distribution width is associated with ST-segment elevation myocardial infarction and

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[106] McRedmond JP, Park SD, Reilly DF, Coppinger JA, Maguire PB, Shields DC, et al. Integration of proteomics and genomics in platelets: A profile of platelet proteins and

[107] Ferroni P, Riondino S, Vazzana N, Santoro N, Guadagni F, Davi G. Biomarkers of platelet activation in acute coronary syndromes. Thrombosis and Haemostasis.

[108] Baccarelli AA, Byun HM. Platelet mitochondrial DNA methylation: A potential new

[109] Kaudewitz D, Skroblin P, Bender LH, Barwari T, Willeit P, Pechlaner R, et al. Association of MicroRNAs and YRNAs with platelet function. Circulation Research.

[110] Dahiya N, Sarachana T, Kulkarni S, Wood WHIII, Zhang Y, Becker KG, et al. miR-570 interacts with mitochondrial ATPase subunit g (ATP5L) encoding mRNA in stored

platelet-specific genes. Molecular & Cellular Proteomics. 2004;**3**(2):133-144

marker of cardiovascular disease. Clinical Epigenetics. 2015;**7**:44

myocardial infarction. Blood Coagulation & Fibrinolysis. 2016;**27**(6):696-701

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thrombolysis failure. Angiology. 2014;**65**(8):737-743

2013;**20**(5):491-498

92 Homeostasis - An Integrated Vision

2012;**108**(6):1109-1123

2016;**118**(3):420-432

platelets. Platelets. 2017;**28**(1):74-81

Cardiovascular disease (CVD) risk factors can be classed as modifiable or non-modifiable. Physical inactivity and obesity represent major behavioural risk factors for the initiation, development and progression of CVD. Platelet dysfunction is pivotal to the aetiology of CVD, a chronic vascular inflammatory condition, which is characterised by a lag time between onset and clinical manifestation. This indicates the role of epigenetic drift, defined by stochastic patterns of gene expression not dependent on dynamic changes in coding DNA. The epigenome, a collection of chemical marks on DNA and histones, is established during embryogenesis and modified by age and lifestyle. Biogenesis and effector function of non-coding RNA, such as microRNA, play a regulatory role in gene expression and thus the epigenetic mechanism. In this chapter, we will focus on the effect of the modifiable risk factors of physical activity/inactivity and overweight/obesity on platelet function, via epigenetic changes in both *megakaryocytopoiesis* and *thrombopoiesis*. We will also discuss the role of acute exercise on platelet function and the impact of cardiorespiratory fitness (CRF) on platelet responses to acute exercise. This chapter will highlight the potential role of platelets as circulating functional biomarkers of epigenetic drift to implement, optimise and monitor CVD preventive management strategies.

**Keywords:** platelets, epigenetics, microRNA, lifestyle, physical activity, physical inactivity, cardiovascular disease, preventive medicine

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

#### **1. Introduction**

#### **1.1. Epigenetics**

Epigenetics describes modifiable changes that occur to genes, *via* chemical modifications and/or varying states of chromatin organisation and structure, which alter the gene expression without altering the DNA sequence itself. Smoking habits, obesity, ageing and physical fitness among others are examples of environmental factors that have been suggested to have a long-term influence on epigenetic changes [1]. Epigenetics may be classed as three distinct but highly interconnected processes; DNA methylation, histone modification and RNA-associated silencing (**Figure 1**). DNA methylation and histone modification alter DNA accessibility for transcriptional machinery and chromatin structure. These changes are heritable and can be passed down between generations through either mitosis or meiosis. DNA methylation involves the addition of a methyl group to the 5-position of cytosine by DNA methyl-transferases, at areas known as CpG islands. Methyl groups control gene expression by binding to promoter sites of the gene. This changes the affinity of methylation-sensitive binding proteins, and is associated with transcriptional gene silencing [2]. Whilst required for normal development, changes in DNA methylation have been linked to CVD conditions such as atherosclerosis. For example, the athero-protective oestrogen receptor genes ESR1 and ESR2, usually expressed in SMCs, are hyper-methylated in atherosclerosis [3, 4].

Unlike the platelet transcriptome and proteome, the investigation of epigenetic processes is an almost completely unexplored area in platelet biology, as analysis of these mechanisms requires DNA [2]. Although anucleate, platelets have functionally active mitochondria, with mitochondrial DNA (mtDNA) that can also be methylated, moderating the control of mitochondrial gene expression. Interestingly, Zhong and colleagues recently reported that *de novo* DNA synthesis in mitochondria, and its subsequent oxidation, plays a key role in triggering the innate immune response. Mitochondria can regulate how immune cells respond to infection and tissue damage, producing pro- or anti-inflammatory signals by regulating Krebs cycle metabolites or the production of reactive oxygen species (ROS). More and more examples are being found of mitochondrial functions being repurposed in unexpected ways to contribute to many biological processes, including inflammatory signalling [5].

Understanding epigenetic regulation of mitochondrial genes in platelets is proving crucial to understanding their implication in CVD development. Novel research by Baccarelli and Byun showed that CVD patients had significantly higher platelet mtDNA methylation than healthy individuals in MT-CO1, MT-C02, MT-CO3 and MT-TL1 genes involved in ATP synthesis [6]. These results suggest that DNA methylation in platelet mitochondria could be a potential contributor to CVD development through the regulation of platelet function.

deacetyltransferases (HDACs). This mechanism alters chromatin structure (heterochromatin

**Figure 1.** Modifiable risk factors covalently alter the static genome by processes involving epigenetic writers, erasers and readers, which fine-tune gene expression in an age and lifestyle dependent manner, a process known as *epigenetic drift*. The term epigenetics refers to stable patterns of gene expression and they are not dependent on dynamic changes in coding DNA. These gene expression states are encoded in the epigenome—a collection of chemical marks on DNA or on histones that are established during embryogenesis and are modified by age and environment over a person's lifetime. Studies have uncovered stochastic DNA methylation drift that reflects imperfect maintenance of epigenetic marks. We hypothesise that *drift* creates epigenetic mosaicism in ageing haemopoietic stem cells in the bone marrow. This in turn impacts on HSC differentiation, maturation and platelet production. This may highlight platelets as a functional diagnostic index of cardiovascular competence. The fact that the initiation and progression of CVD is characterised by a lag time between onset and clinical manifestation provides a window of opportunity for the implementation of intervention strategies to reduce the CVD burden. In addition, such studies will better inform primary, secondary and tertiary preventive strategies—promoting an ageing well paradigm and optimising a person's disease free years.

Platelets: Functional Biomarkers of Epigenetic Drift http://dx.doi.org/10.5772/intechopen.83447 95

RNA-based epigenetic processes involve non-coding RNA (ncRNA) and are factors in the chromatin-based regulation of gene expression [8]. ncRNAs can be classified as either long or short. Whilst long ncRNAs are a major form of RNA-based epigenetic regulation, some small ncRNAs also have a function in chromatin-based silencing. For example, microRNA (miRNA) is a subset of ncRNA that negatively regulates gene transcription by degrading

versus euchromatin) to influence gene expression [3].

Histone, proteins that structure DNA into units known as nucleosomes, can be modified at their amino-acid tails. Histone modifications refer to the post-translational alterations of the N-termini of these tails that subsequently modify histone-DNA interactions [7]. Acetylation is a major type of histone modification involving the addition or removal of an acetyl group. This process is catalysed by proteins known as histone acetyltransferases (HATs) and histone

**1. Introduction**

94 Homeostasis - An Integrated Vision

**1.1. Epigenetics**

Epigenetics describes modifiable changes that occur to genes, *via* chemical modifications and/or varying states of chromatin organisation and structure, which alter the gene expression without altering the DNA sequence itself. Smoking habits, obesity, ageing and physical fitness among others are examples of environmental factors that have been suggested to have a long-term influence on epigenetic changes [1]. Epigenetics may be classed as three distinct but highly interconnected processes; DNA methylation, histone modification and RNA-associated silencing (**Figure 1**). DNA methylation and histone modification alter DNA accessibility for transcriptional machinery and chromatin structure. These changes are heritable and can be passed down between generations through either mitosis or meiosis. DNA methylation involves the addition of a methyl group to the 5-position of cytosine by DNA methyl-transferases, at areas known as CpG islands. Methyl groups control gene expression by binding to promoter sites of the gene. This changes the affinity of methylation-sensitive binding proteins, and is associated with transcriptional gene silencing [2]. Whilst required for normal development, changes in DNA methylation have been linked to CVD conditions such as atherosclerosis. For example, the athero-protective oestrogen receptor genes ESR1

and ESR2, usually expressed in SMCs, are hyper-methylated in atherosclerosis [3, 4].

to contribute to many biological processes, including inflammatory signalling [5].

contributor to CVD development through the regulation of platelet function.

Understanding epigenetic regulation of mitochondrial genes in platelets is proving crucial to understanding their implication in CVD development. Novel research by Baccarelli and Byun showed that CVD patients had significantly higher platelet mtDNA methylation than healthy individuals in MT-CO1, MT-C02, MT-CO3 and MT-TL1 genes involved in ATP synthesis [6]. These results suggest that DNA methylation in platelet mitochondria could be a potential

Histone, proteins that structure DNA into units known as nucleosomes, can be modified at their amino-acid tails. Histone modifications refer to the post-translational alterations of the N-termini of these tails that subsequently modify histone-DNA interactions [7]. Acetylation is a major type of histone modification involving the addition or removal of an acetyl group. This process is catalysed by proteins known as histone acetyltransferases (HATs) and histone

Unlike the platelet transcriptome and proteome, the investigation of epigenetic processes is an almost completely unexplored area in platelet biology, as analysis of these mechanisms requires DNA [2]. Although anucleate, platelets have functionally active mitochondria, with mitochondrial DNA (mtDNA) that can also be methylated, moderating the control of mitochondrial gene expression. Interestingly, Zhong and colleagues recently reported that *de novo* DNA synthesis in mitochondria, and its subsequent oxidation, plays a key role in triggering the innate immune response. Mitochondria can regulate how immune cells respond to infection and tissue damage, producing pro- or anti-inflammatory signals by regulating Krebs cycle metabolites or the production of reactive oxygen species (ROS). More and more examples are being found of mitochondrial functions being repurposed in unexpected ways

**Figure 1.** Modifiable risk factors covalently alter the static genome by processes involving epigenetic writers, erasers and readers, which fine-tune gene expression in an age and lifestyle dependent manner, a process known as *epigenetic drift*. The term epigenetics refers to stable patterns of gene expression and they are not dependent on dynamic changes in coding DNA. These gene expression states are encoded in the epigenome—a collection of chemical marks on DNA or on histones that are established during embryogenesis and are modified by age and environment over a person's lifetime. Studies have uncovered stochastic DNA methylation drift that reflects imperfect maintenance of epigenetic marks. We hypothesise that *drift* creates epigenetic mosaicism in ageing haemopoietic stem cells in the bone marrow. This in turn impacts on HSC differentiation, maturation and platelet production. This may highlight platelets as a functional diagnostic index of cardiovascular competence. The fact that the initiation and progression of CVD is characterised by a lag time between onset and clinical manifestation provides a window of opportunity for the implementation of intervention strategies to reduce the CVD burden. In addition, such studies will better inform primary, secondary and tertiary preventive strategies—promoting an ageing well paradigm and optimising a person's disease free years.

deacetyltransferases (HDACs). This mechanism alters chromatin structure (heterochromatin versus euchromatin) to influence gene expression [3].

RNA-based epigenetic processes involve non-coding RNA (ncRNA) and are factors in the chromatin-based regulation of gene expression [8]. ncRNAs can be classified as either long or short. Whilst long ncRNAs are a major form of RNA-based epigenetic regulation, some small ncRNAs also have a function in chromatin-based silencing. For example, microRNA (miRNA) is a subset of ncRNA that negatively regulates gene transcription by degrading or repressing target mRNA [9]. miRNA can control the expression of important epigenetic regulators such as histone deacetylases and DNA methyl-transferases and similarly, DNA methylation and histone modification can control the expression of some miRNA, thereby forming a feedback loop [10]. This complex crosstalk between miRNA and epigenetic pathways forms an epigenetic-miRNA regulatory circuit, arranging the whole gene expression profile. Disruption of this circuit interferes with normal physiological functions and can contribute to disease process.

Individuals age differently and lifestyle factors such as exercise or smoking have been shown to delay or accelerate the ageing process, respectively [11]. These observations have resulted in the search for molecular markers to predict and monitor age-associated disease. DNA methylation is associated with chronological age over time [12]. *Epigenetic drift* is the term given to epigenetic modifications as they occur as a direct consequence of age [13]. This was previously observed when DNA methylation marks in identical twins differed increasingly as a function of age [1]. Monozygous twins share a common genotype and while this study found that the twins were epigenetically synonymous during childhood, older twins showed significant differences in their total content and dispersal of histone acetylation and DNA methylation. Disparity in these epigenetic marks between twins may be as a result of lifestyle influences such as diet, physical activity levels, stress and smoking.

*Epigenetic drift* affects the majority of the genome over time leading to biological ageing (**Figure 2**). Ageing is a natural process associated with the de-regulation of histone tags, senescence-associated lncRNA, a gradual de-regulation of DNA methylation, in a potential linear fashion depicted by age-predictive linear models [14]. However, an individual exposed to either environmental or genetic risk factors may show signs of premature ageing as a result of either lifestyle or environmental risk factors. De-regulation of DNA methylation can increase the susceptibility to chronic diseases like CVD. Furthermore, it has been hypothesised that a healthy lifestyle may reserve a more intact epigenome, promoting longevity [15]. In a recent compelling study by Horvath and colleagues, a novel, sensitive and highly robust DNAm age estimator (based on 391 CpGs) was developed for human fibroblasts, keratinocytes, buccal cells, endothelial cells, lymphoblastoid cells, skin, blood and saliva samples [16]. This seminal research builds upon and overcomes the limitations of the two ground-breaking studies on the epigenetic clock (biological/molecular age) and its relationship with chronological age. These studies, one a blood-based age estimator [14] and the other a pan-tissue estimator [17], facilitated age estimates (DNAmAge) that are widely used in epidemiological studies. The novel 'skin and blood clock' overcomes the technical and sensitivity limitations of the previous DNAmAge biomarker panels.

determining PLT or changes in histone acetylation with aging have been suggested to play an

**Figure 2.** Schematic representation of ageing well versus accelerated ageing. Poor lifestyle choices such as inactivity and diet are rapidly becoming a global pandemic, accelerating many chronic illnesses such as cardiovascular disease, metabolic disorder, diabetes, Alzheimer's disease and cancer. Understanding their pathophysiology is important for the development of future therapeutic interventions, stratification of clinical trials and to challenge our current perception of engaging with cost effective measures such as lifestyle management. The aetiology of age-related chronic illnesses involves a complex interplay between many biological processes and is modulated by non-modifiable and modifiable risk factors.

Platelets: Functional Biomarkers of Epigenetic Drift http://dx.doi.org/10.5772/intechopen.83447 97

miRNAs are short [18–24] nucleotide long, non-coding RNAs that function in post-transcriptional regulation of gene expression. They inhibit translation by binding with the 3′-untranslated (UTR) regions of their target mRNA. Here, the miRNA promotes silencing of various genes [21, 22], hence now termed 'fine-tuners' of cellular phenotypes. They are thought to be involved in the regulation of ~60% of human genes [23, 24]. miRNA can be classed as intronic, exonic and intergenic miRNA, according to the location of their encoding genes [25]. Intronic miRNAs account for approximately 70% of all transcribed miRNA [26, 27]. Intergenic miRNAs are found between two protein-coding genes and employ their own promoters and regulatory molecules. As miRNAs target mRNA by imperfect binding, each miRNA has multiple targets, enabling miRNAs to regulate over half of the human genome [28]. The miRNA population within a cell can be highly concentrated, with tens of thousands of miRNA copies per cell.

important role in platelet function [20].

DNA methylation undergoes extensive changes during differentiation of self-renewing stem cells [18, 19]. Indeed, DNA methylation is involved in the production of MKs and subsequent transcription. Lifestyle components such as physical inactivity and obesity may incur epigenetic changes in the production of platelets from megakaryocytes. Thus, platelets could signify a marker of megakaryocyte epigenetic drift, holding substantial predictive potential of disease. Epigenetic changes in the megakaryocyte genome such as hypomethylation of genes

or repressing target mRNA [9]. miRNA can control the expression of important epigenetic regulators such as histone deacetylases and DNA methyl-transferases and similarly, DNA methylation and histone modification can control the expression of some miRNA, thereby forming a feedback loop [10]. This complex crosstalk between miRNA and epigenetic pathways forms an epigenetic-miRNA regulatory circuit, arranging the whole gene expression profile. Disruption of this circuit interferes with normal physiological functions and can con-

Individuals age differently and lifestyle factors such as exercise or smoking have been shown to delay or accelerate the ageing process, respectively [11]. These observations have resulted in the search for molecular markers to predict and monitor age-associated disease. DNA methylation is associated with chronological age over time [12]. *Epigenetic drift* is the term given to epigenetic modifications as they occur as a direct consequence of age [13]. This was previously observed when DNA methylation marks in identical twins differed increasingly as a function of age [1]. Monozygous twins share a common genotype and while this study found that the twins were epigenetically synonymous during childhood, older twins showed significant differences in their total content and dispersal of histone acetylation and DNA methylation. Disparity in these epigenetic marks between twins may be as a result of lifestyle

*Epigenetic drift* affects the majority of the genome over time leading to biological ageing (**Figure 2**). Ageing is a natural process associated with the de-regulation of histone tags, senescence-associated lncRNA, a gradual de-regulation of DNA methylation, in a potential linear fashion depicted by age-predictive linear models [14]. However, an individual exposed to either environmental or genetic risk factors may show signs of premature ageing as a result of either lifestyle or environmental risk factors. De-regulation of DNA methylation can increase the susceptibility to chronic diseases like CVD. Furthermore, it has been hypothesised that a healthy lifestyle may reserve a more intact epigenome, promoting longevity [15]. In a recent compelling study by Horvath and colleagues, a novel, sensitive and highly robust DNAm age estimator (based on 391 CpGs) was developed for human fibroblasts, keratinocytes, buccal cells, endothelial cells, lymphoblastoid cells, skin, blood and saliva samples [16]. This seminal research builds upon and overcomes the limitations of the two ground-breaking studies on the epigenetic clock (biological/molecular age) and its relationship with chronological age. These studies, one a blood-based age estimator [14] and the other a pan-tissue estimator [17], facilitated age estimates (DNAmAge) that are widely used in epidemiological studies. The novel 'skin and blood clock' overcomes the technical and sensitivity limitations

DNA methylation undergoes extensive changes during differentiation of self-renewing stem cells [18, 19]. Indeed, DNA methylation is involved in the production of MKs and subsequent transcription. Lifestyle components such as physical inactivity and obesity may incur epigenetic changes in the production of platelets from megakaryocytes. Thus, platelets could signify a marker of megakaryocyte epigenetic drift, holding substantial predictive potential of disease. Epigenetic changes in the megakaryocyte genome such as hypomethylation of genes

influences such as diet, physical activity levels, stress and smoking.

of the previous DNAmAge biomarker panels.

tribute to disease process.

96 Homeostasis - An Integrated Vision

**Figure 2.** Schematic representation of ageing well versus accelerated ageing. Poor lifestyle choices such as inactivity and diet are rapidly becoming a global pandemic, accelerating many chronic illnesses such as cardiovascular disease, metabolic disorder, diabetes, Alzheimer's disease and cancer. Understanding their pathophysiology is important for the development of future therapeutic interventions, stratification of clinical trials and to challenge our current perception of engaging with cost effective measures such as lifestyle management. The aetiology of age-related chronic illnesses involves a complex interplay between many biological processes and is modulated by non-modifiable and modifiable risk factors.

determining PLT or changes in histone acetylation with aging have been suggested to play an important role in platelet function [20].

miRNAs are short [18–24] nucleotide long, non-coding RNAs that function in post-transcriptional regulation of gene expression. They inhibit translation by binding with the 3′-untranslated (UTR) regions of their target mRNA. Here, the miRNA promotes silencing of various genes [21, 22], hence now termed 'fine-tuners' of cellular phenotypes. They are thought to be involved in the regulation of ~60% of human genes [23, 24]. miRNA can be classed as intronic, exonic and intergenic miRNA, according to the location of their encoding genes [25]. Intronic miRNAs account for approximately 70% of all transcribed miRNA [26, 27]. Intergenic miRNAs are found between two protein-coding genes and employ their own promoters and regulatory molecules. As miRNAs target mRNA by imperfect binding, each miRNA has multiple targets, enabling miRNAs to regulate over half of the human genome [28]. The miRNA population within a cell can be highly concentrated, with tens of thousands of miRNA copies per cell. They possess a long half-life (a half-life of between 28 and 220 h has been reported) and are very stable [29]. Turnover of mature miRNA is required for rapid changes in miRNA expression profiles. Regulation of miRNA maturation occurs during various steps throughout their biogenesis at both a transcriptional and post-transcriptional level [30]. Transcriptional regulation involves alterations to the expression of a host gene such as epigenetic regulation (where miRNA genes located near CpG islands in the genome are found to be hyper-methylated). Post-transcriptional mechanisms define modifications in miRNA processing and stability [31].

for miRNA in the regulation of platelet function. Most notably, research by Nagalla et al. [38], who focused on the roles of miRNA as biomarkers of platelet reactivity and controllers of platelet mRNA disparity, demonstrated that miRNA profiles of healthy subjects (n = 19) were associated with the response of platelet aggregation to epinephrine [38]. They also employed a computational approach to produce possible miRNA-mRNA pairs (miR-200b: PRKAR2B, miR-495: KLHL5 and miR-107: CLOCK), pairings which were experimentally validated in cell lines. Networks of miRNA-mRNA pairs also associated with age, gender and race [39, 40]. Other reports on agonist-induced platelet activation by thrombin (and ADP) show differential

Platelets: Functional Biomarkers of Epigenetic Drift http://dx.doi.org/10.5772/intechopen.83447 99

Progression in miRNA detection techniques has led to the revelation of 40 new miRNA sequences, expanding the total amount of platelet expressed miRNAs to more than twice that (544) of the initial finding [23, 35, 42]. Transcriptomic approaches show that miRNA make up the majority (80%) of all small RNAs in platelets. Furthermore, comparison of RNA and miRNA by cell type showed that despite low RNA yields, platelets express high quantities of

A number of highly expressed miRNA have been characterised in human platelets, some of which are involved in myeloid cell differentiation, megakaryocytopoiesis and thrombopoiesis. miR-223 has been identified as the most highly expressed platelet miRNA [38, 43, 44] and has roles in thrombopoiesis and megakaryocyte differentiation [24]. miR-223 regulates ADP P2Y12, a target for existing anti-platelet drug therapy. The 3′-UTR of P2Y12 mRNA has been identified as complementary to the miR-223 seed region. Platelet miR-223 has also been observed to be decreased in subjects who show high levels of platelet activation whilst on clopidogrel therapy. Furthermore, miR-223-deficient mice show reduced bleeding times, larger thrombi and elevated sensitivity to low doses of thrombin, suggesting an important role of miR-223 in modulating platelet function [45]. miR-126 plays central roles in vascular inflammation and is thought to be the second most highly expressed miRNA in platelets [46]. miR-126 was found to correlate with circulating P-Selectin levels in T2DM subjects and this level was sensitive to aspirin treatment, signifying a platelet origin. miR-126 is postulated to regulate ADAM9 and P2Y12 receptor expression in platelets and inhibition of miR-126 in mice

Existence of miRNA in platelets is multifaceted. Besides their obvious function as regulators of platelet protein expression, platelet miRNAs have been labelled as biomarkers of disease and platelet activation, markers of mature megakaryocyte miRNA and as a means of understanding megakaryocyte/platelet gene expression [48]. The majority of platelet miRNA are supposedly formed in the megakaryocyte and packaged into platelets upon formation. For example, miR-146b positively regulates megakaryopoieses by targeting and down regulating the megakaryopoieses inhibitor PDGFRA [49, 50]. miR-142 has also been reported to inhibit megakaryocyte production. In miR-142 knockout mice, platelet counts are decreased and MK differentiation is modified, including reduced proplatelet network establishment [51]. The total extent to which MK and platelet mature miRNA patterns correlate remains an area of active investigation. A significant correlation between the miRNA levels was found using three separate studies [34, 52, 53].

expression of platelet miRNA compared to resting platelets [41].

miRNA compared to their nucleated counterparts.

distinctly reduces platelet aggregation [47].

#### **2. Platelet Epigenetics**

#### **2.1. Platelet miRNA**

Platelet function is a highly regulated process. Despite their anucleate nature, platelets accommodate a small but competent transcriptome that is employed for translation of various proteins with significant physiological functions. Platelets have been shown to retain genetic material derived from their megakaryocyte precursor. Approximately 32% of all human genes are present in platelets at the mRNA level [23]. It is well accepted that platelets contain the necessary splicing machinery, rough ER and polyribosomes that allow the synthesis of proteins required for their functioning [32]. Perhaps due to the requirement of sustaining a proteome over an ~8-day life span, the fact that the average half-life of a cellular protein is 46 h, or the necessity to adapt to environmental stimuli, it is equitable to assume that the platelet must also retain its transcriptome, as well as processes of nucleated cells such as splicing, translating and post-transcriptional RNA mechanisms. The fact that platelets contain mRNA and are capable of protein synthesis has raised the issue of how these mRNAs are regulated. Notably, stored platelets in blood banks can synthesis integrin β3 [33]. The existence and functionality of a miRNA pathway in the anucleate human platelet was first described in a landmark study by Landry and co-workers [34], who showed by locked nucleic acid (LNA) microarray profiling, that platelets harboured an impressive number (219) of miRNA. Further analysis discovered the presence of functional processing miRNA machinery in platelets—Dicer and Ago2 suggesting that partial biogenesis of mature miRNA from pre-miRNA could occur within platelets themselves [34, 35] as pre-miRNAs have been identified at low levels (21 transcripts) in platelets [23]. Star or passenger strand miRNA have also been identified [36]. Accordingly, the detection of nuclear miRNA microprocessor Drosha and DGCR8 in platelets has not been observed, consistent with their anucleate nature. Moreover, miRNA-associated Ago2 complexes were identified, in addition to the presence of P2Y12 in Ago2 precipitates, suggesting a regulation of P2Y12 by miRNAs [34].

The next breakthrough study in platelet miRNA biology revealed that a protein involved in platelet granule release, platelet vesicle-associated membrane protein 8 (VAMP8), was associated with distinctly different platelet aggregation responses to epinephrine in healthy donors, and that VAMP8 was regulated by miR-86 [37]. Since then, the platelet miRNA field has grown exponentially, whereby a number of studies have suggested a physiological role for miRNA in the regulation of platelet function. Most notably, research by Nagalla et al. [38], who focused on the roles of miRNA as biomarkers of platelet reactivity and controllers of platelet mRNA disparity, demonstrated that miRNA profiles of healthy subjects (n = 19) were associated with the response of platelet aggregation to epinephrine [38]. They also employed a computational approach to produce possible miRNA-mRNA pairs (miR-200b: PRKAR2B, miR-495: KLHL5 and miR-107: CLOCK), pairings which were experimentally validated in cell lines. Networks of miRNA-mRNA pairs also associated with age, gender and race [39, 40]. Other reports on agonist-induced platelet activation by thrombin (and ADP) show differential expression of platelet miRNA compared to resting platelets [41].

They possess a long half-life (a half-life of between 28 and 220 h has been reported) and are very stable [29]. Turnover of mature miRNA is required for rapid changes in miRNA expression profiles. Regulation of miRNA maturation occurs during various steps throughout their biogenesis at both a transcriptional and post-transcriptional level [30]. Transcriptional regulation involves alterations to the expression of a host gene such as epigenetic regulation (where miRNA genes located near CpG islands in the genome are found to be hyper-methylated). Post-transcriptional mechanisms define modifications in miRNA processing and stability [31].

Platelet function is a highly regulated process. Despite their anucleate nature, platelets accommodate a small but competent transcriptome that is employed for translation of various proteins with significant physiological functions. Platelets have been shown to retain genetic material derived from their megakaryocyte precursor. Approximately 32% of all human genes are present in platelets at the mRNA level [23]. It is well accepted that platelets contain the necessary splicing machinery, rough ER and polyribosomes that allow the synthesis of proteins required for their functioning [32]. Perhaps due to the requirement of sustaining a proteome over an ~8-day life span, the fact that the average half-life of a cellular protein is 46 h, or the necessity to adapt to environmental stimuli, it is equitable to assume that the platelet must also retain its transcriptome, as well as processes of nucleated cells such as splicing, translating and post-transcriptional RNA mechanisms. The fact that platelets contain mRNA and are capable of protein synthesis has raised the issue of how these mRNAs are regulated. Notably, stored platelets in blood banks can synthesis integrin β3 [33]. The existence and functionality of a miRNA pathway in the anucleate human platelet was first described in a landmark study by Landry and co-workers [34], who showed by locked nucleic acid (LNA) microarray profiling, that platelets harboured an impressive number (219) of miRNA. Further analysis discovered the presence of functional processing miRNA machinery in platelets—Dicer and Ago2 suggesting that partial biogenesis of mature miRNA from pre-miRNA could occur within platelets themselves [34, 35] as pre-miRNAs have been identified at low levels (21 transcripts) in platelets [23]. Star or passenger strand miRNA have also been identified [36]. Accordingly, the detection of nuclear miRNA microprocessor Drosha and DGCR8 in platelets has not been observed, consistent with their anucleate nature. Moreover, miRNA-associated Ago2 complexes were identified, in addition to the presence of P2Y12 in Ago2 precipitates, suggesting a

The next breakthrough study in platelet miRNA biology revealed that a protein involved in platelet granule release, platelet vesicle-associated membrane protein 8 (VAMP8), was associated with distinctly different platelet aggregation responses to epinephrine in healthy donors, and that VAMP8 was regulated by miR-86 [37]. Since then, the platelet miRNA field has grown exponentially, whereby a number of studies have suggested a physiological role

**2. Platelet Epigenetics**

98 Homeostasis - An Integrated Vision

regulation of P2Y12 by miRNAs [34].

**2.1. Platelet miRNA**

Progression in miRNA detection techniques has led to the revelation of 40 new miRNA sequences, expanding the total amount of platelet expressed miRNAs to more than twice that (544) of the initial finding [23, 35, 42]. Transcriptomic approaches show that miRNA make up the majority (80%) of all small RNAs in platelets. Furthermore, comparison of RNA and miRNA by cell type showed that despite low RNA yields, platelets express high quantities of miRNA compared to their nucleated counterparts.

A number of highly expressed miRNA have been characterised in human platelets, some of which are involved in myeloid cell differentiation, megakaryocytopoiesis and thrombopoiesis. miR-223 has been identified as the most highly expressed platelet miRNA [38, 43, 44] and has roles in thrombopoiesis and megakaryocyte differentiation [24]. miR-223 regulates ADP P2Y12, a target for existing anti-platelet drug therapy. The 3′-UTR of P2Y12 mRNA has been identified as complementary to the miR-223 seed region. Platelet miR-223 has also been observed to be decreased in subjects who show high levels of platelet activation whilst on clopidogrel therapy. Furthermore, miR-223-deficient mice show reduced bleeding times, larger thrombi and elevated sensitivity to low doses of thrombin, suggesting an important role of miR-223 in modulating platelet function [45]. miR-126 plays central roles in vascular inflammation and is thought to be the second most highly expressed miRNA in platelets [46]. miR-126 was found to correlate with circulating P-Selectin levels in T2DM subjects and this level was sensitive to aspirin treatment, signifying a platelet origin. miR-126 is postulated to regulate ADAM9 and P2Y12 receptor expression in platelets and inhibition of miR-126 in mice distinctly reduces platelet aggregation [47].

Existence of miRNA in platelets is multifaceted. Besides their obvious function as regulators of platelet protein expression, platelet miRNAs have been labelled as biomarkers of disease and platelet activation, markers of mature megakaryocyte miRNA and as a means of understanding megakaryocyte/platelet gene expression [48]. The majority of platelet miRNA are supposedly formed in the megakaryocyte and packaged into platelets upon formation. For example, miR-146b positively regulates megakaryopoieses by targeting and down regulating the megakaryopoieses inhibitor PDGFRA [49, 50]. miR-142 has also been reported to inhibit megakaryocyte production. In miR-142 knockout mice, platelet counts are decreased and MK differentiation is modified, including reduced proplatelet network establishment [51]. The total extent to which MK and platelet mature miRNA patterns correlate remains an area of active investigation. A significant correlation between the miRNA levels was found using three separate studies [34, 52, 53].

Perhaps, the most intriguing feature regarding platelet miRNA is their extracellular function. miRNA can be packaged and delivered to distant cells in the form of platelet microvesicles (PMV) and/or microparticles (PMP), fulfilling novel processes of gene regulation in target cells [54]. Initial studies by Laffont and Gidlöf demonstrated the functionality of platelet miRNA [24, 55]. Functional complexes of miR-223 and Argonaute 2 protein (Ago2) packaged in MVs from activated platelets were found to modulate the expression of targeted endothelial cell endogenous mRNA transcripts FBXW7 and EFNA1. This miR-223/Ago2 complex has also been shown to reduce expression levels of insulin-like growth factor 1 receptor in endothelial cells, and to promote human umbilical vein endothelial cell (HUVEC) apoptosis [56].

[65]. TLR signalling may also play a role in the progression of atherosclerosis by binding of lipopolysaccharides (LPS) to TLR4 on platelets and also mediating platelet-neutrophil

Platelets: Functional Biomarkers of Epigenetic Drift http://dx.doi.org/10.5772/intechopen.83447 101

Direct cell-cell communication through platelet P-Selectin and CD40 ligand (CD40L) encourages inflammatory processes [66]. CD40L is thought to be at the heart of the atherosclerotic process, with 90% of circulating CD40L residing in platelets. CD40L is sent to the platelet surface upon activation, where it can initiate numerous inflammatory processes. The release of CD40L is intrinsically linked to αIIbβ3 as αIIbβ3 antagonists can block the release of sCD40L from activated platelets *in vitro*. Recently, platelet CD40 was shown to mediate the formation of platelet-leukocyte aggregates (stimulates leukocyte activation) and release inflammatory chemokines that activate endothelial cells, supporting atherosclerosis [66]. The significance of P-Selectin in atherosclerosis has been demonstrated in P-Selectin deficient animals that were protected from the disease. The role of platelet P-Selectin was clarified further by Huo et al., who illustrated that the introduction of P-Selectin expressing platelets into ApoE (−/−) mice accelerated atherosclerosis, whereas mice injected with platelets lacking P-Selectin formed

Platelet-derived microparticles released upon activation may further amplify the progression of atherosclerosis through processes of adhesion, coagulation, inflammation and lipid metabolism [68]. Platelets also provide a huge repertoire of additional inflammatory mediators including a vast array of chemokines and cytokines that contribute to the crosstalk of platelets with other inflammatory cells—e.g., endothelial cells, monocytes, neutrophils, dendritic cells and T-cells [66]. The major function of platelets in atherosclerosis is the recruitment of leukocytes through direct receptor-ligand interactions or amplification of leukocyte recruitment through chemokine release. This bidirectional relationship is extremely important as platelets encourage leukocyte differentiation into a pro-adhesive and pro-migratory phenotype, and the leuko-

Following atherosclerotic plaque rupture in severe CVD states, the exposure of thrombogenic substrates to circulating platelets instantly triggers platelet adhesion, activation and aggregation, forming a prothrombotic surface and subsequently encouraging thrombosis, vasoconstriction and vascular occlusion. Activated platelets expose phospholipids on their surface, which also promotes the coagulation cascade and subsequent fibrin production [64]. Given the critical roles of platelets in the pathogenesis of atherosclerosis and the development of acute thrombotic events, anti-platelet therapy has been widely employed in the primary and secondary prevention of CVD. Some of the current anti-platelet therapy drugs include

Aspirin, which irreversibly inhibits cyclooxygenase to subsequently decrease TxA<sup>2</sup>

terase inhibitors (block degradation of cyclic nucleotides) [69, 70].

tion and limit platelet aggregation. Clopidogrel and Prasugrel are examples of P2Y12 receptor antagonists that inhibit the soluble agonist ADP, whilst Tirofiban and Abciximab block αIIbβ3-ligand interactions. Other anti-platelet therapies include thrombin and phosphodies-

Given the impact of miRNA gene regulation, it is unsurprising that the dysregulation of miRNA is implicated in CVD. miRNAs are central players in modulating gene expression of cells/platelets collectively involved in CVD, and mediate inflammation, lipid uptake and cell differentiation

produc-

cytes secrete mediators that reciprocally activate platelets.

interactions.

smaller plaques [67].

Gidlof et al. suggested that platelet miRNA could modulate vascular endothelial inflammatory responses [55]. They described a down regulation of intercellular adhesion molecule 1 (ICAM-1) gene expression in cultured human microvascular endothelial cells after exposure to miR-320b, which is secreted upon platelet activation and reduced in platelet thrombi aspirated from patients with ST-segment elevation myocardial infarction (STEMI). The relevance of this intercellular transfer was further reinforced when Liang et al., showed that plateletreleased miR-223 through platelet MPs can encourage lung cancer cell invasion by targeting the tumour suppressor EPB41L3 [57].

Novel research has shown that platelet MPs containing miRNA can also be internalised by primary human macrophages and deliver functional miR-126-3p. miR-126-3p caused a downregulation in the expression of four predicted mRNA targets of miR-126-3p and a reduction in macrophage cytokine release. This suggests that platelet miRNA-containing MPs can modify the macrophage transcriptome and potentially reprogram their function [58]. Finally, plateletderived exosomes have recently been shown to carry miR-223, miR-339 and miR-21, which can be transferred to SMCs affecting PDGRFβ [13].

#### **2.2. Platelets, lifestyle and miRNA in the aetiology of CVD**

Efforts in coping with CVD require further understanding of its aetiology in order to develop effective management strategies. Epidemiological studies in adults have acknowledged a set of characteristic risk factors that predict the probability of a person developing clinical manifestations of disease [59, 60]. CVD risk factors are classed as modifiable or non-modifiable. Non-modifiable risk factors include age, ethnicity, gender and family history. Modifiable risk factors include hypertension, smoking, diabetes, unhealthy diet, cholesterol, physical inactivity (PI, sedentary lifestyle and low cardiorespiratory fitness) and overweight/obesity. Risk factors for CVD track from childhood into adulthood [61] and are strong predictors of subclinical atherosclerosis in early adulthood. The majority of CVD is caused by modifiable risk factors and up to 80% of CVD may be prevented if risk factors are avoided [62]. Physical inactivity and obesity are primary potent risk factors, both of which can severely impact platelet physiology.

Platelets have central roles in CVD [63] contributing to both early stages of endothelial dysfunction and advanced stages of the plaque rupture [64]. Platelets participate in early stage disease initiation through multiple mechanisms enabling adhesion to dysfunctional endothelium. Activated platelets express high levels of adhesion receptors (e.g., ICAM1, P-Selectin, CD40L) associated with oxidised-LDL (ox-LDL) that contributes to vascular inflammation [65]. TLR signalling may also play a role in the progression of atherosclerosis by binding of lipopolysaccharides (LPS) to TLR4 on platelets and also mediating platelet-neutrophil interactions.

Perhaps, the most intriguing feature regarding platelet miRNA is their extracellular function. miRNA can be packaged and delivered to distant cells in the form of platelet microvesicles (PMV) and/or microparticles (PMP), fulfilling novel processes of gene regulation in target cells [54]. Initial studies by Laffont and Gidlöf demonstrated the functionality of platelet miRNA [24, 55]. Functional complexes of miR-223 and Argonaute 2 protein (Ago2) packaged in MVs from activated platelets were found to modulate the expression of targeted endothelial cell endogenous mRNA transcripts FBXW7 and EFNA1. This miR-223/Ago2 complex has also been shown to reduce expression levels of insulin-like growth factor 1 receptor in endothelial

cells, and to promote human umbilical vein endothelial cell (HUVEC) apoptosis [56].

the tumour suppressor EPB41L3 [57].

100 Homeostasis - An Integrated Vision

can be transferred to SMCs affecting PDGRFβ [13].

**2.2. Platelets, lifestyle and miRNA in the aetiology of CVD**

Gidlof et al. suggested that platelet miRNA could modulate vascular endothelial inflammatory responses [55]. They described a down regulation of intercellular adhesion molecule 1 (ICAM-1) gene expression in cultured human microvascular endothelial cells after exposure to miR-320b, which is secreted upon platelet activation and reduced in platelet thrombi aspirated from patients with ST-segment elevation myocardial infarction (STEMI). The relevance of this intercellular transfer was further reinforced when Liang et al., showed that plateletreleased miR-223 through platelet MPs can encourage lung cancer cell invasion by targeting

Novel research has shown that platelet MPs containing miRNA can also be internalised by primary human macrophages and deliver functional miR-126-3p. miR-126-3p caused a downregulation in the expression of four predicted mRNA targets of miR-126-3p and a reduction in macrophage cytokine release. This suggests that platelet miRNA-containing MPs can modify the macrophage transcriptome and potentially reprogram their function [58]. Finally, plateletderived exosomes have recently been shown to carry miR-223, miR-339 and miR-21, which

Efforts in coping with CVD require further understanding of its aetiology in order to develop effective management strategies. Epidemiological studies in adults have acknowledged a set of characteristic risk factors that predict the probability of a person developing clinical manifestations of disease [59, 60]. CVD risk factors are classed as modifiable or non-modifiable. Non-modifiable risk factors include age, ethnicity, gender and family history. Modifiable risk factors include hypertension, smoking, diabetes, unhealthy diet, cholesterol, physical inactivity (PI, sedentary lifestyle and low cardiorespiratory fitness) and overweight/obesity. Risk factors for CVD track from childhood into adulthood [61] and are strong predictors of subclinical atherosclerosis in early adulthood. The majority of CVD is caused by modifiable risk factors and up to 80% of CVD may be prevented if risk factors are avoided [62]. Physical inactivity and obesity are primary potent risk factors, both of which can severely impact platelet physiology. Platelets have central roles in CVD [63] contributing to both early stages of endothelial dysfunction and advanced stages of the plaque rupture [64]. Platelets participate in early stage disease initiation through multiple mechanisms enabling adhesion to dysfunctional endothelium. Activated platelets express high levels of adhesion receptors (e.g., ICAM1, P-Selectin, CD40L) associated with oxidised-LDL (ox-LDL) that contributes to vascular inflammation Direct cell-cell communication through platelet P-Selectin and CD40 ligand (CD40L) encourages inflammatory processes [66]. CD40L is thought to be at the heart of the atherosclerotic process, with 90% of circulating CD40L residing in platelets. CD40L is sent to the platelet surface upon activation, where it can initiate numerous inflammatory processes. The release of CD40L is intrinsically linked to αIIbβ3 as αIIbβ3 antagonists can block the release of sCD40L from activated platelets *in vitro*. Recently, platelet CD40 was shown to mediate the formation of platelet-leukocyte aggregates (stimulates leukocyte activation) and release inflammatory chemokines that activate endothelial cells, supporting atherosclerosis [66]. The significance of P-Selectin in atherosclerosis has been demonstrated in P-Selectin deficient animals that were protected from the disease. The role of platelet P-Selectin was clarified further by Huo et al., who illustrated that the introduction of P-Selectin expressing platelets into ApoE (−/−) mice accelerated atherosclerosis, whereas mice injected with platelets lacking P-Selectin formed smaller plaques [67].

Platelet-derived microparticles released upon activation may further amplify the progression of atherosclerosis through processes of adhesion, coagulation, inflammation and lipid metabolism [68]. Platelets also provide a huge repertoire of additional inflammatory mediators including a vast array of chemokines and cytokines that contribute to the crosstalk of platelets with other inflammatory cells—e.g., endothelial cells, monocytes, neutrophils, dendritic cells and T-cells [66]. The major function of platelets in atherosclerosis is the recruitment of leukocytes through direct receptor-ligand interactions or amplification of leukocyte recruitment through chemokine release. This bidirectional relationship is extremely important as platelets encourage leukocyte differentiation into a pro-adhesive and pro-migratory phenotype, and the leukocytes secrete mediators that reciprocally activate platelets.

Following atherosclerotic plaque rupture in severe CVD states, the exposure of thrombogenic substrates to circulating platelets instantly triggers platelet adhesion, activation and aggregation, forming a prothrombotic surface and subsequently encouraging thrombosis, vasoconstriction and vascular occlusion. Activated platelets expose phospholipids on their surface, which also promotes the coagulation cascade and subsequent fibrin production [64]. Given the critical roles of platelets in the pathogenesis of atherosclerosis and the development of acute thrombotic events, anti-platelet therapy has been widely employed in the primary and secondary prevention of CVD. Some of the current anti-platelet therapy drugs include Aspirin, which irreversibly inhibits cyclooxygenase to subsequently decrease TxA<sup>2</sup> production and limit platelet aggregation. Clopidogrel and Prasugrel are examples of P2Y12 receptor antagonists that inhibit the soluble agonist ADP, whilst Tirofiban and Abciximab block αIIbβ3-ligand interactions. Other anti-platelet therapies include thrombin and phosphodiesterase inhibitors (block degradation of cyclic nucleotides) [69, 70].

Given the impact of miRNA gene regulation, it is unsurprising that the dysregulation of miRNA is implicated in CVD. miRNAs are central players in modulating gene expression of cells/platelets collectively involved in CVD, and mediate inflammation, lipid uptake and cell differentiation in atherosclerosis. Platelet miRNA signatures (miR-25-3p, miR-221-3p and miR-374b-5) alter between patients with ST-segment elevation myocardial infarction (STEMI) and those with non-STEMI [71] suggesting that levels of platelet miRNA could impact platelet thrombogenicity and type of infarction. Furthermore, circulating miRNAs associated with the risk of MI (miR-126, miR-150, miR-223 and miR-197) are abundantly expressed in platelets. Platelet miRNA are implicated in premature CAD as two miRNAs in platelets are up-regulated in patients compared to controls (miR-340\* and miR-624\*), although whether or not they are the cause or consequence is currently unknown [72]. Besides their roles as mediators and biomarkers of CVD, platelet miRNA act as novel surrogate measures of the responsiveness to anti-platelet therapies used in CVD [73]. miR-223 levels are significantly down regulated in low responders to anti-platelet therapy [45, 74]. Furthermore, expression of platelet miR-26a has been linked with clopidogrel resistance during coronary stenting [75]. This theory is strengthened by research demonstrating how the switch from dual anti-platelet treatment with clopidogrel to ticagrelor is linked with significant changes in the level of platelet-specific circulating miRNAs, namely miR-223, miR-126 and miR-150 and miR-96 [76]. Other research investigating the effects of anti-platelet therapy on platelet miRNA levels showed that *in vitro* platelet activation resulted in transfer of miR-126 from platelets to plasma, whereas in aspirin-treated platelets, this process was not observed. *In vivo*, aspirin intake resulted in platelet inhibition and lower circulating platelet-derived miR-126 levels than were seen in untreated subjects [77]. Greater understanding of the meaning of platelet miRNA in CVD patients could aid in the diagnosis and treatment of these diseases.

αIIbβ3 and CD40L expression [90, 91] and stimulating intraplatelet F<sup>2</sup>

aggregatory effects of PGI<sup>2</sup>

activation in obesity [36].

duce energy. VO<sup>2</sup>

**2.4. Role of physical activity on platelet function**

partially due to alterations of platelet function [103].

Aerobic fitness is measured by maximal oxygen uptake (VO<sup>2</sup>

tion. Both decreased NO synthesis and bioavailability from ECs and platelets contribute to the pathogenesis of obesity, likely promoting thrombosis. Research by Leite et al. describes a decrease of nitric oxide synthase (NOS) activity and cGMP levels with simultaneous platelet hyper-aggregability in obese subjects compared to healthy controls with impaired antioxidant responses as potential contributors [92]. Anfossi et al. showed that platelet sensitivity to anti-

obese subjects marks a reduction in platelet activation markers and can potentially reverse the platelet responsiveness to NO and prostacyclin [93, 94]. A 10% weight reduction in obese subjects resulted in significant reductions in BMI, endothelial dysfunction and platelet aggregation. The changes in platelet function were associated with improvement in insulin sensitivity, indicating a tight relationship between the two. Weight loss also resulted in reduction in lipid

Although an association between obesity and platelet activation is evident, the molecular mechanisms responsible have only begun to surface [97]. Platelet RNA is reflective of pathological disease states where inflammatory transcript profiles (e.g., INFG, IL1R1, IL6 and TLR2) correlate significantly with increasing BMI [98], supporting the hypothesis that surplus fat could unfavourably alter the inflammatory potential of platelets. However, obesity can also cause dysregulation of other factors that control haemostasis such as microRNA (miRNA). There is increasing evidence to show that miRNA is involved in the pathogenesis of obesity [99], where plasma levels of miR-223 are reduced in obese compared to lean subjects, suggesting that the miR-223/P2Y12 alliance could signify a contributing mechanism of platelet

Those who engage in regular physical activity or exercise have a reduced prevalence of CVD. PA has been extensively studied due to its beneficial effects on all-cause mortality. Evidence to support the inverse relationship between PA and either CVD, cancer or depression continues to accumulate. With regard to CVD, regular PA/exercise reduces blood pressure, serum triglycerides, total body fat and visceral fat and LDL cholesterol [100]. Differences in these known factors have been demonstrated to explain a large proportion of the inverse relationship between physical activity and CVD risk [101, 102]. However, over 40% of the inverse association remains unexplained. Although the beneficial effects of regular exercise on blood lipids and blood pressure have been well documented, research focusing on platelet function has only recently gained greater attention. Since platelets play a key role in the pathogenesis of CVD, the protective effect of exercise against CVD may be

cise and is globally acknowledged as the best assessment of cardiovascular fitness [104]. VO<sup>2</sup> max represents the maximal amount of oxygen that an individual can take in and use to pro-

and oxygen to skeletal muscle, and the ability of skeletal muscle to extract this oxygen and use it to produce energy. Exercise effects on platelet function in both diseased and healthy

max is a function of the ability of the cardiovascular system to deliver blood

peroxidation markers [95] and P-Selectin expression in overweight CAD patients [96].

and NO is reduced in obesity [84]. Importantly, weight loss in


103

Platelets: Functional Biomarkers of Epigenetic Drift http://dx.doi.org/10.5772/intechopen.83447

max) during incremental exer-

#### **2.3. Effect of obesity on platelet function**

Obesity is a multifactorial condition involving a plethora of interrelated processes such as alterations in lipid metabolism, insulin resistance, inflammation, endothelial dysfunction, adipokine imbalance and oxidative stress. These metabolic aberrations have been postulated to be involved in platelet hyper-aggregability. Indeed, platelet activation markers are described as elevated in obesity, contributing to the inflammatory and prothrombotic state [78]. Subjects with overweight and obesity display increased platelet activation markers urinary-11-dehydro-TXB2 [79], MPV [80] and PLT [81]. Greater platelet activation (P-Selectin and PMP) is also linked to central arterial stiffness and carotid wall thickness amongst other atherosclerotic risk factors in overweight and obese subjects [82, 83]. The major mechanisms behind platelet function in obesity include a reduced sensitivity to insulin and resistance to their main inhibitory mediators PGI<sup>2</sup> and NO, elevated oxidative stress and an altered intracellular environment with increased cytosolic Ca2+ [84]. Platelets have insulin receptors which impact platelet function by regulating platelet response and sensitisation of platelets to inhibitory mechanism of PGI<sup>2</sup> and NO. In obese subjects, the anti-aggregating effect of insulin is diminished [85, 86].

Elevated oxidative stress also plays important roles in obesity-related platelet dysfunction. Oxidative stress results from an imbalance between the generation of free radicals and antioxidant enzymes [87]. High reactive oxygen species (ROS) generation by excess adipose tissue reduces NO bioavailability, enhancing surface expression of adhesion molecules, and enabling platelet activation and adhesion. Increased ROS also converts arachidonic acid into F2 -isoprostanes such as 8-iso-PGF<sup>2</sup><sup>α</sup> that can modulate platelet adhesive function [88]. Activated platelets also produce ROS [89], amplifying their own aggregatory potential by increasing αIIbβ3 and CD40L expression [90, 91] and stimulating intraplatelet F<sup>2</sup> -isoprostanes production. Both decreased NO synthesis and bioavailability from ECs and platelets contribute to the pathogenesis of obesity, likely promoting thrombosis. Research by Leite et al. describes a decrease of nitric oxide synthase (NOS) activity and cGMP levels with simultaneous platelet hyper-aggregability in obese subjects compared to healthy controls with impaired antioxidant responses as potential contributors [92]. Anfossi et al. showed that platelet sensitivity to antiaggregatory effects of PGI<sup>2</sup> and NO is reduced in obesity [84]. Importantly, weight loss in obese subjects marks a reduction in platelet activation markers and can potentially reverse the platelet responsiveness to NO and prostacyclin [93, 94]. A 10% weight reduction in obese subjects resulted in significant reductions in BMI, endothelial dysfunction and platelet aggregation. The changes in platelet function were associated with improvement in insulin sensitivity, indicating a tight relationship between the two. Weight loss also resulted in reduction in lipid peroxidation markers [95] and P-Selectin expression in overweight CAD patients [96].

Although an association between obesity and platelet activation is evident, the molecular mechanisms responsible have only begun to surface [97]. Platelet RNA is reflective of pathological disease states where inflammatory transcript profiles (e.g., INFG, IL1R1, IL6 and TLR2) correlate significantly with increasing BMI [98], supporting the hypothesis that surplus fat could unfavourably alter the inflammatory potential of platelets. However, obesity can also cause dysregulation of other factors that control haemostasis such as microRNA (miRNA). There is increasing evidence to show that miRNA is involved in the pathogenesis of obesity [99], where plasma levels of miR-223 are reduced in obese compared to lean subjects, suggesting that the miR-223/P2Y12 alliance could signify a contributing mechanism of platelet activation in obesity [36].

#### **2.4. Role of physical activity on platelet function**

in atherosclerosis. Platelet miRNA signatures (miR-25-3p, miR-221-3p and miR-374b-5) alter between patients with ST-segment elevation myocardial infarction (STEMI) and those with non-STEMI [71] suggesting that levels of platelet miRNA could impact platelet thrombogenicity and type of infarction. Furthermore, circulating miRNAs associated with the risk of MI (miR-126, miR-150, miR-223 and miR-197) are abundantly expressed in platelets. Platelet miRNA are implicated in premature CAD as two miRNAs in platelets are up-regulated in patients compared to controls (miR-340\* and miR-624\*), although whether or not they are the cause or consequence is currently unknown [72]. Besides their roles as mediators and biomarkers of CVD, platelet miRNA act as novel surrogate measures of the responsiveness to anti-platelet therapies used in CVD [73]. miR-223 levels are significantly down regulated in low responders to anti-platelet therapy [45, 74]. Furthermore, expression of platelet miR-26a has been linked with clopidogrel resistance during coronary stenting [75]. This theory is strengthened by research demonstrating how the switch from dual anti-platelet treatment with clopidogrel to ticagrelor is linked with significant changes in the level of platelet-specific circulating miRNAs, namely miR-223, miR-126 and miR-150 and miR-96 [76]. Other research investigating the effects of anti-platelet therapy on platelet miRNA levels showed that *in vitro* platelet activation resulted in transfer of miR-126 from platelets to plasma, whereas in aspirin-treated platelets, this process was not observed. *In vivo*, aspirin intake resulted in platelet inhibition and lower circulating platelet-derived miR-126 levels than were seen in untreated subjects [77]. Greater understanding of the meaning of plate-

let miRNA in CVD patients could aid in the diagnosis and treatment of these diseases.

Obesity is a multifactorial condition involving a plethora of interrelated processes such as alterations in lipid metabolism, insulin resistance, inflammation, endothelial dysfunction, adipokine imbalance and oxidative stress. These metabolic aberrations have been postulated to be involved in platelet hyper-aggregability. Indeed, platelet activation markers are described as elevated in obesity, contributing to the inflammatory and prothrombotic state [78]. Subjects with overweight and obesity display increased platelet activation markers urinary-11-dehydro-TXB2 [79], MPV [80] and PLT [81]. Greater platelet activation (P-Selectin and PMP) is also linked to central arterial stiffness and carotid wall thickness amongst other atherosclerotic risk factors in overweight and obese subjects [82, 83]. The major mechanisms behind platelet function in obesity include a reduced sensitivity to insulin and resistance to their main inhibi-

ment with increased cytosolic Ca2+ [84]. Platelets have insulin receptors which impact platelet function by regulating platelet response and sensitisation of platelets to inhibitory mechanism

Elevated oxidative stress also plays important roles in obesity-related platelet dysfunction. Oxidative stress results from an imbalance between the generation of free radicals and antioxidant enzymes [87]. High reactive oxygen species (ROS) generation by excess adipose tissue reduces NO bioavailability, enhancing surface expression of adhesion molecules, and enabling platelet activation and adhesion. Increased ROS also converts arachidonic acid into

platelets also produce ROS [89], amplifying their own aggregatory potential by increasing

and NO. In obese subjects, the anti-aggregating effect of insulin is diminished [85, 86].

and NO, elevated oxidative stress and an altered intracellular environ-

that can modulate platelet adhesive function [88]. Activated

**2.3. Effect of obesity on platelet function**

tory mediators PGI<sup>2</sup>

102 Homeostasis - An Integrated Vision


of PGI<sup>2</sup>

F2

Those who engage in regular physical activity or exercise have a reduced prevalence of CVD. PA has been extensively studied due to its beneficial effects on all-cause mortality. Evidence to support the inverse relationship between PA and either CVD, cancer or depression continues to accumulate. With regard to CVD, regular PA/exercise reduces blood pressure, serum triglycerides, total body fat and visceral fat and LDL cholesterol [100]. Differences in these known factors have been demonstrated to explain a large proportion of the inverse relationship between physical activity and CVD risk [101, 102]. However, over 40% of the inverse association remains unexplained. Although the beneficial effects of regular exercise on blood lipids and blood pressure have been well documented, research focusing on platelet function has only recently gained greater attention. Since platelets play a key role in the pathogenesis of CVD, the protective effect of exercise against CVD may be partially due to alterations of platelet function [103].

Aerobic fitness is measured by maximal oxygen uptake (VO<sup>2</sup> max) during incremental exercise and is globally acknowledged as the best assessment of cardiovascular fitness [104]. VO<sup>2</sup> max represents the maximal amount of oxygen that an individual can take in and use to produce energy. VO<sup>2</sup> max is a function of the ability of the cardiovascular system to deliver blood and oxygen to skeletal muscle, and the ability of skeletal muscle to extract this oxygen and use it to produce energy. Exercise effects on platelet function in both diseased and healthy populations have elicited profound interest in the last decade. The majority of research surrounding platelet function and physical activity/exercise has focused on acute (single bout) aerobic exercise. Potential effects of acute exercise on platelet function (mainly aggregation) have been investigated through various studies in adult subjects with varying intra- and inter-individual results, making interpretation problematic. Differences in population type (e.g., CVD versus healthy), methods employed to assess platelet function and techniques to examine reactivity are the main reasons for discrepancies and lack of consistency between research groups [105]. Different platelet adhesion experimental protocols have provided no definitive consensus on the platelet response to acute exercise in healthy adult subjects [106–110]. High levels of plasma fibrinogen after exercise result in elevated blood viscosity and this along with increased vWF binding, αIIbβ3 and P-Selectin expression all contribute to the increased platelet aggregation after acute exercise [111]. In general, it appears that acute vigorous exercise induces a hyper-reactive haemostatic state [112] and a transient increase in agonist-induced platelet adhesion and aggregation *in vitro* and *ex vivo*. However, there is no definitive consensus regarding the short-term effects of exercise on platelet function.

heighten platelet reactivity. Regular exercise can improve this response, seems to have an antithrombotic effect on platelets and could represent a portion of the protective effects of exercise on CVD risk factors. Moreover, effects of exercise are not maintained with cessation of training. Of importance, all of these studies discussed were performed in adults and not adolescents when the CVD risk factors and atherosclerotic process has begun. Platelet function and exercise in children or adolescents is in its infancy, an area that requires urgent research [122].

Platelets: Functional Biomarkers of Epigenetic Drift http://dx.doi.org/10.5772/intechopen.83447 105

In contrast to physical activity, physical inactivity/sedentary behaviour is a universal leading cause of death and independent CVD risk factor [123, 124]. Sedentary behaviour refers to any waking activity characterised by an energy expenditure ≤1.5 metabolic equivalents in a sitting or reclining posture [125]. However, in contrast to the evidence supporting the benefits of acute and chronic exercise, relatively little is understood about the mechanisms underlying the physiological, cellular and molecular responses to physical inactivity. Incomplete understanding of this relationship is a huge barrier to combating the development of CVD and its ancillary risk factors. Our knowledge of physical inactivity is somewhat indirect and is mainly based on the positive effects of exercise training on the sedentary population. As a sedentary lifestyle is often associated with obesity [126], some mechanisms involved in the pathogenesis of physical inactivity are similar to that of obesity such as insulin resistance [127], hypertension and increased inflammation [128]. However, distinct factors associated with sedentary behaviour include reduced muscular activity of lower extremities, decreased blood flow and reduction of shear stress, which increases oxidative stress, endothelial dysfunction [129] and arterial remodelling [130, 131].

The plasticity of platelets and other blood cells is vital for responding to environmental changes in response to physical (in)activity patterns. However, the molecular factors influencing platelet function/response/adaptation to physical (in)activity remain poorly understood. Recently identified miRNAs have gained attention as modulators of platelet function [34]. Evidence for miRNA involvement in exercise-associated gene expression changes in a number of cell types including peripheral blood mononuclear cell, neutrophil and skeletal muscle in non-trained and trained subjects has been illustrated [132–134]. Work by Baggish et al. showed altered expression of circulating miRNA (c-miRNA) in response to acute and chronic exercise in athletes [135]. Eight c-miRNA involved in cellular processes related to exercise adaptation (muscle contractility, inflammation, and angiogenesis) were examined. They observed four distinctive signatures of c-miRNA; c-miRNA up-regulated by acute exhaustive exercise pre- and post-exercise intervention, c-miRNA responsive to acute exercise pre- but not post-intervention, c-miRNA only responsive to exercise intervention and non-responsive miRNA. Moreover, evidence of these physical activity-specific microRNA signatures [136–138] has ingrained concepts of physical inactivityspecific miRNA profiles. Epigenetic variation could therefore be a potential mechanism allowing for independent or synergistic effects of physical inactivity on platelet function. Hibler et al. recently described indications for epigenetic variation (by miRNA expression) as a link between physical activity and sedentary lifestyle [139]. An epigenetic adaptation to habitual exercise has been described [140, 141]. Similarly, an epigenetic adaptation to physical inactivity may exist.

**2.5. Role of physical inactivity and sedentary lifestyle on platelet function**

*2.5.1. Physical activity/inactivity-specific miRNA*

Cardiorespiratory fitness (CRF) is the ability to perform large muscle, moderate to high intensity exercise for prolonged periods and depends on the respiratory, cardiovascular and skeletal systems. CRF represents the adaptation to long-term exercise. High CRF levels are also linked with reduced CVD risk factors such as hypertension, obesity in the general population and CVD patients [113–116]. CRF was first postulated as a significant determinant for changes in platelet function in response to acute exercise after observations that acute strenuous exercise increased platelet activation in sedentary, but not physically active, subjects [106, 117]. The actual relationship between CRF and platelet function has been referred to in a recent breakthrough study by Heber et al., who investigated platelet function and CRF in 62 young women [118]. Platelet function was assessed by determination of P-Selectin and CD40L expression and quantification of platelet ROS generation in platelet-rich plasma (PRP). Basal platelet activation (reflected by CD62P expression) and agonist-induced platelet activation (ROS, CD62P and CD40L) were higher in the LF compared to the MF and HF. The group found no difference between basal CD40L expressions (non-agonist induced). Interestingly, basal platelet function in the MF and HF were almost equal, indicating a definite influence of CRF on platelet function. A high CRF level is a result of exercise training and habitual physical activity. Therefore, research on the effects of longitudinal exercise training on platelet function has mainly shown that habitual exercise has favourable effects on platelet function. Eight weeks of exercise training (60% VO<sup>2</sup> max 5×/week 30 min/day), reduced shear stress-induced platelet activation and ox-LDL-potentiated platelet function [109, 111]. Importantly, after 12 weeks of de-conditioning, the beneficial effects of exercise on platelets were non-existent and platelet function returned to its pre-training state.

De Meirelles et al. reported that chronic physical activity had favourable effects on platelet activation in hypertensive patients at rest [119]. Twelve weeks of regular exercise (75–85% VO<sup>2</sup> max 5×/week for 45–60 min) reduced platelet aggregation in response to collagen. Santilli et al. investigated the effects of regular high intensity (60–75%) aerobic exercise for 2 months in low and intermediate CVD risk sedentary subjects [120]. Exercise training was associated with reductions in TxA<sup>2</sup> , plasma P-Selectin and platelet-derived CD40L, despite no reduction in CRP (representing systemic inflammation). Evidently, physical activity and exercise affects nearly all facets of platelet function [121]. Studies on the effects of acute exercise appear to heighten platelet reactivity. Regular exercise can improve this response, seems to have an antithrombotic effect on platelets and could represent a portion of the protective effects of exercise on CVD risk factors. Moreover, effects of exercise are not maintained with cessation of training. Of importance, all of these studies discussed were performed in adults and not adolescents when the CVD risk factors and atherosclerotic process has begun. Platelet function and exercise in children or adolescents is in its infancy, an area that requires urgent research [122].

#### **2.5. Role of physical inactivity and sedentary lifestyle on platelet function**

In contrast to physical activity, physical inactivity/sedentary behaviour is a universal leading cause of death and independent CVD risk factor [123, 124]. Sedentary behaviour refers to any waking activity characterised by an energy expenditure ≤1.5 metabolic equivalents in a sitting or reclining posture [125]. However, in contrast to the evidence supporting the benefits of acute and chronic exercise, relatively little is understood about the mechanisms underlying the physiological, cellular and molecular responses to physical inactivity. Incomplete understanding of this relationship is a huge barrier to combating the development of CVD and its ancillary risk factors. Our knowledge of physical inactivity is somewhat indirect and is mainly based on the positive effects of exercise training on the sedentary population. As a sedentary lifestyle is often associated with obesity [126], some mechanisms involved in the pathogenesis of physical inactivity are similar to that of obesity such as insulin resistance [127], hypertension and increased inflammation [128]. However, distinct factors associated with sedentary behaviour include reduced muscular activity of lower extremities, decreased blood flow and reduction of shear stress, which increases oxidative stress, endothelial dysfunction [129] and arterial remodelling [130, 131].

#### *2.5.1. Physical activity/inactivity-specific miRNA*

populations have elicited profound interest in the last decade. The majority of research surrounding platelet function and physical activity/exercise has focused on acute (single bout) aerobic exercise. Potential effects of acute exercise on platelet function (mainly aggregation) have been investigated through various studies in adult subjects with varying intra- and inter-individual results, making interpretation problematic. Differences in population type (e.g., CVD versus healthy), methods employed to assess platelet function and techniques to examine reactivity are the main reasons for discrepancies and lack of consistency between research groups [105]. Different platelet adhesion experimental protocols have provided no definitive consensus on the platelet response to acute exercise in healthy adult subjects [106–110]. High levels of plasma fibrinogen after exercise result in elevated blood viscosity and this along with increased vWF binding, αIIbβ3 and P-Selectin expression all contribute to the increased platelet aggregation after acute exercise [111]. In general, it appears that acute vigorous exercise induces a hyper-reactive haemostatic state [112] and a transient increase in agonist-induced platelet adhesion and aggregation *in vitro* and *ex vivo*. However, there is no

definitive consensus regarding the short-term effects of exercise on platelet function.

favourable effects on platelet function. Eight weeks of exercise training (60% VO<sup>2</sup>

VO<sup>2</sup>

with reductions in TxA<sup>2</sup>

104 Homeostasis - An Integrated Vision

cise on platelets were non-existent and platelet function returned to its pre-training state.

30 min/day), reduced shear stress-induced platelet activation and ox-LDL-potentiated platelet function [109, 111]. Importantly, after 12 weeks of de-conditioning, the beneficial effects of exer-

De Meirelles et al. reported that chronic physical activity had favourable effects on platelet activation in hypertensive patients at rest [119]. Twelve weeks of regular exercise (75–85%

in CRP (representing systemic inflammation). Evidently, physical activity and exercise affects nearly all facets of platelet function [121]. Studies on the effects of acute exercise appear to

 max 5×/week for 45–60 min) reduced platelet aggregation in response to collagen. Santilli et al. investigated the effects of regular high intensity (60–75%) aerobic exercise for 2 months in low and intermediate CVD risk sedentary subjects [120]. Exercise training was associated

, plasma P-Selectin and platelet-derived CD40L, despite no reduction

max 5×/week

Cardiorespiratory fitness (CRF) is the ability to perform large muscle, moderate to high intensity exercise for prolonged periods and depends on the respiratory, cardiovascular and skeletal systems. CRF represents the adaptation to long-term exercise. High CRF levels are also linked with reduced CVD risk factors such as hypertension, obesity in the general population and CVD patients [113–116]. CRF was first postulated as a significant determinant for changes in platelet function in response to acute exercise after observations that acute strenuous exercise increased platelet activation in sedentary, but not physically active, subjects [106, 117]. The actual relationship between CRF and platelet function has been referred to in a recent breakthrough study by Heber et al., who investigated platelet function and CRF in 62 young women [118]. Platelet function was assessed by determination of P-Selectin and CD40L expression and quantification of platelet ROS generation in platelet-rich plasma (PRP). Basal platelet activation (reflected by CD62P expression) and agonist-induced platelet activation (ROS, CD62P and CD40L) were higher in the LF compared to the MF and HF. The group found no difference between basal CD40L expressions (non-agonist induced). Interestingly, basal platelet function in the MF and HF were almost equal, indicating a definite influence of CRF on platelet function. A high CRF level is a result of exercise training and habitual physical activity. Therefore, research on the effects of longitudinal exercise training on platelet function has mainly shown that habitual exercise has

The plasticity of platelets and other blood cells is vital for responding to environmental changes in response to physical (in)activity patterns. However, the molecular factors influencing platelet function/response/adaptation to physical (in)activity remain poorly understood. Recently identified miRNAs have gained attention as modulators of platelet function [34]. Evidence for miRNA involvement in exercise-associated gene expression changes in a number of cell types including peripheral blood mononuclear cell, neutrophil and skeletal muscle in non-trained and trained subjects has been illustrated [132–134]. Work by Baggish et al. showed altered expression of circulating miRNA (c-miRNA) in response to acute and chronic exercise in athletes [135]. Eight c-miRNA involved in cellular processes related to exercise adaptation (muscle contractility, inflammation, and angiogenesis) were examined. They observed four distinctive signatures of c-miRNA; c-miRNA up-regulated by acute exhaustive exercise pre- and post-exercise intervention, c-miRNA responsive to acute exercise pre- but not post-intervention, c-miRNA only responsive to exercise intervention and non-responsive miRNA. Moreover, evidence of these physical activity-specific microRNA signatures [136–138] has ingrained concepts of physical inactivityspecific miRNA profiles. Epigenetic variation could therefore be a potential mechanism allowing for independent or synergistic effects of physical inactivity on platelet function. Hibler et al. recently described indications for epigenetic variation (by miRNA expression) as a link between physical activity and sedentary lifestyle [139]. An epigenetic adaptation to habitual exercise has been described [140, 141]. Similarly, an epigenetic adaptation to physical inactivity may exist.

#### **2.6. Physical activity/inactivity and platelet epigenetic drift**

It has been well recognised that regular exercise may reduce risk of major vascular thrombotic events and protect against CVD [123]. Differences in known factors explain a large percentage of the inverse relationship between physical activity and CVD risk [101, 102]. Nevertheless, over 40% of the inverse association remains unexplained. Although the beneficial effects of regular exercise on blood lipids and blood pressure have been well accepted, research focusing on platelet function has only recently gained greater attention. Whilst it is known that platelet function and platelet indices (markers of platelet activation) are altered in pathological states such as CVD, only a minority of studies have solely examined the relationship between overall physiological health and platelet function in healthy subjects [120, 142]. Therefore, it is imperative that future studies explore the feasibility of platelet indices and whole blood platelet function measurements, as useful, non-invasive initial biomarkers of early/subclinical CVD risk and lifestyle parameters.

sufficient to normalise platelet function in low fit females [118]. Larger studies incorporating exercise interventions at low doses over a lengthy period of time and examining more extensive aspects of platelet function in low fit subjects would develop this knowledge. The prescription of anti-platelet therapy is frequently used to treat CVD patients. However, the other residual risks (oxidative stress, inflammation etc.), which occur due to associations between CVD risk factors, are not eliminated efficiently by these therapies. In this sense, physical activity has been emphasised as it promotes favourable physiological adaptations, which may attenuate the cardiovascular risk factors and residual risks. Regular exercise may also impact platelet function in CVD patients. Exercise interventions in these populations could be beneficial in terms of reducing anti-platelet therapy dosage or combining anti-platelet therapy with exercise [147],

Platelets: Functional Biomarkers of Epigenetic Drift http://dx.doi.org/10.5772/intechopen.83447 107

The investigation of epigenetic processes is almost a completely unexplored area in platelet biology as analyses of these mechanisms require DNA [2]. Platelets have functionally active mitochondria [148]. Like nuclear DNA, mitochondrial DNA (mtDNA) can also be methylated, moderating control of mitochondrial gene expression. Understanding epigenetic regulation of mitochondrial genes in platelets is proving crucial to understanding their implication in CVD development [6]. Furthermore, miRNA have recently been linked with platelet mitochondrial health in stored platelets [149]. Platelets contain the machinery to process pre-miRNA to mature miRNA [34]. Platelets contain higher levels of pre-miRNA than other blood cells [56], and the maturation of pre-miRNA could contributed to altered miRNA profiles due to physical activity and inactivity. This may represent a more focused and efficient method of monitoring platelet function. Targeting levels of other biogenesis molecules in the miRNA pathway would also be an interesting avenue of platelet miRNA biology. Recently, Elgheznawy et al. showed that Dicer was decreased in patients with TD2M compared to healthy controls, whereas interestingly, Argonaute 2 levels did not differ [150]. Experiments investigating levels of miRNA processing machinery such as Dicer and Argonaute 2 in physically active and sedentary populations would be of major interest. Long-term lifestyle choices such as physical inactivity may incur epigenetic penalties in megakaryocytes, and in the biological processes of *megakaryocytopoiesis* and *thrombopoiesis*. Thus, platelet miRNA could reflect these epigenetic changes, holding substantial predictive potential of both health and disease. Epigenetic changes in the megakaryocyte genome such as methylation of genes determining platelet biogenesis or changes in histone acetylation with aging have been suggested to play an important role in platelet function [20]. Prescribed exercise could induce epigenetic changes in megakaryocytes to produce a healthier phenotype of

It is evident that lifestyle factors such as physical activity, physical inactivity and overweight do impact platelet function. Platelets are indeed reflective of physiological and lifestyle changes, making them sensitive biomarkers of human health. Platelets represent a tangible link to physiological and pathological changes within the body. Future research in this area, will no doubt contribute to a greater mechanistic understanding of the relationship between

epigenetics, cardiovascular health, lifestyle factors and platelet biology.

i.e., prescriptive exercise medicine as an adjuvant management strategy/therapy.

platelets with a direct change in platelet reactivity.

**4. Conclusion**

Low cardiorespiratory fitness is associated with physical inactivity [143]. This has major health effects globally, with approximately 3.2 million deaths each year attributable to inadequate physical activity. Evidence has shown that physical inactivity and sedentary behaviour have direct effects on CVD risk factors [144]. Moreover, in contrast to the accumulating evidence supporting the benefits of regular exercise, relatively little is understood about the deleterious mechanisms underlying the physiological, cellular and molecular responses to PI, specifically with regard to platelet function.

#### **3. Future research avenues**

Our group, in collaboration with the European Space Agency (ESA), the Centre National d'Etudes Spatiales (*CNES*) and *MEDES* (Institute for Space Medicine and Physiology, Toulouse, France) have employed ground-based models of microgravity, i.e., dry water immersion (DI) to study the effects of spaceflight on human physiology in a precisely controlled environment. DI involves immersing a subject in a bath of thermoneutral water covered by a waterproof fabric [145]. Several factors act simultaneously on the human body during immersion, including hydrostatic compression, supportlessness and extensive physical inactivity. Hypokinesia and hypodynamia are the major characteristics of physical inactivity induced by dry immersion. Hypodynamia involves a reduction in postural muscle load, whereas hypokinesia is a decline in motor activity. For these reasons, DI has been well accepted as a valuable tool to study physical inactivity [146]. DI presents a unique opportunity to analyse the specific effects of physical inactivity on platelet physiology/function and related biomarkers.

Recent studies reflect the first comprehensive attempts to evaluate the relationship between platelet function and physical activity, physical inactivity and overweight. While exploratory in nature to date, several questions remain unanswered and so further studies are warranted. The search for simple biomarkers that allow for early identification of subclinical/CVD risk is ongoing. Platelets can reflect changes in unhealthy lifestyle patterns. The Impact-R test is a relatively inexpensive test that can reliably detect changes in platelet adhesion and could be employed for CVD risk evaluation amongst subjects who are asymptomatic. Platelet indices and function markers should be further tested in larger populations to determine their reliability as surrogate markers for evaluating physiological health and to test during either pharmacological and lifestyle interventions. A relatively low dose of exercise has been shown to be sufficient to normalise platelet function in low fit females [118]. Larger studies incorporating exercise interventions at low doses over a lengthy period of time and examining more extensive aspects of platelet function in low fit subjects would develop this knowledge. The prescription of anti-platelet therapy is frequently used to treat CVD patients. However, the other residual risks (oxidative stress, inflammation etc.), which occur due to associations between CVD risk factors, are not eliminated efficiently by these therapies. In this sense, physical activity has been emphasised as it promotes favourable physiological adaptations, which may attenuate the cardiovascular risk factors and residual risks. Regular exercise may also impact platelet function in CVD patients. Exercise interventions in these populations could be beneficial in terms of reducing anti-platelet therapy dosage or combining anti-platelet therapy with exercise [147], i.e., prescriptive exercise medicine as an adjuvant management strategy/therapy.

The investigation of epigenetic processes is almost a completely unexplored area in platelet biology as analyses of these mechanisms require DNA [2]. Platelets have functionally active mitochondria [148]. Like nuclear DNA, mitochondrial DNA (mtDNA) can also be methylated, moderating control of mitochondrial gene expression. Understanding epigenetic regulation of mitochondrial genes in platelets is proving crucial to understanding their implication in CVD development [6]. Furthermore, miRNA have recently been linked with platelet mitochondrial health in stored platelets [149]. Platelets contain the machinery to process pre-miRNA to mature miRNA [34]. Platelets contain higher levels of pre-miRNA than other blood cells [56], and the maturation of pre-miRNA could contributed to altered miRNA profiles due to physical activity and inactivity. This may represent a more focused and efficient method of monitoring platelet function. Targeting levels of other biogenesis molecules in the miRNA pathway would also be an interesting avenue of platelet miRNA biology. Recently, Elgheznawy et al. showed that Dicer was decreased in patients with TD2M compared to healthy controls, whereas interestingly, Argonaute 2 levels did not differ [150]. Experiments investigating levels of miRNA processing machinery such as Dicer and Argonaute 2 in physically active and sedentary populations would be of major interest.

Long-term lifestyle choices such as physical inactivity may incur epigenetic penalties in megakaryocytes, and in the biological processes of *megakaryocytopoiesis* and *thrombopoiesis*. Thus, platelet miRNA could reflect these epigenetic changes, holding substantial predictive potential of both health and disease. Epigenetic changes in the megakaryocyte genome such as methylation of genes determining platelet biogenesis or changes in histone acetylation with aging have been suggested to play an important role in platelet function [20]. Prescribed exercise could induce epigenetic changes in megakaryocytes to produce a healthier phenotype of platelets with a direct change in platelet reactivity.

#### **4. Conclusion**

**2.6. Physical activity/inactivity and platelet epigenetic drift**

**3. Future research avenues**

106 Homeostasis - An Integrated Vision

It has been well recognised that regular exercise may reduce risk of major vascular thrombotic events and protect against CVD [123]. Differences in known factors explain a large percentage of the inverse relationship between physical activity and CVD risk [101, 102]. Nevertheless, over 40% of the inverse association remains unexplained. Although the beneficial effects of regular exercise on blood lipids and blood pressure have been well accepted, research focusing on platelet function has only recently gained greater attention. Whilst it is known that platelet function and platelet indices (markers of platelet activation) are altered in pathological states such as CVD, only a minority of studies have solely examined the relationship between overall physiological health and platelet function in healthy subjects [120, 142]. Therefore, it is imperative that future studies explore the feasibility of platelet indices and whole blood platelet function measurements, as useful, non-invasive initial biomarkers of early/subclinical CVD risk and lifestyle parameters. Low cardiorespiratory fitness is associated with physical inactivity [143]. This has major health effects globally, with approximately 3.2 million deaths each year attributable to inadequate physical activity. Evidence has shown that physical inactivity and sedentary behaviour have direct effects on CVD risk factors [144]. Moreover, in contrast to the accumulating evidence supporting the benefits of regular exercise, relatively little is understood about the deleterious mechanisms underlying the physiological, cellular and molecular responses to PI, specifically with regard to platelet function.

Our group, in collaboration with the European Space Agency (ESA), the Centre National d'Etudes Spatiales (*CNES*) and *MEDES* (Institute for Space Medicine and Physiology, Toulouse, France) have employed ground-based models of microgravity, i.e., dry water immersion (DI) to study the effects of spaceflight on human physiology in a precisely controlled environment. DI involves immersing a subject in a bath of thermoneutral water covered by a waterproof fabric [145]. Several factors act simultaneously on the human body during immersion, including hydrostatic compression, supportlessness and extensive physical inactivity. Hypokinesia and hypodynamia are the major characteristics of physical inactivity induced by dry immersion. Hypodynamia involves a reduction in postural muscle load, whereas hypokinesia is a decline in motor activity. For these reasons, DI has been well accepted as a valuable tool to study physical inactivity [146]. DI presents a unique opportunity to analyse the specific effects

Recent studies reflect the first comprehensive attempts to evaluate the relationship between platelet function and physical activity, physical inactivity and overweight. While exploratory in nature to date, several questions remain unanswered and so further studies are warranted. The search for simple biomarkers that allow for early identification of subclinical/CVD risk is ongoing. Platelets can reflect changes in unhealthy lifestyle patterns. The Impact-R test is a relatively inexpensive test that can reliably detect changes in platelet adhesion and could be employed for CVD risk evaluation amongst subjects who are asymptomatic. Platelet indices and function markers should be further tested in larger populations to determine their reliability as surrogate markers for evaluating physiological health and to test during either pharmacological and lifestyle interventions. A relatively low dose of exercise has been shown to be

of physical inactivity on platelet physiology/function and related biomarkers.

It is evident that lifestyle factors such as physical activity, physical inactivity and overweight do impact platelet function. Platelets are indeed reflective of physiological and lifestyle changes, making them sensitive biomarkers of human health. Platelets represent a tangible link to physiological and pathological changes within the body. Future research in this area, will no doubt contribute to a greater mechanistic understanding of the relationship between epigenetics, cardiovascular health, lifestyle factors and platelet biology.

#### **Acknowledgements**

This work was supported through the Irish Higher Education Authority Programme for Research in Third Level Institutes (HEA-PRTLI Cycle 4: T3 Targeted Therapeutics & Theranostics), the Health Research Board of Ireland (HRB-RP/2005/184, HRA/2009/122, HRA/ 2009/122/R), Science Foundation Ireland (SFI-04/BR/B0577, 11/TIDA/B1927) to Dr. R.P. Murphy, and the International Visitor Award, Dublin City University, Dublin, Ireland, to Drs. B. Degryse and R.P. Murphy, ASU-DCU Transatlantic Research Fund to M.M and R.P.M. R.P. Murphy is a management committee member of the EU H2020 COST action-CM1406, EpiChemBio. The ground-based protocol and studies of microgravity (3-Day Dry Immersion and 5-Day Dry Immersion) were undertaken in MEDES, the Institute for Space Medicine and Physiology, *Toulouse* University Hospital, France, and was supported and funded by the Centre National d'Etudes Spatiales (*CNES-*National Centre for Space Studies) to M-A.C., R.P.M., N.N., and L.T.

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#### **Conflict of interest**

We wish to confirm that there are no known conflicts of interest associated with this publication, and there has been no significant financial support for this work that could have influenced its outcome.

## **Author details**

Laura Twomey<sup>1</sup> , Robert G. Wallace<sup>1</sup> , Marco Mangone<sup>2</sup> , Bernard Degryse<sup>1</sup> , Sinead Sheridan<sup>3</sup> , Michael Harrison<sup>4</sup> , Niall Moyna<sup>1</sup> , Gerardene Meade-Murphy<sup>5</sup> , Nastassia Navasiolava6 , Marc-Antoine Custaud<sup>6</sup> and Ronan P. Murphy<sup>1</sup> \*

\*Address all correspondence to: ronan.murphy@dcu.ie

1 School of Health & Human Performance, Dublin City University, Glasnevin, Dublin, Ireland

2 School of Life Sciences, Arizona State University, Tempe Campus, USA

3 School of Public Health, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong

4 School of Health Sciences—Health Sport and Exercise Science, Waterford Institute of Technology, Ireland

5 Department of Pharmacology and Therapeutics, University College Cork, Cork, Ireland

6 Centre de Recherche Clinique du CHU d'Angers, Angers, France

#### **References**

**Acknowledgements**

108 Homeostasis - An Integrated Vision

to M-A.C., R.P.M., N.N., and L.T.

, Robert G. Wallace<sup>1</sup>

, Niall Moyna<sup>1</sup>

\*Address all correspondence to: ronan.murphy@dcu.ie

**Conflict of interest**

influenced its outcome.

**Author details**

Laura Twomey<sup>1</sup>

Ireland

Hong Kong

Technology, Ireland

Michael Harrison<sup>4</sup>

Marc-Antoine Custaud<sup>6</sup>

This work was supported through the Irish Higher Education Authority Programme for Research in Third Level Institutes (HEA-PRTLI Cycle 4: T3 Targeted Therapeutics & Theranostics), the Health Research Board of Ireland (HRB-RP/2005/184, HRA/2009/122, HRA/ 2009/122/R), Science Foundation Ireland (SFI-04/BR/B0577, 11/TIDA/B1927) to Dr. R.P. Murphy, and the International Visitor Award, Dublin City University, Dublin, Ireland, to Drs. B. Degryse and R.P. Murphy, ASU-DCU Transatlantic Research Fund to M.M and R.P.M. R.P. Murphy is a management committee member of the EU H2020 COST action-CM1406, EpiChemBio. The ground-based protocol and studies of microgravity (3-Day Dry Immersion and 5-Day Dry Immersion) were undertaken in MEDES, the Institute for Space Medicine and Physiology, *Toulouse* University Hospital, France, and was supported and funded by the Centre National d'Etudes Spatiales (*CNES-*National Centre for Space Studies)

We wish to confirm that there are no known conflicts of interest associated with this publication, and there has been no significant financial support for this work that could have

, Marco Mangone<sup>2</sup>

1 School of Health & Human Performance, Dublin City University, Glasnevin, Dublin,

3 School of Public Health, Li Ka Shing Faculty of Medicine, The University of Hong Kong,

4 School of Health Sciences—Health Sport and Exercise Science, Waterford Institute of

5 Department of Pharmacology and Therapeutics, University College Cork, Cork, Ireland

and Ronan P. Murphy<sup>1</sup>

2 School of Life Sciences, Arizona State University, Tempe Campus, USA

6 Centre de Recherche Clinique du CHU d'Angers, Angers, France

, Gerardene Meade-Murphy<sup>5</sup>

\*

, Bernard Degryse<sup>1</sup>

, Sinead Sheridan<sup>3</sup>

,

, Nastassia Navasiolava6

,


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**Section 4**

**Homeostasis and Disease**

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## **Homeostasis and Disease**

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120 Homeostasis - An Integrated Vision

**Chapter 7**

Provisional chapter

**Reactive Oxygen Species, Cellular Redox Homeostasis**

DOI: 10.5772/intechopen.76096

Reactive Oxygen Species, Cellular Redox Homeostasis

Redox homeostasis is attained by the cautious regulation of both reactive oxygen species (ROS) formation and removal from the body system. A shift in ROS balance promotes oxidative injury and tumour development by inflicting damage to DNA and inducing inconsistencies in the genome. The sources of endogenous ROS in a cell include mETC, NOX, LOX, cytochrome P450 and XO. The exogenous risk factors of ROS are pollutants, chemicals/drugs, radiation and heavy metals. Oxidative phosphorylation in the mitochondria produces ROS with unpaired electrons. Superoxide anion is the major ROS produced in the human mitochondria. Bulk of the ROS generation in the mitochondria occurs at the electron transport chain as derivatives of respiration. Cancer cells sustain ROS production by suppressing the antioxidant-generation system. Balance between ROS production and subsequent detoxification is regulated by scavenging enzymes and antioxidant agents. Failure in sirtuin-3 (SIRT3), ATM and p53 activities elevates the intracellular levels of ROS. PKCα induces the expression of NOX (DUOX) during cancer development and the consequent increase in ROS production. The PI3K/AKT signalling pathway activates NOX with consequent ROS production and subsequent induction of instability in the genome, leading to cancer. In conclusion, the interruption of the redox pathways that regulate ROS and its redox signalling activities affects cell physiology and can ultimately result in abnormal

Keywords: homeostasis, cancer, reactive oxygen species, mitochondrial electron transport chain (ETC), NOX, GSH, glutathione oxidase (GPX), superoxide dismutase (SOD),

> © 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 eproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

signalling, uncontrolled oxidative impairment and tumorigenesis.

Rabiatul Basria S.M.N. Mydin and Simon I. Okekpa

Rabiatul Basria S.M.N. Mydin and Simon I. Okekpa

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.76096

thioredoxin (TRX), sirtuins

**and Cancer**

and Cancer

Abstract

#### **Reactive Oxygen Species, Cellular Redox Homeostasis** Provisional chapter

DOI: 10.5772/intechopen.76096

#### **and Cancer** Reactive Oxygen Species, Cellular Redox Homeostasis

and Cancer

Rabiatul Basria S.M.N. Mydin and Simon I. Okekpa Rabiatul Basria S.M.N. Mydin and Simon I. Okekpa

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

http://dx.doi.org/10.5772/intechopen.76096

#### Abstract

Redox homeostasis is attained by the cautious regulation of both reactive oxygen species (ROS) formation and removal from the body system. A shift in ROS balance promotes oxidative injury and tumour development by inflicting damage to DNA and inducing inconsistencies in the genome. The sources of endogenous ROS in a cell include mETC, NOX, LOX, cytochrome P450 and XO. The exogenous risk factors of ROS are pollutants, chemicals/drugs, radiation and heavy metals. Oxidative phosphorylation in the mitochondria produces ROS with unpaired electrons. Superoxide anion is the major ROS produced in the human mitochondria. Bulk of the ROS generation in the mitochondria occurs at the electron transport chain as derivatives of respiration. Cancer cells sustain ROS production by suppressing the antioxidant-generation system. Balance between ROS production and subsequent detoxification is regulated by scavenging enzymes and antioxidant agents. Failure in sirtuin-3 (SIRT3), ATM and p53 activities elevates the intracellular levels of ROS. PKCα induces the expression of NOX (DUOX) during cancer development and the consequent increase in ROS production. The PI3K/AKT signalling pathway activates NOX with consequent ROS production and subsequent induction of instability in the genome, leading to cancer. In conclusion, the interruption of the redox pathways that regulate ROS and its redox signalling activities affects cell physiology and can ultimately result in abnormal signalling, uncontrolled oxidative impairment and tumorigenesis.

Keywords: homeostasis, cancer, reactive oxygen species, mitochondrial electron transport chain (ETC), NOX, GSH, glutathione oxidase (GPX), superoxide dismutase (SOD), thioredoxin (TRX), sirtuins

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

© 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 eproduction in any medium, provided the original work is properly cited.

#### 1. Introduction

Reactive oxygen species (ROS) are known as oxygen free radicals, which greatly contribute in complex cellular pathways, such as metabolism, immune system regulation, proliferation, differentiation and vascular transformations [1, 2]. ROS have a short life span and possess unpaired electrons [3]. Oxidative stress, DNA damage and cancer occur as a result of ROS imbalance due to dysregulated generation of free radicals (ROS) from oxygen and inability to neutralise and detoxify the harmful effects caused by the free radicals in the body through counteracting their oxidative effect by antioxidants [4–6]. Under normal and healthy circumstances, ROS production and removal are strictly regulated and controlled by very effective defensive machinery that blocks excessive ROS production. Some ROS, such as superoxide anion radical (O2 ) and hydrogen peroxide (H2O2), are necessary life functions because they play a vital role in the regulation of cell defence mechanisms necessary for signalling, steroid synthesis, G-protein-coupled-receptor activation, gene expression and transcription factor regulation [7, 8]. Therefore, ROS can act both as good and bad molecules because of their dual nature and can either induce regulation of cellular physiology or promote the induction of cytotoxicity depending on generation levels, site of generation and magnitude of generation [9]. However, high ROS levels make cells vulnerable to damage. The derivatives from oxygen contain free radicals, such as superoxide anion (O2 ) and hydroxyl radical (OH) plus nonradical molecules, such as hypochlorous acid and H2O2 [3], which have been linked to oxidative injury due to their high reactivity potential against proteins, lipids and DNA [10].

inducible factor-1 and subsequent metabolic reprogramming [13, 14]. The ROS detoxification mechanism has provided selective advantage for its survival during pro-oxidation situations. Balance between ROS production and its quick detoxification are regulated by scavenging

Mitochondria generates 90% of the energy required for cells and tissues to function effectively and serves as the core site for energy metabolism in the cells, since it is involved in the generation of ATP via oxidative phosphorylation (OXPHOS) [15]. This process liberates electron from reducing substrates and delivers the electron to O2 leading to the establishment of electrochemical gradient which triggers the ATP synthesis. Oxidative phosphorylation in the mitochondria produces ROS with unpaired electrons due to electron reduction from the

dria which is formed due to monoelectronic O2 reduction. Most ROS originate from the

oxygenases, NADPH oxidase and xanthine oxidase or non-enzymatically by transferring an

ously or through superoxide dismutases (SODs) catalysed reaction to generate hydrogen peroxide (H2O2) [20–22]. The mitochondrial generated H2O2 has numerous probable fates. H2O2 is fairly stable and permeable to the membrane, and therefore, it can diffuse inside the cell and get eliminated by mitochondrial or cytosolic antioxidant systems, which are catalase, thioredoxin-peroxidase and glutathione-peroxidase [23]. Mitochondrially produced H2O2, also function as a cytosolic signalling molecule, thereby, affecting the networks that control energy metabolism, stress response, redox balance and cell cycle [24–26]. None metabolised H2O2 in the mitochondria undergoes Fenton reaction and then transformed subsequently into hydroxyl radical (•OH) which is naturally a very strong oxidant with high damaging impact on molecules due to its high reactive nature [27]. The above reason has made researchers to believe that mitochondria have developed competent systems for H2O2 removal and also mechanisms for metal chelating (chaperone proteins) which prevents the formation of radical. Bulk of the ROS generation in the mitochondria occurs at the electron transport chain (ETC) as derivatives of respiration [17, 18, 28]. The ETC terminal component known as cytochrome c oxidase (Complex IV) acquires four (4) electrons from cytochrome c and then reduces one molecule of O2 to form two H2O. All the intermediates that is partially reduced are retained

High exposure of the mitochondria to ROS results to injurious consequences such as inflicting oxidative mitochondrial DNA damage. It has also been suggested that ROS is deeply involved

•) is the major ROS produced in the human mitochon-

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• dismutation can occur spontane-

• could either be synthesised enzymatically by cyP450-dependent

• is capable of reacting with free radicals such as nitric oxide

enzymes and antioxidant agents that limit the accumulation of ROS in the body.

2. Roles of mitochondria in ROS formation

superoxide anion which also mediates oxidative chain reactions.

(NO•) to produce reactive nitrogen species (RNS) [19]. O2

oxygen [16–18]. Superoxide anion (O2

In vivo production of O2

electron directly to O2 [18]. O2

until reduction is fully achieved [16].

2.1. ROS and mitochondrial activation of apoptosis

The generation of ROS can be activated by either various endogenous or exogenous factors. The major source of endogenous ROS in cells of mammals is the mitochondrial electron transport chain (ETC). Other endogenous ROS sources are from the activities of NADPH oxidases (NOX), lipoxygenases (LOX), cytochrome P450 and xanthine oxidase (XO) [11]. Exogenous factors that contribute to ROS production are pollutants, chemicals/drugs, radiation and heavy metals [12]. Redox homeostasis is attained by cautious regulation of both ROS formation and removal from the body system [10]. Maintenance of homeostasis and signalling event of redox require the significant regulation of synthesis and detoxification. An interruption to the redox route that regulates ROS and its redox signalling activities affects cell physiology and can ultimately result in abnormal signalling, uncontrolled toxic by-product accumulation, oxidative impairment and cytotoxicity [2]. High oxidative stress levels are normally linked to abnormalities, which characterise tumour-specific modification that exposes the cancer cells to additional raise of ROS depending on the strength of their antioxidant defence system [13].

Cancer is one of the leading causes of death globally. Recent evidence suggests altered redox stability and dysregulated redox signalling as the two frequent hallmarks of cancers, which are implicated in the progression of malignancy and treatment resistance [13]. Cancer cells have been postulated to persistently exhibit high levels of reactive oxygen species (ROS) as a result of alterations in microenvironment, genetic mutations and dysregulation of metabolic processes [13]. This shift in pro-oxidant balance promotes tumour development by inflicting damage to DNA and causing inconsistency in the genome [1]. The DNA damage and instability induced to the genome activate an inflammatory reaction leading to stability of hypoxia inducible factor-1 and subsequent metabolic reprogramming [13, 14]. The ROS detoxification mechanism has provided selective advantage for its survival during pro-oxidation situations. Balance between ROS production and its quick detoxification are regulated by scavenging enzymes and antioxidant agents that limit the accumulation of ROS in the body.

#### 2. Roles of mitochondria in ROS formation

1. Introduction

124 Homeostasis - An Integrated Vision

anion radical (O2

contain free radicals, such as superoxide anion (O2

Reactive oxygen species (ROS) are known as oxygen free radicals, which greatly contribute in complex cellular pathways, such as metabolism, immune system regulation, proliferation, differentiation and vascular transformations [1, 2]. ROS have a short life span and possess unpaired electrons [3]. Oxidative stress, DNA damage and cancer occur as a result of ROS imbalance due to dysregulated generation of free radicals (ROS) from oxygen and inability to neutralise and detoxify the harmful effects caused by the free radicals in the body through counteracting their oxidative effect by antioxidants [4–6]. Under normal and healthy circumstances, ROS production and removal are strictly regulated and controlled by very effective defensive machinery that blocks excessive ROS production. Some ROS, such as superoxide

play a vital role in the regulation of cell defence mechanisms necessary for signalling, steroid synthesis, G-protein-coupled-receptor activation, gene expression and transcription factor regulation [7, 8]. Therefore, ROS can act both as good and bad molecules because of their dual nature and can either induce regulation of cellular physiology or promote the induction of cytotoxicity depending on generation levels, site of generation and magnitude of generation [9]. However, high ROS levels make cells vulnerable to damage. The derivatives from oxygen

radical molecules, such as hypochlorous acid and H2O2 [3], which have been linked to oxida-

The generation of ROS can be activated by either various endogenous or exogenous factors. The major source of endogenous ROS in cells of mammals is the mitochondrial electron transport chain (ETC). Other endogenous ROS sources are from the activities of NADPH oxidases (NOX), lipoxygenases (LOX), cytochrome P450 and xanthine oxidase (XO) [11]. Exogenous factors that contribute to ROS production are pollutants, chemicals/drugs, radiation and heavy metals [12]. Redox homeostasis is attained by cautious regulation of both ROS formation and removal from the body system [10]. Maintenance of homeostasis and signalling event of redox require the significant regulation of synthesis and detoxification. An interruption to the redox route that regulates ROS and its redox signalling activities affects cell physiology and can ultimately result in abnormal signalling, uncontrolled toxic by-product accumulation, oxidative impairment and cytotoxicity [2]. High oxidative stress levels are normally linked to abnormalities, which characterise tumour-specific modification that exposes the cancer cells to additional raise of ROS depending on the strength of their antioxidant defence system [13].

Cancer is one of the leading causes of death globally. Recent evidence suggests altered redox stability and dysregulated redox signalling as the two frequent hallmarks of cancers, which are implicated in the progression of malignancy and treatment resistance [13]. Cancer cells have been postulated to persistently exhibit high levels of reactive oxygen species (ROS) as a result of alterations in microenvironment, genetic mutations and dysregulation of metabolic processes [13]. This shift in pro-oxidant balance promotes tumour development by inflicting damage to DNA and causing inconsistency in the genome [1]. The DNA damage and instability induced to the genome activate an inflammatory reaction leading to stability of hypoxia

tive injury due to their high reactivity potential against proteins, lipids and DNA [10].

) and hydrogen peroxide (H2O2), are necessary life functions because they

) and hydroxyl radical (OH) plus non-

Mitochondria generates 90% of the energy required for cells and tissues to function effectively and serves as the core site for energy metabolism in the cells, since it is involved in the generation of ATP via oxidative phosphorylation (OXPHOS) [15]. This process liberates electron from reducing substrates and delivers the electron to O2 leading to the establishment of electrochemical gradient which triggers the ATP synthesis. Oxidative phosphorylation in the mitochondria produces ROS with unpaired electrons due to electron reduction from the oxygen [16–18]. Superoxide anion (O2 •) is the major ROS produced in the human mitochondria which is formed due to monoelectronic O2 reduction. Most ROS originate from the superoxide anion which also mediates oxidative chain reactions.

In vivo production of O2 • could either be synthesised enzymatically by cyP450-dependent oxygenases, NADPH oxidase and xanthine oxidase or non-enzymatically by transferring an electron directly to O2 [18]. O2 • is capable of reacting with free radicals such as nitric oxide (NO•) to produce reactive nitrogen species (RNS) [19]. O2 • dismutation can occur spontaneously or through superoxide dismutases (SODs) catalysed reaction to generate hydrogen peroxide (H2O2) [20–22]. The mitochondrial generated H2O2 has numerous probable fates. H2O2 is fairly stable and permeable to the membrane, and therefore, it can diffuse inside the cell and get eliminated by mitochondrial or cytosolic antioxidant systems, which are catalase, thioredoxin-peroxidase and glutathione-peroxidase [23]. Mitochondrially produced H2O2, also function as a cytosolic signalling molecule, thereby, affecting the networks that control energy metabolism, stress response, redox balance and cell cycle [24–26]. None metabolised H2O2 in the mitochondria undergoes Fenton reaction and then transformed subsequently into hydroxyl radical (•OH) which is naturally a very strong oxidant with high damaging impact on molecules due to its high reactive nature [27]. The above reason has made researchers to believe that mitochondria have developed competent systems for H2O2 removal and also mechanisms for metal chelating (chaperone proteins) which prevents the formation of radical. Bulk of the ROS generation in the mitochondria occurs at the electron transport chain (ETC) as derivatives of respiration [17, 18, 28]. The ETC terminal component known as cytochrome c oxidase (Complex IV) acquires four (4) electrons from cytochrome c and then reduces one molecule of O2 to form two H2O. All the intermediates that is partially reduced are retained until reduction is fully achieved [16].

#### 2.1. ROS and mitochondrial activation of apoptosis

High exposure of the mitochondria to ROS results to injurious consequences such as inflicting oxidative mitochondrial DNA damage. It has also been suggested that ROS is deeply involved in the extrinsic pathway of apoptosis. Extrinsic receptor-mediated pathway for cell death requires active engagement of the death receptors on the cell membrane surface with their corresponding ligands [29]. Receptor-mediated apoptotic pathway comprises of death receptors such as CD95 (Fas), TNF-related apoptosis-inducing ligand (TRAIL) receptors and TNF. Activation of Fas as well as TNFR1 generate ROS due to superoxide (O2•) production and formation of NADPH oxidase daises derived from lipid raft. Induction of apoptosis or necrosis is linked with lipid raft-mediated downstream ROS generation [30, 31]. Downregulation of FLIP (FLICE inhibitory protein), a strong inhibitor of apoptosis is mediated by ROS via ubiquitination and consequent proteasome degradation or by scavenging of nitric oxide (NO) to prevent FLIP S-nitrosation and cytoprotection [32]. ROS sensitises cancer cells to apoptosis induced by TRAIL [33]. CD95 and TRAIL death receptors have been observed to be highly upregulated in reaction in the presence of hydrogen peroxide via NF-kappa B activation [34]. ROS promote apoptosis via JNK activation, inducing either intrinsic or extrinsic apoptotic signalling [35]. TNFα induced ROS perpetrates oxidation of JNK, thereby, inactivatingphosphatases via catalytic transformation of their cysteine into sulfenic acid resulting to prolonged activation of JNK which is necessary for the release of cytochrome c and cleavage of caspase 3 as well as cell death [36]. TNFα activates MAPK cascade. ASK1, a redox-sensitive MAPK kinase, is located at the JNK upstream. Reduced thioredoxin1 (Trx1) binds to ASK1 during non-oxidising circumstances to form a complex known as ASK1 signalosome (Trx1/ ASK1 complex) which perform redox switch functions. Persistent cellular ROS causes detachment of the oxidised Trx1 from the Trx1/ASK1 complex leading to full ASK1 activation through TRAF2/6 recruitment [37]. ASK2 a member of ASK family attaches to ASK1 and stabilises it in mitochondria, nucleus and cytosol. Saxena et al. have revealed that redox protein known as thioredoxin interacting protein (TXNIP) with apoptosis promoting potential under oxidative stress, shuttles from the nucleus to the mitochondria leading to the removal of TXNIP from ASK1 and formation of a compound with mitochondrial Trx2. This suppression of the inhibition is mediated by Trx2 results in ASK1phosphorylation and induction of the mitochondrial pathway for apoptosis with caspase-3 cleavage and cytochrome c release [38]. The major target for ROS inside the mitochondria is the permeability transition pore (mPTP) in which the oxidative modification of its proteins has significant influence on the anion fluxes within the mitochondria [39]. This could cause overload of Ca2+ and ROS in reaction to proapoptotic stimuli causing mPTP to assume a very high state of conductance allowing unrestrained entry of solutes along the electrochemical gradient into the matrix of the mitochondria. The above phenomenon is termed mitochondrial permeability transition (MPT), which results in mitochondrial membrane potential dissipation and consequent osmotic swelling of the matrix of the mitochondria due to fluid influx [40]. The early phase of the mitochondrial swelling involves water movement from inter-cristae spaces into the mitochondrial matrix. Persistent movement of this water exacts pressure on the outer membrane due to increased volume of the matrix leading to mPTP opening and/or rupturing of the outer membrane of the mitochondria allowing the matrix to expand further [41]. This causes cytochrome c to be released with consequent activation of the downstream effector caspases by Apaf-1-procaspase 9-apoptosome complex.

3. Antioxidant system responsible for the redox homeostasis

glutathione peroxidase (GPX) [42]. SOD functions to convert O2

and ascorbic acid [42–45].

cancer [48–51].

3.2. Glutathione oxidase (GPX)

3.3. Thioredoxins (Trx)

3.1. Superoxide dismutases (SOD)

The antioxidant systems are either enzymatic or non-enzymatic. The enzymatic antioxidant system consists of peroxiredoxin (Prx) system, catalase, SOD and the glutathione peroxidase (GPx) system, while the non-enzymatic antioxidant systems consist of α-tocopherol, lipoic acid

Intracellular ROS levels are regulated by the balance between ROS generating enzymes and antioxidant enzymes, which include superoxide dismutases (SOD), catalase, thioredoxin and

converted into water by glutathione peroxidase or catalase. Human cells express three types of SOD: MnSOD (manganese SOD) expressed by the mitochondria, CuZnSOD (copper-zinc SOD) expressed by the cytoplasm and third is the extracellular SOD. A study has demonstrated that lack of MnSOD in mice generated excessive oxidative stress causing their mortality [46]. Another study also revealed that mice with a deficiency of CuZnSOD developed hepatocellular carcinoma due to sustained oxidative damage [47]. Lack of MnSOD has also been linked to elevated risk of lung cancer, prostate cancer, non-Hodgkin's lymphoma and ovarian

GPX is a selenium-dependent antioxidant enzyme, which regulates hydrogen and lipid peroxide levels. Lack of GPX in the body increases tissue damage by ROS [43] and low GPX levels, which results in increased LDL oxidation [44]. GPX catalyses the reduction of hydrogen peroxide to form glutathione disulphide (GSSG) with glutathione (GSH) functioning as the substrate. An increased risk of bladder cancer, lung cancer and breast cancer has been associ-

The protective function of thioredoxins (Trxs) in cells against oxidative stress is via the reaction between their active site known as 2-cysteine and ROS resulting in reduction of oxidised proteins. Trxs also function as hydrogen donors to thioredoxin-dependent peroxide reductases. Trx possesses a Cys-Gly-Pro-Cys active site, which is essential for redox regulatory functions of Trx. Trx, when combined with Trx reductase and NADPH, forms a redox-sensitive machinery, which controls the levels of oxidised cysteine on proteins. The antioxidant properties of Trx can be attributed to the reduction of the oxidised form of Trx peroxidase by Trx, while the reduced peroxidase scavenges H2O2 [45]. The two isoforms of Trx are Trx1 (expressed in the cytoplasm and the nucleus) and Trx2 (expressed in the mitochondria), which are very crucial for cell survival [56]. Trx1 is a redox-sensitive binding protein that controls the

ated with the substitution of proline-leucine at codon 198 in human GPX [52–55].

into H2O2, which is later

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### 3. Antioxidant system responsible for the redox homeostasis

The antioxidant systems are either enzymatic or non-enzymatic. The enzymatic antioxidant system consists of peroxiredoxin (Prx) system, catalase, SOD and the glutathione peroxidase (GPx) system, while the non-enzymatic antioxidant systems consist of α-tocopherol, lipoic acid and ascorbic acid [42–45].

#### 3.1. Superoxide dismutases (SOD)

in the extrinsic pathway of apoptosis. Extrinsic receptor-mediated pathway for cell death requires active engagement of the death receptors on the cell membrane surface with their corresponding ligands [29]. Receptor-mediated apoptotic pathway comprises of death receptors such as CD95 (Fas), TNF-related apoptosis-inducing ligand (TRAIL) receptors and TNF. Activation of Fas as well as TNFR1 generate ROS due to superoxide (O2•) production and formation of NADPH oxidase daises derived from lipid raft. Induction of apoptosis or necrosis is linked with lipid raft-mediated downstream ROS generation [30, 31]. Downregulation of FLIP (FLICE inhibitory protein), a strong inhibitor of apoptosis is mediated by ROS via ubiquitination and consequent proteasome degradation or by scavenging of nitric oxide (NO) to prevent FLIP S-nitrosation and cytoprotection [32]. ROS sensitises cancer cells to apoptosis induced by TRAIL [33]. CD95 and TRAIL death receptors have been observed to be highly upregulated in reaction in the presence of hydrogen peroxide via NF-kappa B activation [34]. ROS promote apoptosis via JNK activation, inducing either intrinsic or extrinsic apoptotic signalling [35]. TNFα induced ROS perpetrates oxidation of JNK, thereby, inactivatingphosphatases via catalytic transformation of their cysteine into sulfenic acid resulting to prolonged activation of JNK which is necessary for the release of cytochrome c and cleavage of caspase 3 as well as cell death [36]. TNFα activates MAPK cascade. ASK1, a redox-sensitive MAPK kinase, is located at the JNK upstream. Reduced thioredoxin1 (Trx1) binds to ASK1 during non-oxidising circumstances to form a complex known as ASK1 signalosome (Trx1/ ASK1 complex) which perform redox switch functions. Persistent cellular ROS causes detachment of the oxidised Trx1 from the Trx1/ASK1 complex leading to full ASK1 activation through TRAF2/6 recruitment [37]. ASK2 a member of ASK family attaches to ASK1 and stabilises it in mitochondria, nucleus and cytosol. Saxena et al. have revealed that redox protein known as thioredoxin interacting protein (TXNIP) with apoptosis promoting potential under oxidative stress, shuttles from the nucleus to the mitochondria leading to the removal of TXNIP from ASK1 and formation of a compound with mitochondrial Trx2. This suppression of the inhibition is mediated by Trx2 results in ASK1phosphorylation and induction of the mitochondrial pathway for apoptosis with caspase-3 cleavage and cytochrome c release [38]. The major target for ROS inside the mitochondria is the permeability transition pore (mPTP) in which the oxidative modification of its proteins has significant influence on the anion fluxes within the mitochondria [39]. This could cause overload of Ca2+ and ROS in reaction to proapoptotic stimuli causing mPTP to assume a very high state of conductance allowing unrestrained entry of solutes along the electrochemical gradient into the matrix of the mitochondria. The above phenomenon is termed mitochondrial permeability transition (MPT), which results in mitochondrial membrane potential dissipation and consequent osmotic swelling of the matrix of the mitochondria due to fluid influx [40]. The early phase of the mitochondrial swelling involves water movement from inter-cristae spaces into the mitochondrial matrix. Persistent movement of this water exacts pressure on the outer membrane due to increased volume of the matrix leading to mPTP opening and/or rupturing of the outer membrane of the mitochondria allowing the matrix to expand further [41]. This causes cytochrome c to be released with consequent activation of the downstream effector caspases by Apaf-1-pro-

caspase 9-apoptosome complex.

126 Homeostasis - An Integrated Vision

Intracellular ROS levels are regulated by the balance between ROS generating enzymes and antioxidant enzymes, which include superoxide dismutases (SOD), catalase, thioredoxin and glutathione peroxidase (GPX) [42]. SOD functions to convert O2 into H2O2, which is later converted into water by glutathione peroxidase or catalase. Human cells express three types of SOD: MnSOD (manganese SOD) expressed by the mitochondria, CuZnSOD (copper-zinc SOD) expressed by the cytoplasm and third is the extracellular SOD. A study has demonstrated that lack of MnSOD in mice generated excessive oxidative stress causing their mortality [46]. Another study also revealed that mice with a deficiency of CuZnSOD developed hepatocellular carcinoma due to sustained oxidative damage [47]. Lack of MnSOD has also been linked to elevated risk of lung cancer, prostate cancer, non-Hodgkin's lymphoma and ovarian cancer [48–51].

#### 3.2. Glutathione oxidase (GPX)

GPX is a selenium-dependent antioxidant enzyme, which regulates hydrogen and lipid peroxide levels. Lack of GPX in the body increases tissue damage by ROS [43] and low GPX levels, which results in increased LDL oxidation [44]. GPX catalyses the reduction of hydrogen peroxide to form glutathione disulphide (GSSG) with glutathione (GSH) functioning as the substrate. An increased risk of bladder cancer, lung cancer and breast cancer has been associated with the substitution of proline-leucine at codon 198 in human GPX [52–55].

#### 3.3. Thioredoxins (Trx)

The protective function of thioredoxins (Trxs) in cells against oxidative stress is via the reaction between their active site known as 2-cysteine and ROS resulting in reduction of oxidised proteins. Trxs also function as hydrogen donors to thioredoxin-dependent peroxide reductases. Trx possesses a Cys-Gly-Pro-Cys active site, which is essential for redox regulatory functions of Trx. Trx, when combined with Trx reductase and NADPH, forms a redox-sensitive machinery, which controls the levels of oxidised cysteine on proteins. The antioxidant properties of Trx can be attributed to the reduction of the oxidised form of Trx peroxidase by Trx, while the reduced peroxidase scavenges H2O2 [45]. The two isoforms of Trx are Trx1 (expressed in the cytoplasm and the nucleus) and Trx2 (expressed in the mitochondria), which are very crucial for cell survival [56]. Trx1 is a redox-sensitive binding protein that controls the activity of NF-κB through the reduction of cys62 on the p50 of NF-κB [57]. Trx1 has been linked to breast tumours, colon cancer, cervical cancer, gastric cancer, lung cancer, liver cancer and melanoma and carcinomas of the pancreas [58–62].

elements of GSH, such as glutamate, cysteine and glycine [64, 65]. The negative regulator of cysteine/glutamate known as SLC7A11 is always upregulated in human tumours [66]. Glutamate cysteine ligase modifier subunit (GCLM) is also upregulated in many types of human cancer but also requisite for effective GSH synthesis [67]. The cellular levels of GSH and its regeneration are modulated by NADPH and GR catalysing the reduction of oxidised GSSG back to GSH, in a process facilitated by the upregulation of NADPH production by cancer cells (Figure 1). Maintenance and elevation of GSH levels in cells are critical for the initiation and proliferation of tumours [67, 68]. Loss of GSH, or decrease in the ratio of glutathione to glutathione disulphide (GSH:GSSG), results in increased oxidative stress susceptibility and cancer development. Also, elevated levels of GSH increase antioxidant activities against numerous cancer cells, thereby enhancing the resistance of the cancer cells against oxidative

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Prxs are made up of six isoenzyme families capable of reducing H2O2 and alkyl hydroperoxides to their resultant H2O or alcohol. Prxs are essential antioxidants that mediate the balancing mechanism of cellular H2O2 production, which is necessary for signalling and cell metabolism [70]. Nrf2 upregulates Prxs in oxidative stress circumstances [71]. PRDX1 plays the role of tumour suppressor in the development of breast cancer by interacting with oncogene (c-Myc) suppressing its transcriptional action [72, 73]. Contrarily, PRDX1 has promotional activities on pancreatic carcinoma, hepatocellular cancer, oesophageal cancer, oral cancer and lung cancer via upregulation of heme-oxygenase 1 and NF-κB pathway activation [74–77]. PRDX2 stimulates colorectal carcinoma by upregulating Wnt/β catenin levels, while it stimulates

Some of the enzymes associated with redox homeostasis are NADPH oxidase, ATM kinase

NADPH oxidase is hetero-proteins, which consist of seven isoforms. ROS production by NADPH oxidase is via the NOX protein. NADPH oxidases are referred collectively as the NOX family. The NOX family is comprised of NOX (NOX1, NOX2, NOX3, NOX4 andNOX5) and dual oxidases (DUOX1 and DUOX2) [80–82]. The isoforms DUOX1 and DUOX2 contain additional peroxidase domains, which exert the catalytic dismutation of superoxide anion to yield H2O2 [74]. Cytosolic electron transfer from NADPH across the cell membrane is catalysed by NADPH oxidase, thereby oxidising the molecular oxygen, which is later reduced

mitochondria plays a critical role in metastasis. In many tumour cells, reductive carboxylation in the mitochondria to generate NADPH is powered by mitochondrial citrate transporter

). Generation of NADPH in the

prostate cancer by upregulating the receptive activities of androgen [78, 79].

4. Enzymes responsible for the redox homeostasis

to generate ROS species called superoxide anion radical (O2

stress [69].

3.5. Peroxiredoxins (Prxs)

and sirtuin-3 among others.

4.1. NADPH oxidase

#### 3.4. GSH

GSH is a thiol protein consisting of cysteine, glutamine and glycine, which functions as an important antioxidant in detoxification of metabolic processes [63]. Elevated levels of GSH in the cancerous patient tissue are reinforced by improved accessibility to the biosynthetic

Figure 1. Mechanism of ROS formation and disposal. Enzymatically or non-enzymatically generated superoxide reacts with other radicals. The generated superoxide and H2O2 form hydroxyl radicals and singlet oxygen. SODs catalyse the dismutation of the superoxide, while H2O2 decomposition to water is catalysed by catalases in conjunction with glutathione peroxidase/GPx and Prxs. Thiol, a component of cysteine found in Prx, is oxidatively converted into Cys-sulfenic acid by (Prxox) and subsequently reduced by thioredoxin (Trxred). H2O2 further oxidises Cys-sulfenic acid (Prxox) into Cyssulfinic acid and reduced subsequently back by ATP and reduced sulfiredoxin (Srxred) to Cys-sulfenic acid (Cys-SOH). Cys-SOH formed functions to regulate protein activities by absorbing the oxidative insults leading to the deflection of injurious oxidative impairment [24]. Cys-SOH can be converted into oxidative post-translational modification (Ox-PTM) in redox environment. This modification reacts with tripeptide glutathione (Glu-Cys-Gly) to form S-glutathionylated Cys (GSSG) or react with thiol to form disulphide bond. Formation of GSSG protects the Cys of the host from further oxidative reactions.

elements of GSH, such as glutamate, cysteine and glycine [64, 65]. The negative regulator of cysteine/glutamate known as SLC7A11 is always upregulated in human tumours [66]. Glutamate cysteine ligase modifier subunit (GCLM) is also upregulated in many types of human cancer but also requisite for effective GSH synthesis [67]. The cellular levels of GSH and its regeneration are modulated by NADPH and GR catalysing the reduction of oxidised GSSG back to GSH, in a process facilitated by the upregulation of NADPH production by cancer cells (Figure 1). Maintenance and elevation of GSH levels in cells are critical for the initiation and proliferation of tumours [67, 68]. Loss of GSH, or decrease in the ratio of glutathione to glutathione disulphide (GSH:GSSG), results in increased oxidative stress susceptibility and cancer development. Also, elevated levels of GSH increase antioxidant activities against numerous cancer cells, thereby enhancing the resistance of the cancer cells against oxidative stress [69].

#### 3.5. Peroxiredoxins (Prxs)

activity of NF-κB through the reduction of cys62 on the p50 of NF-κB [57]. Trx1 has been linked to breast tumours, colon cancer, cervical cancer, gastric cancer, lung cancer, liver cancer and

GSH is a thiol protein consisting of cysteine, glutamine and glycine, which functions as an important antioxidant in detoxification of metabolic processes [63]. Elevated levels of GSH in the cancerous patient tissue are reinforced by improved accessibility to the biosynthetic

Figure 1. Mechanism of ROS formation and disposal. Enzymatically or non-enzymatically generated superoxide reacts with other radicals. The generated superoxide and H2O2 form hydroxyl radicals and singlet oxygen. SODs catalyse the dismutation of the superoxide, while H2O2 decomposition to water is catalysed by catalases in conjunction with glutathione peroxidase/GPx and Prxs. Thiol, a component of cysteine found in Prx, is oxidatively converted into Cys-sulfenic acid by (Prxox) and subsequently reduced by thioredoxin (Trxred). H2O2 further oxidises Cys-sulfenic acid (Prxox) into Cyssulfinic acid and reduced subsequently back by ATP and reduced sulfiredoxin (Srxred) to Cys-sulfenic acid (Cys-SOH). Cys-SOH formed functions to regulate protein activities by absorbing the oxidative insults leading to the deflection of injurious oxidative impairment [24]. Cys-SOH can be converted into oxidative post-translational modification (Ox-PTM) in redox environment. This modification reacts with tripeptide glutathione (Glu-Cys-Gly) to form S-glutathionylated Cys (GSSG) or react with thiol to form disulphide bond. Formation of GSSG protects the Cys of the host from further oxidative

melanoma and carcinomas of the pancreas [58–62].

3.4. GSH

128 Homeostasis - An Integrated Vision

reactions.

Prxs are made up of six isoenzyme families capable of reducing H2O2 and alkyl hydroperoxides to their resultant H2O or alcohol. Prxs are essential antioxidants that mediate the balancing mechanism of cellular H2O2 production, which is necessary for signalling and cell metabolism [70]. Nrf2 upregulates Prxs in oxidative stress circumstances [71]. PRDX1 plays the role of tumour suppressor in the development of breast cancer by interacting with oncogene (c-Myc) suppressing its transcriptional action [72, 73]. Contrarily, PRDX1 has promotional activities on pancreatic carcinoma, hepatocellular cancer, oesophageal cancer, oral cancer and lung cancer via upregulation of heme-oxygenase 1 and NF-κB pathway activation [74–77]. PRDX2 stimulates colorectal carcinoma by upregulating Wnt/β catenin levels, while it stimulates prostate cancer by upregulating the receptive activities of androgen [78, 79].

#### 4. Enzymes responsible for the redox homeostasis

Some of the enzymes associated with redox homeostasis are NADPH oxidase, ATM kinase and sirtuin-3 among others.

#### 4.1. NADPH oxidase

NADPH oxidase is hetero-proteins, which consist of seven isoforms. ROS production by NADPH oxidase is via the NOX protein. NADPH oxidases are referred collectively as the NOX family. The NOX family is comprised of NOX (NOX1, NOX2, NOX3, NOX4 andNOX5) and dual oxidases (DUOX1 and DUOX2) [80–82]. The isoforms DUOX1 and DUOX2 contain additional peroxidase domains, which exert the catalytic dismutation of superoxide anion to yield H2O2 [74]. Cytosolic electron transfer from NADPH across the cell membrane is catalysed by NADPH oxidase, thereby oxidising the molecular oxygen, which is later reduced to generate ROS species called superoxide anion radical (O2 ). Generation of NADPH in the mitochondria plays a critical role in metastasis. In many tumour cells, reductive carboxylation in the mitochondria to generate NADPH is powered by mitochondrial citrate transporter (mCTP), cytosolic isocitrate dehydrogenase (IDH1) and mitochondrial isocitrate dehydrogenase (IDH2). This development assists cells to retain redox balance in the mitochondria averting the oxidative trauma received as a result of the detachment from the extracellular matrix [75].

5.1. PKC pathways

influencing the differentiation of tumour cells [90].

the process leading to invasion/metastasis.

PKC has isoenzymes, such as PKCα, PKCβ and PKCδ, with conflicting actions in different cancers [89]. PKCβ is the isoenzyme of PKC responsible for the stimulation and phosphorylation of p66/shc, which binds to cytochrome c in order to activate ROS generation [90]. Recent studies demonstrated that PKCα induces the expression of DUOX (a member of NOX family) during cancer development and subsequent ROS production [91, 92]. PKCδ has been demonstrated to be involved in the activation of NOX through the alteration of redox balance, thereby

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Figure 2. Effect of ROS imbalance in some pathways. Imbalance in the levels of ROS in the cell causes inhibition of PTEN and PTP dependent phosphorylation and consequent inactivation of FAK. The PI3K/Akt and PKC signals are activated in

#### 4.2. ATM kinase

ROS production can be inhibited by ATM kinase and the same ATM kinase also serves as the function of redox-regulated DNA damage sensing protein [76]. ATM-regulated tumour suppressor works by interfering with KEAP1-facilitated NRF2 ubiquitination, thereby activating and stabilising the major regulators of antioxidants [77]. ATM facilitates the upregulation of glucose-6-phosphate dehydrogenase in order to promote NADPH production, thereby suppressing ROS levels [83].

#### 4.3. Sirtuin-3

ROS levels are inherently elevated in cancer cells owing to mitochondrial defective oxidative metabolism [84]. Upregulated oxidative signals are implicated in the development and advancement of different cancer types [85]. Raised levels of ROS contribute to the initiation of cancer, transformation to malignancy and therapy resistance. ROS inflicted damage is more frequently seen in mitochondrial DNA than nuclear DNA because of its closeness to the main ROS source of generation and inadequate restoring capacities. For instance, silent information regulators of gene transcription-3 (sirtuin-3) are crucial in ROS regulation and effective flow of electrons via ETC. Failure in sirtuin-3 activities elevates intracellular levels of ROS, thereby inducing instability to the DNA of the mitochondria [86]. Sirtuins are an enzyme family, which is dependent on NAD-class III histone deacetylase. Seven homologues of Sirtuins (SIRT1–7) exist in mammals [87]. SIRT1 deacetylate gene regulates proteins like p53, forkhead proteins and NF-κB, which modulate resistance of cells to stress [36]. The deacetylase function of sirtuin proteins depends on the intracellular or endogenous content of NAD<sup>+</sup> [87]. Sirtuins are involved in the catalysis of exclusive reactions that lead to the formation of deacetylated substrate, acetyl ADP-ribose (AADPR) and nicotinamide [87]. Also, SIRT1 interrupts apoptosis, rescuing vulnerable cells after repetitive oxidative stress exposure [60]. SIRT2 deacetylate cytoskeletal proteins, such as forkhead proteins, histones, etc. SIRT3 reacts to redox status changes in the mitochondria by influencing the enzyme activities of manganese superoxide dismutase (MnSOD), which in turn scavenges ROS in the mitochondria and thus modulating the levels of ROS and metabolic homeostatic reliability [87].

#### 5. Pathways implicated in redox homeostasis

Dysregulations associated with various tumour proliferations, autophagy and apoptosis depend on the activation of targets sensitive to redox reactions, such as PKC, Akt, PTEN, p53, etc. [88].

#### 5.1. PKC pathways

(mCTP), cytosolic isocitrate dehydrogenase (IDH1) and mitochondrial isocitrate dehydrogenase (IDH2). This development assists cells to retain redox balance in the mitochondria averting the oxidative trauma received as a result of the detachment from the extracellular

ROS production can be inhibited by ATM kinase and the same ATM kinase also serves as the function of redox-regulated DNA damage sensing protein [76]. ATM-regulated tumour suppressor works by interfering with KEAP1-facilitated NRF2 ubiquitination, thereby activating and stabilising the major regulators of antioxidants [77]. ATM facilitates the upregulation of glucose-6-phosphate dehydrogenase in order to promote NADPH production, thereby

ROS levels are inherently elevated in cancer cells owing to mitochondrial defective oxidative metabolism [84]. Upregulated oxidative signals are implicated in the development and advancement of different cancer types [85]. Raised levels of ROS contribute to the initiation of cancer, transformation to malignancy and therapy resistance. ROS inflicted damage is more frequently seen in mitochondrial DNA than nuclear DNA because of its closeness to the main ROS source of generation and inadequate restoring capacities. For instance, silent information regulators of gene transcription-3 (sirtuin-3) are crucial in ROS regulation and effective flow of electrons via ETC. Failure in sirtuin-3 activities elevates intracellular levels of ROS, thereby inducing instability to the DNA of the mitochondria [86]. Sirtuins are an enzyme family, which is dependent on NAD-class III histone deacetylase. Seven homologues of Sirtuins (SIRT1–7) exist in mammals [87]. SIRT1 deacetylate gene regulates proteins like p53, forkhead proteins and NF-κB, which modulate resistance of cells to stress [36]. The deacetylase function of sirtuin proteins depends on the intracellular or endogenous content of NAD<sup>+</sup> [87]. Sirtuins are involved in the catalysis of exclusive reactions that lead to the formation of deacetylated substrate, acetyl ADP-ribose (AADPR) and nicotinamide [87]. Also, SIRT1 interrupts apoptosis, rescuing vulnerable cells after repetitive oxidative stress exposure [60]. SIRT2 deacetylate cytoskeletal proteins, such as forkhead proteins, histones, etc. SIRT3 reacts to redox status changes in the mitochondria by influencing the enzyme activities of manganese superoxide dismutase (MnSOD), which in turn scavenges ROS in the mitochondria and thus modulating

Dysregulations associated with various tumour proliferations, autophagy and apoptosis depend on the activation of targets sensitive to redox reactions, such as PKC, Akt, PTEN, p53,

the levels of ROS and metabolic homeostatic reliability [87].

5. Pathways implicated in redox homeostasis

matrix [75].

4.2. ATM kinase

130 Homeostasis - An Integrated Vision

4.3. Sirtuin-3

etc. [88].

suppressing ROS levels [83].

PKC has isoenzymes, such as PKCα, PKCβ and PKCδ, with conflicting actions in different cancers [89]. PKCβ is the isoenzyme of PKC responsible for the stimulation and phosphorylation of p66/shc, which binds to cytochrome c in order to activate ROS generation [90]. Recent studies demonstrated that PKCα induces the expression of DUOX (a member of NOX family) during cancer development and subsequent ROS production [91, 92]. PKCδ has been demonstrated to be involved in the activation of NOX through the alteration of redox balance, thereby influencing the differentiation of tumour cells [90].

Figure 2. Effect of ROS imbalance in some pathways. Imbalance in the levels of ROS in the cell causes inhibition of PTEN and PTP dependent phosphorylation and consequent inactivation of FAK. The PI3K/Akt and PKC signals are activated in the process leading to invasion/metastasis.

#### 5.2. PI3K/AKT pathway

PI3K/AKT signalling pathway activates NOX with consequent ROS production and subsequent induction of instability to the genome of cancer cells [91]. Upregulation of PTEN suppresses ROS synthesis, thus regulating PI3K/AKT pathway [92]. ROS-mediated PTEN inactivity alters kinasephosphatase stability favouring the signalling of tumorigenic-tyrosine kinase receptor via Akt (Figure 2) leading to the inhibition of apoptosis due to phosphorylation and inactivation of Bad and caspase-9 [93]. Akt improves cell survival by negatively regulating the activities of Bcl-2 homology domain 3 (BH3)-only proteins via binding and inactivation of pro-survival Bcl-2 family members. Akt survival effects on cells depend on the S136 phosphorylation on BAD [94]. Akt-mediated BAD phosphorylation is stimulated by survival factors on S136 leading to the creation of 14-3-3 protein binding site causing BAD to miss its protein target [94]. Akt phosphorylates FOXO proteins (FOXO1, FOXO3a and FOXO4) on T24 and S256 attaching onto 14-3-3 proteins in the cell nucleus causing displacement of transcription factors of FOXO from their gene target and consequent export out of the nucleus. This results in the blocking FOXO facilitated transcription of genes that can stimulate apoptotic processes and cell-cycle arrest, thereby encouraging cell survival. Akt also promotes survival by targeting HDM2 causing inhibition of BH3 only proteins by triggering degradation of p53. Akt induces phosphorylation of HDM2 on S166 and S186, causing HDM2 translocation to the nucleus to regulate p53 function negatively [94]. Deficiency of p53 in cancer results in higher cytokine transcription and consequent accumulation of ROS [95]. p53 is another tumour suppressor that has the potential to activate NRF2 and elevate antioxidant enzyme (SOD, GPX1 and NADPH) expression, thereby reactivating the antioxidant system (Figure 1) [10, 96]. Previous study has shown that 53 plays a pro oxidant role through the reduction of SLC7A11 expression, which is responsible for cysteine uptake during GSH synthesis [87]. Thus, the antioxidant activity of p53 is necessary because of its ability to avert cancer, thus implicating loss of tumour suppressors in upregulated intracellular ROS expression.

Conflict of interest

Abbreviations

All authors declared that there is no conflict of interests.

Reactive Oxygen Species, Cellular Redox Homeostasis and Cancer

http://dx.doi.org/10.5772/intechopen.76096

133

mETC mitochondrial electron transport chain

GCLM glutamate cysteine ligase modifier subunit

GCLM glutamate cysteine ligase modifier subunit Ox-PTM oxidative post-translational modification

Sirtuin-3 silent information regulators of gene transcription-3

mCTP mitochondrial citrate transporter

IDH isocitrate dehydrogenase

FAK focal adhesion kinase BAD Bcl-2-associated death

ATM ataxia telangiectasia mutated

ROS reactive oxygen species SOD superoxide dismutases

GPX glutathione oxidase

NOX NADPH oxidases

XO xanthine oxidase

MnSOD manganese SOD CuZnSOD copper-zinc SOD

PRDXs peroxiredoxins

DUOX dual oxidases

GSSG S-glutathionylated Cys

GR glutathione reductase

LOX lipoxygenases

Prx peroxiredoxin

TRX thioredoxin

GSH glutathione

#### 6. Conclusion

Redox homeostasis is achieved by the regulation of both ROS formation and removal. Shifts in ROS balance induce oxidative injury and tumour development. The balance between ROS production and subsequent detoxification is regulated by scavenging enzymes and antioxidant agents. Targeting the ROS generation pathway with anticancer medications can aid patient recuperation. Therefore, the modulation of ROS levels is a modern anticancer therapy. Further studies are needed to determine when ROS inhibition and activation can be applied in clinical cancer treatment.

#### Acknowledgements

The authors would like to thank Universiti Sains Malaysia Bridging Grant: 304/CIPPT/6316184 and Dr. Norehan Mokhtar, The Director of Advanced Medical and Dental Institute, Universiti Sains Malaysia for supporting this work.

#### Conflict of interest

5.2. PI3K/AKT pathway

132 Homeostasis - An Integrated Vision

6. Conclusion

cancer treatment.

Acknowledgements

Sains Malaysia for supporting this work.

PI3K/AKT signalling pathway activates NOX with consequent ROS production and subsequent induction of instability to the genome of cancer cells [91]. Upregulation of PTEN suppresses ROS synthesis, thus regulating PI3K/AKT pathway [92]. ROS-mediated PTEN inactivity alters kinasephosphatase stability favouring the signalling of tumorigenic-tyrosine kinase receptor via Akt (Figure 2) leading to the inhibition of apoptosis due to phosphorylation and inactivation of Bad and caspase-9 [93]. Akt improves cell survival by negatively regulating the activities of Bcl-2 homology domain 3 (BH3)-only proteins via binding and inactivation of pro-survival Bcl-2 family members. Akt survival effects on cells depend on the S136 phosphorylation on BAD [94]. Akt-mediated BAD phosphorylation is stimulated by survival factors on S136 leading to the creation of 14-3-3 protein binding site causing BAD to miss its protein target [94]. Akt phosphorylates FOXO proteins (FOXO1, FOXO3a and FOXO4) on T24 and S256 attaching onto 14-3-3 proteins in the cell nucleus causing displacement of transcription factors of FOXO from their gene target and consequent export out of the nucleus. This results in the blocking FOXO facilitated transcription of genes that can stimulate apoptotic processes and cell-cycle arrest, thereby encouraging cell survival. Akt also promotes survival by targeting HDM2 causing inhibition of BH3 only proteins by triggering degradation of p53. Akt induces phosphorylation of HDM2 on S166 and S186, causing HDM2 translocation to the nucleus to regulate p53 function negatively [94]. Deficiency of p53 in cancer results in higher cytokine transcription and consequent accumulation of ROS [95]. p53 is another tumour suppressor that has the potential to activate NRF2 and elevate antioxidant enzyme (SOD, GPX1 and NADPH) expression, thereby reactivating the antioxidant system (Figure 1) [10, 96]. Previous study has shown that 53 plays a pro oxidant role through the reduction of SLC7A11 expression, which is responsible for cysteine uptake during GSH synthesis [87]. Thus, the antioxidant activity of p53 is necessary because of its ability to avert cancer, thus

implicating loss of tumour suppressors in upregulated intracellular ROS expression.

Redox homeostasis is achieved by the regulation of both ROS formation and removal. Shifts in ROS balance induce oxidative injury and tumour development. The balance between ROS production and subsequent detoxification is regulated by scavenging enzymes and antioxidant agents. Targeting the ROS generation pathway with anticancer medications can aid patient recuperation. Therefore, the modulation of ROS levels is a modern anticancer therapy. Further studies are needed to determine when ROS inhibition and activation can be applied in clinical

The authors would like to thank Universiti Sains Malaysia Bridging Grant: 304/CIPPT/6316184 and Dr. Norehan Mokhtar, The Director of Advanced Medical and Dental Institute, Universiti All authors declared that there is no conflict of interests.

#### Abbreviations



#### Author details

Rabiatul Basria S.M.N. Mydin<sup>1</sup> \* and Simon I. Okekpa1,2

\*Address all correspondence to: rabiatulbasria@usm.my

1 Oncological and Radiological Sciences Cluster, Advanced Medical and Dental Institute, Universiti Sains Malaysia, Kepala Batas, Pulau Pinang Malaysia

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**Chapter 8**

**Provisional chapter**

**S6 Kinase: A Compelling Prospect for Therapeutic**

**S6 Kinase: A Compelling Prospect for Therapeutic** 

DOI: 10.5772/intechopen.75209

S6 kinase, a member of AGC family of protein kinases and a downstream effector of mTORC1 pathway has over the years found much relevance in maintaining a normal cellular state by virtue of its established role in regulation of cell growth and proliferation. S6 kinase activity has been linked to different cellular processes like glucose homeostasis, translational and transcriptional regulation. Hence any dysregulation in S6K1 leads to the emergence of various pathological conditions like diabetes, cancer and obesity. It is as such S6 kinase has emerged as a potential target for therapeutic interventions employed in curing such diseases. The Present Chapter reviews the regulation of S6K1, its structural organization and functions, besides highlighting its potential to act as an alternative therapeutic target for various cancerous situations exhibiting deranged mTOR signaling so as to overcome the possibility of relapses observed otherwise while using conven-

**Keywords:** S6 kinase, mTOR, rapamycin, cancer, cell growth and proliferation

Cell signaling pathways function as cohorts to contribute in maintaining cellular homeostasis-a phenomenon that remains pivotal in regulating cell growth and survival. The unique but immensely coordinated response of these diverse signals is primarily regulated by the availability of nutrients, growth factors and energy status of the cell. The signals destined to activate certain cellular functions may simultaneously be antagonistic for other functions; to strike a much required balance for proper functioning of the cell. Relentless efforts

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

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

**Interventions**

and Khurshid I Andrabi

**Abstract**

tional drugs

**1. Introduction**

**Interventions**

Sheikh Tahir Majeed, Rabiya Majeed, Ghazia Shah and Khurshid I Andrabi

http://dx.doi.org/10.5772/intechopen.75209

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

Sheikh Tahir Majeed, Rabiya Majeed, Ghazia Shah

#### **S6 Kinase: A Compelling Prospect for Therapeutic Interventions S6 Kinase: A Compelling Prospect for Therapeutic Interventions**

DOI: 10.5772/intechopen.75209

Sheikh Tahir Majeed, Rabiya Majeed, Ghazia Shah and Khurshid I Andrabi Sheikh Tahir Majeed, Rabiya Majeed, Ghazia Shah and Khurshid I Andrabi

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

http://dx.doi.org/10.5772/intechopen.75209

#### **Abstract**

S6 kinase, a member of AGC family of protein kinases and a downstream effector of mTORC1 pathway has over the years found much relevance in maintaining a normal cellular state by virtue of its established role in regulation of cell growth and proliferation. S6 kinase activity has been linked to different cellular processes like glucose homeostasis, translational and transcriptional regulation. Hence any dysregulation in S6K1 leads to the emergence of various pathological conditions like diabetes, cancer and obesity. It is as such S6 kinase has emerged as a potential target for therapeutic interventions employed in curing such diseases. The Present Chapter reviews the regulation of S6K1, its structural organization and functions, besides highlighting its potential to act as an alternative therapeutic target for various cancerous situations exhibiting deranged mTOR signaling so as to overcome the possibility of relapses observed otherwise while using conventional drugs

**Keywords:** S6 kinase, mTOR, rapamycin, cancer, cell growth and proliferation

#### **1. Introduction**

Cell signaling pathways function as cohorts to contribute in maintaining cellular homeostasis-a phenomenon that remains pivotal in regulating cell growth and survival. The unique but immensely coordinated response of these diverse signals is primarily regulated by the availability of nutrients, growth factors and energy status of the cell. The signals destined to activate certain cellular functions may simultaneously be antagonistic for other functions; to strike a much required balance for proper functioning of the cell. Relentless efforts

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

over the last several decades have led to recognize the ability of these signaling networks to promote cell growth and proliferation by way of their potential to phosphorylate one another to distinguish themselves as kinases. The kinases specifically transfer the phosphoryl group from ATP to their substrates such as to bring about a change in their function that helps transduce the upstream signals for the accomplishment of functions. Intrigued by the properties of these kinases, the scientific community over the years has shown great interest in identifying more kinases that lead to the identification of a family of protein kinases known as ribosomal S6 kinases (RSKs) that respond to nutrients and growth factors. The RSKs came to fore after studying the inducible phosphorylations on ribosomal protein S6 (rps6) and accordingly p90 ribosomal S6 kinase was identified from Xenopus oocytes. The homologous counterparts of p90 S6 kinase in humans were soon identified and grouped into family of four enzymes termed as RSK1-4. It was later on observed that the RSKs may play a minor role in rps6 phosphorylation [1] to give way for the emergence of a major rps6 kinase identified as 70 kDa ribosomal S6 kinase [1–4]. The 70 kDa ribosomal S6 kinase was purified from mitogen stimulated Swiss mouse 3T3 cells [5]. Cloning of 70 kDa ribosomal S6 kinase was soon accomplished after protein sequencing of a rat and rabbit cDNA [6–8]. Subsequent identification and cloning of human orthologs of S6K genes RPS6KB1 and RPS6KB2 encoding S6K1 and S6K2 thus, respectively, led to the emergence of a second family of S6 kinases, a principal kinase of rps6 in somatic cells [2, 9–14].

them to exhibit more or less a similar mode of regulation. These kinases fundamentally exhibit bi-lobal structural organization around the kinase domain. This structural arrangement enables their amino and carboxyl termini to coordinate ATP binding [17, 18]. Further these kinases comprise of an activation loop (commonly known as T-loop) that precedes their C-terminus lobe. The phosphorylation of T-loop brings in conformational changes important for phosphoryl transfer. Two other important phosphorylation sites, the turn motif (TM) (sonamed due to its location at the cusp of a structural turn in the PKA tail) and hydrophobic motif (HM) sequentially follow the kinase domain. The phosphorylated HM site engages a hydrophobic pocket within the N-lobe. The phosphorylated TM site stabilizes phospho-HM binding to the N-lobe hydrophobic pocket. Together, these three phosphorylations stand criti-

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http://dx.doi.org/10.5772/intechopen.75209

145

RPS6KB1, human ortholog of S6K1 genes, encodes two isoforms, p70S6K1 and p85S6K1 (formerly known as p70S6KII and p70S6KI, respectively) through alternative translational start sites [10]. p70S6K1, a 502 amino acid protein and its larger isoform p85S6K1, having nuclear localization sequence (NLS) within the 23 amino acid N-terminal extension, were earlier believed to remain localized in the cytoplasm and nucleus, respectively. However, the data generated of late, contests the nuclear localization of p85S6K1 while showing its sub-cellular localization to be cytoplasmic and at the same time claims p70S6K1 localization to be both nuclear and cytoplasmic [19]. S6K2, encoded by RPS6KB2 gene, constitutes

**Figure 1.** S6K isoforms, structure and domain organization with potential phosphorylating kinases along with their sites of action. S6K1 isoforms include p70-, p85- and p31-S6K1; alternative start site usage lengthens the p85- and p31- S6K1 N-termini by 23 amino acids (note that p31-S6K1 lacks most of the kinase domain). S6K2 isoforms include p54 and p56-S6K2; alternative start site usage lengthens the p56-S6K2 N-terminus by 13 amino acids. NLSs lie within the N-terminal extensions of p85-S6K1 and p56-S6K2, whereas S6K2 additionally contains an NLS within the C-terminus as well as a proline-rich domain (Pro). S6Ks contain an acidic N-terminal domain (NTD), kinase domain (KD), linker region and acidic C-terminal domain (CTD). The N-terminal domain contains the TOS motif, whereas the CTD contains the auto-inhibitory pseudosubstrate domain and RSPRR motif. mTORC1 phosphorylates the HM (hydrophobic motif) site (Thr412) in the linker region and PDK1 phosphorylates the T-loop site (Thr252) within the kinase domain. Other

regulatory phosphorylation (P) sites, including the TM (turn motif) site (Ser394), are shown.

cal in stabilizing the catalytically active state [17, 18].

S6 kinases figure among important signaling molecules required for propagation of cell growth and proliferation. Adding to its importance is the fact that S6 kinase (S6K1) is a downstream effector of mTORC1 (mechanistic target of rapamycin), a master regulator of cell growth and proliferation. Evidences are strong enough to suggest that the mTOR-S6K1 axis controls fundamental cellular processes including transcription, translation and lipid synthesis. Further, this nexus partly regulates glucose homoeostasis, controls insulin sensitivity, adipocyte metabolism, tissue and organ size, learning, memory and aging [15]. S6K1 has therefore been considered to play a pivotal role in regulating cellular physiology. Any dysregulation of this signaling axis has deleterious implications ranging from metabolic disorders to various cancers. mTOR/ S6K1 signaling network has hence remained central target for various medical interventions employed over the years for treating the pathological conditions. However, so far the therapeutic interventions involving mTOR as a target molecule for treating cancers, particularly renal carcinomas have reported a fair share of relapses. The resistance shown to these drugs post chronic exposures remain one of the major concerns the scientific community is faced upto. One of the reasons for any such observation would be the incomplete understanding visa-vis the regulation of mTOR/S6K1 axis. It is as such conceivable that improved understanding of S6K1 regulation would help develop novel therapeutics that should take care of these challenges. The present chapter reviews the regulation of S6K, its structure and functions.

#### **2. S6K protein family; domain architecture and cellular localization**

Based on the sequence alignment of their catalytic domains, S6Ks have been placed in AGC family of protein kinases that mainly comprise of protein kinase A (PKA) protein kinase G (PKG) and protein kinase C (PKC) [16]. The structural resemblance in AGC kinases allows them to exhibit more or less a similar mode of regulation. These kinases fundamentally exhibit bi-lobal structural organization around the kinase domain. This structural arrangement enables their amino and carboxyl termini to coordinate ATP binding [17, 18]. Further these kinases comprise of an activation loop (commonly known as T-loop) that precedes their C-terminus lobe. The phosphorylation of T-loop brings in conformational changes important for phosphoryl transfer. Two other important phosphorylation sites, the turn motif (TM) (sonamed due to its location at the cusp of a structural turn in the PKA tail) and hydrophobic motif (HM) sequentially follow the kinase domain. The phosphorylated HM site engages a hydrophobic pocket within the N-lobe. The phosphorylated TM site stabilizes phospho-HM binding to the N-lobe hydrophobic pocket. Together, these three phosphorylations stand critical in stabilizing the catalytically active state [17, 18].

over the last several decades have led to recognize the ability of these signaling networks to promote cell growth and proliferation by way of their potential to phosphorylate one another to distinguish themselves as kinases. The kinases specifically transfer the phosphoryl group from ATP to their substrates such as to bring about a change in their function that helps transduce the upstream signals for the accomplishment of functions. Intrigued by the properties of these kinases, the scientific community over the years has shown great interest in identifying more kinases that lead to the identification of a family of protein kinases known as ribosomal S6 kinases (RSKs) that respond to nutrients and growth factors. The RSKs came to fore after studying the inducible phosphorylations on ribosomal protein S6 (rps6) and accordingly p90 ribosomal S6 kinase was identified from Xenopus oocytes. The homologous counterparts of p90 S6 kinase in humans were soon identified and grouped into family of four enzymes termed as RSK1-4. It was later on observed that the RSKs may play a minor role in rps6 phosphorylation [1] to give way for the emergence of a major rps6 kinase identified as 70 kDa ribosomal S6 kinase [1–4]. The 70 kDa ribosomal S6 kinase was purified from mitogen stimulated Swiss mouse 3T3 cells [5]. Cloning of 70 kDa ribosomal S6 kinase was soon accomplished after protein sequencing of a rat and rabbit cDNA [6–8]. Subsequent identification and cloning of human orthologs of S6K genes RPS6KB1 and RPS6KB2 encoding S6K1 and S6K2 thus, respectively, led to the emergence of a second

144 Homeostasis - An Integrated Vision

family of S6 kinases, a principal kinase of rps6 in somatic cells [2, 9–14].

S6 kinases figure among important signaling molecules required for propagation of cell growth and proliferation. Adding to its importance is the fact that S6 kinase (S6K1) is a downstream effector of mTORC1 (mechanistic target of rapamycin), a master regulator of cell growth and proliferation. Evidences are strong enough to suggest that the mTOR-S6K1 axis controls fundamental cellular processes including transcription, translation and lipid synthesis. Further, this nexus partly regulates glucose homoeostasis, controls insulin sensitivity, adipocyte metabolism, tissue and organ size, learning, memory and aging [15]. S6K1 has therefore been considered to play a pivotal role in regulating cellular physiology. Any dysregulation of this signaling axis has deleterious implications ranging from metabolic disorders to various cancers. mTOR/ S6K1 signaling network has hence remained central target for various medical interventions employed over the years for treating the pathological conditions. However, so far the therapeutic interventions involving mTOR as a target molecule for treating cancers, particularly renal carcinomas have reported a fair share of relapses. The resistance shown to these drugs post chronic exposures remain one of the major concerns the scientific community is faced upto. One of the reasons for any such observation would be the incomplete understanding visa-vis the regulation of mTOR/S6K1 axis. It is as such conceivable that improved understanding of S6K1 regulation would help develop novel therapeutics that should take care of these chal-

lenges. The present chapter reviews the regulation of S6K, its structure and functions.

**2. S6K protein family; domain architecture and cellular localization**

Based on the sequence alignment of their catalytic domains, S6Ks have been placed in AGC family of protein kinases that mainly comprise of protein kinase A (PKA) protein kinase G (PKG) and protein kinase C (PKC) [16]. The structural resemblance in AGC kinases allows RPS6KB1, human ortholog of S6K1 genes, encodes two isoforms, p70S6K1 and p85S6K1 (formerly known as p70S6KII and p70S6KI, respectively) through alternative translational start sites [10]. p70S6K1, a 502 amino acid protein and its larger isoform p85S6K1, having nuclear localization sequence (NLS) within the 23 amino acid N-terminal extension, were earlier believed to remain localized in the cytoplasm and nucleus, respectively. However, the data generated of late, contests the nuclear localization of p85S6K1 while showing its sub-cellular localization to be cytoplasmic and at the same time claims p70S6K1 localization to be both nuclear and cytoplasmic [19]. S6K2, encoded by RPS6KB2 gene, constitutes

**Figure 1.** S6K isoforms, structure and domain organization with potential phosphorylating kinases along with their sites of action. S6K1 isoforms include p70-, p85- and p31-S6K1; alternative start site usage lengthens the p85- and p31- S6K1 N-termini by 23 amino acids (note that p31-S6K1 lacks most of the kinase domain). S6K2 isoforms include p54 and p56-S6K2; alternative start site usage lengthens the p56-S6K2 N-terminus by 13 amino acids. NLSs lie within the N-terminal extensions of p85-S6K1 and p56-S6K2, whereas S6K2 additionally contains an NLS within the C-terminus as well as a proline-rich domain (Pro). S6Ks contain an acidic N-terminal domain (NTD), kinase domain (KD), linker region and acidic C-terminal domain (CTD). The N-terminal domain contains the TOS motif, whereas the CTD contains the auto-inhibitory pseudosubstrate domain and RSPRR motif. mTORC1 phosphorylates the HM (hydrophobic motif) site (Thr412) in the linker region and PDK1 phosphorylates the T-loop site (Thr252) within the kinase domain. Other regulatory phosphorylation (P) sites, including the TM (turn motif) site (Ser394), are shown.

another member of S6 kinase family that, via alternative start sites, produces two nuclear isoforms, owing to the presence of nuclear localization sequence at the C-terminus [11]. The longer isoform, p56S6K2 comprises a 13 amino acid extension at N-terminus than its shorter isoform, p54S6K2 [9]. Moreover S6K2 isoforms have been characterized by the presence of proline-rich domain at the C-terminal [20]. S6K1 and S6K2 have been structurally dissected into several regulatory domains, namely an acidic N-terminus that contains the TOR signaling (TOS) motif; the kinase domain that contains the activation/T-loop; a linker region that contains the TM and HM sites; and a basic C-terminus containing an auto-inhibitory pseudosubstrate domain (**Figure 1**). These proteins are homologous to each other within appreciable limits; their catalytic domains share about 83% identity at the amino acid level [9]. However, the differences observed in the extreme N- and C-terminal regions, direct these kinases to distinct compartments or to different molecular targets. Thus, for example, the C-terminal PDZ binding domain in S6K1 allows recruitment to the actin cytoskeleton via binding to neurabin [21] and S6K2 containing a proline-rich region in its C-terminus facilitates interaction with SH3 domain or WW repeat containing protein [9]. Besides full length forms of S6K1 and S6K2, a novel kinase domain truncated splice variant, p31S6K (**Figure 1**) with potential oncogenic properties, has also been reported [22].

kinase(s) play a dominant role in-vivo [32]. Scientific observations during early 1990's propounded the theory that mTOR controls the in-vivo activation of S6K1 [35]. These observations were further augmented during 1998–1999 when mTORC1 was shown to directly phosphorylate the HM site (Thr412) of S6K1 in vitro [36, 37]. The indispensible nature of HM site phosphorylation is further supported by the evidence that mutation of Thr 412 to alanine (T412A) abolishes the S6K1 activity, whereas the phosphor mimicked variant (T412E) enhances basal activity even in absence of mitogens [20, 33, 38]. The findings put forward hence strengthened the notion that phosphorylation of Thr 412 at HM site remains pivotal for S6K1 activation. Co-ordinate phosphorylation of Thr252 in the activation/T-loop and Thr412 at HM site leads to maximal activation of S6K1 [29, 33, 39]. Set out to identify the potential kinases responsible for Thr 252 phosphorylation, in vitro followed by in-vivo studies show PDK1, also corroborated as a kinase for AKT, to directly phosphorylate Thr252 at T-loop site [29, 39, 40]. This was supported by the findings that PDK1 null embryonic stem cells, PDK1−/− or T252A mutation rendered S6K1 enzymatically dead [33, 41]. Further it is observed that PDK1 mediated phosphorylation of C-Terminus truncated variant of S6K1, S6K1-∆CT remains significantly higher than full length S6K1 while mutation in c-terminal phosphorylation sites to alanine results in poor S6K1 activation [29]. Moreover, PDK1 poorly activates S6K1 T412A-∆CT or T394A-∆CT in vitro. All these findings reveal that unphosphorylated c-terminus puts restraint toward full activation of S6K1 by blocking the access of PDK1 to activation loop and the importance of HM and TM sites toward bringing PDK1 mediated S6K1 activation [29]. Phosphorylation of Ser394 (or Ser371 in P70S6K isoform) at turn motif site also remains of much significance for complete activation of S6K1, as S394A substitution of S6K1 renders it completely inactive [42]. Notwithstanding the importance of turn motif site, the data accumulated on regulation and function of this phosphorylation event has progressed, albeit rather slowly. Whatever little the quantum of data may be available for regulation around TM site phosphorylation till date, it concordantly reflects that this site does not represent an autophosphorylation event [42]. Some more interesting observations reveal that even the addition of T412E substitution fails to restore the activity of S394A mutant, thus supporting the notion that this site plays an important yet independent role in regulating the activity of S6K1 [42]. Evidences, though scant, have also emerged which corroborate mTOR in promoting Ser394 phosphorylation in vitro [43]. However, since data accumulated in certain other cases does not correlate phospho-Ser394 well with mTORC1 activity, intermediation of some other kinase besides mTORC1 is also speculated. Reports based on analogy to co-translational phosphorylation of TM site (Thr450) in AKT are tempting to speculate that S6K1 TM site phosphorylation also represents an early event that occurs co-translationally prior to T-loop and HM site phosphorylation [44]. Indeed, a recent report supports such an idea, as Ser394 phosphorylation occurs simultaneously with the production of S6K1 protein from a transfected plasmid [31]. Since the phosphorylations at the HM and T-loop sites exhibit strong cooperativity, the temporal order of these two phosphorylations has not yet been convincingly deciphered. However, the data available till date has put forth two models for S6K1 activation wherein the conventional and widely accepted model suggests mTORC1 mediated phosphorylation at Thr 412 to precede PDK1 mediated phosphorylation at Thr 252 [29, 38, 39]. An alternate model however, suggests that the phosphorylation at T-loop (Thr

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252) precedes HM phosphorylation (Thr 412) [31, 33] (**Figure 2**).

#### **3. S6 kinase regulation; a historical perspective for prevalent mode of activation**

Research carried out over the past 20 years identified diverse growth factors and mitogens with the ability to activate the S6 kinases [23, 24]. Seemingly among these multiple inputs, insulin/IGF pathway, which signals via phosphoinositide 3-kinase (PI3K) and mTORC1 appears to be the best studied activator of S6K1 [25, 26]. Besides PI3K, other independent signaling pathways like Ras/MAPK (mitogen activated protein kinase) have also been implicated in S6K1 activation [27, 28]. S6K1's contribution in controlling cell growth and proliferation being paramount made the basis for studying its structure and function. The data accumulated over a period of time, hence revealed the molecular insights that govern activation of S6K1 by mitogens and identified, to a large extent, the complex interactions between its specific domains and phosphorylation sites. These observations eventually became the reason for putting forth the models that explain the activation of S6K1 by stepwise multisite phosphorylations [29–33]. These models propose that during inactive state, the basic C-terminal pseudosubstrate domain of S6 K1 interacts with its acidic N-terminus. This interaction obstructs the phosphorylation at kinase domain and renders it inactive [6, 34]. Stimulation by growth factors besides other mitogens result in the phosphorylation of four proline-directed sites at C-terminal auto-inhibitory pseudosubstrate domain (Ser434, Ser441, Ser444 and Ser447) [30, 32]. These phosphorylations induce a conformational change thereby releasing the interaction between the two termini and enables access to the HM and T-loop sites. Phosphorylation at these two critical sites ultimately leads to the full activation of the kinase [6, 34]. Several proline-directed kinases, including ERK1/2, JNK1/2 and CDK1 have been implicated in phosphorylation of these sites; it however remains unclear as to which kinase(s) play a dominant role in-vivo [32]. Scientific observations during early 1990's propounded the theory that mTOR controls the in-vivo activation of S6K1 [35]. These observations were further augmented during 1998–1999 when mTORC1 was shown to directly phosphorylate the HM site (Thr412) of S6K1 in vitro [36, 37]. The indispensible nature of HM site phosphorylation is further supported by the evidence that mutation of Thr 412 to alanine (T412A) abolishes the S6K1 activity, whereas the phosphor mimicked variant (T412E) enhances basal activity even in absence of mitogens [20, 33, 38]. The findings put forward hence strengthened the notion that phosphorylation of Thr 412 at HM site remains pivotal for S6K1 activation. Co-ordinate phosphorylation of Thr252 in the activation/T-loop and Thr412 at HM site leads to maximal activation of S6K1 [29, 33, 39]. Set out to identify the potential kinases responsible for Thr 252 phosphorylation, in vitro followed by in-vivo studies show PDK1, also corroborated as a kinase for AKT, to directly phosphorylate Thr252 at T-loop site [29, 39, 40]. This was supported by the findings that PDK1 null embryonic stem cells, PDK1−/− or T252A mutation rendered S6K1 enzymatically dead [33, 41]. Further it is observed that PDK1 mediated phosphorylation of C-Terminus truncated variant of S6K1, S6K1-∆CT remains significantly higher than full length S6K1 while mutation in c-terminal phosphorylation sites to alanine results in poor S6K1 activation [29]. Moreover, PDK1 poorly activates S6K1 T412A-∆CT or T394A-∆CT in vitro. All these findings reveal that unphosphorylated c-terminus puts restraint toward full activation of S6K1 by blocking the access of PDK1 to activation loop and the importance of HM and TM sites toward bringing PDK1 mediated S6K1 activation [29]. Phosphorylation of Ser394 (or Ser371 in P70S6K isoform) at turn motif site also remains of much significance for complete activation of S6K1, as S394A substitution of S6K1 renders it completely inactive [42]. Notwithstanding the importance of turn motif site, the data accumulated on regulation and function of this phosphorylation event has progressed, albeit rather slowly. Whatever little the quantum of data may be available for regulation around TM site phosphorylation till date, it concordantly reflects that this site does not represent an autophosphorylation event [42]. Some more interesting observations reveal that even the addition of T412E substitution fails to restore the activity of S394A mutant, thus supporting the notion that this site plays an important yet independent role in regulating the activity of S6K1 [42]. Evidences, though scant, have also emerged which corroborate mTOR in promoting Ser394 phosphorylation in vitro [43]. However, since data accumulated in certain other cases does not correlate phospho-Ser394 well with mTORC1 activity, intermediation of some other kinase besides mTORC1 is also speculated. Reports based on analogy to co-translational phosphorylation of TM site (Thr450) in AKT are tempting to speculate that S6K1 TM site phosphorylation also represents an early event that occurs co-translationally prior to T-loop and HM site phosphorylation [44]. Indeed, a recent report supports such an idea, as Ser394 phosphorylation occurs simultaneously with the production of S6K1 protein from a transfected plasmid [31]. Since the phosphorylations at the HM and T-loop sites exhibit strong cooperativity, the temporal order of these two phosphorylations has not yet been convincingly deciphered. However, the data available till date has put forth two models for S6K1 activation wherein the conventional and widely accepted model suggests mTORC1 mediated phosphorylation at Thr 412 to precede PDK1 mediated phosphorylation at Thr 252 [29, 38, 39]. An alternate model however, suggests that the phosphorylation at T-loop (Thr 252) precedes HM phosphorylation (Thr 412) [31, 33] (**Figure 2**).

another member of S6 kinase family that, via alternative start sites, produces two nuclear isoforms, owing to the presence of nuclear localization sequence at the C-terminus [11]. The longer isoform, p56S6K2 comprises a 13 amino acid extension at N-terminus than its shorter isoform, p54S6K2 [9]. Moreover S6K2 isoforms have been characterized by the presence of proline-rich domain at the C-terminal [20]. S6K1 and S6K2 have been structurally dissected into several regulatory domains, namely an acidic N-terminus that contains the TOR signaling (TOS) motif; the kinase domain that contains the activation/T-loop; a linker region that contains the TM and HM sites; and a basic C-terminus containing an auto-inhibitory pseudosubstrate domain (**Figure 1**). These proteins are homologous to each other within appreciable limits; their catalytic domains share about 83% identity at the amino acid level [9]. However, the differences observed in the extreme N- and C-terminal regions, direct these kinases to distinct compartments or to different molecular targets. Thus, for example, the C-terminal PDZ binding domain in S6K1 allows recruitment to the actin cytoskeleton via binding to neurabin [21] and S6K2 containing a proline-rich region in its C-terminus facilitates interaction with SH3 domain or WW repeat containing protein [9]. Besides full length forms of S6K1 and S6K2, a novel kinase domain truncated splice variant,

p31S6K (**Figure 1**) with potential oncogenic properties, has also been reported [22].

**3. S6 kinase regulation; a historical perspective for prevalent mode** 

Research carried out over the past 20 years identified diverse growth factors and mitogens with the ability to activate the S6 kinases [23, 24]. Seemingly among these multiple inputs, insulin/IGF pathway, which signals via phosphoinositide 3-kinase (PI3K) and mTORC1 appears to be the best studied activator of S6K1 [25, 26]. Besides PI3K, other independent signaling pathways like Ras/MAPK (mitogen activated protein kinase) have also been implicated in S6K1 activation [27, 28]. S6K1's contribution in controlling cell growth and proliferation being paramount made the basis for studying its structure and function. The data accumulated over a period of time, hence revealed the molecular insights that govern activation of S6K1 by mitogens and identified, to a large extent, the complex interactions between its specific domains and phosphorylation sites. These observations eventually became the reason for putting forth the models that explain the activation of S6K1 by stepwise multisite phosphorylations [29–33]. These models propose that during inactive state, the basic C-terminal pseudosubstrate domain of S6 K1 interacts with its acidic N-terminus. This interaction obstructs the phosphorylation at kinase domain and renders it inactive [6, 34]. Stimulation by growth factors besides other mitogens result in the phosphorylation of four proline-directed sites at C-terminal auto-inhibitory pseudosubstrate domain (Ser434, Ser441, Ser444 and Ser447) [30, 32]. These phosphorylations induce a conformational change thereby releasing the interaction between the two termini and enables access to the HM and T-loop sites. Phosphorylation at these two critical sites ultimately leads to the full activation of the kinase [6, 34]. Several proline-directed kinases, including ERK1/2, JNK1/2 and CDK1 have been implicated in phosphorylation of these sites; it however remains unclear as to which

**of activation**

146 Homeostasis - An Integrated Vision

mediated by another member of mTORC1 complex, a scaffold protein called Raptor [46, 47]. For phosphorylation of S6K1 and 4EBP-1, the other mTORC1 effector molecule, binding with raptor has been unequivocally stated to be critical. However, the precise mechanism by which raptor mediates efficient phosphorylation of S6K1 and 4EBP-1 remains largely debatable. Two models have however been proposed to explain this mechanism. The first model suggests that raptor and mTOR associate in two states with varying affinities governed by the nutrient availability. During nutrient starved state Raptor binds tightly to mTOR and renders it inactive. While as the loose-binding complex, formed during nutrient sufficiency, activates mTOR and promotes efficient phosphorylation of mTOR targets [48]. Furthermore, overexpression of raptor increases the amount of mTOR found in the tightbinding complex, thereby explaining the observation that overexpression of raptor inhibits mTOR activity. However, it is interesting to note that rapamycin is able to disrupt the raptor-mTOR interaction regardless of nutrient status [49], but it is phosphate dependent. The second model supports the existence of Raptor as a scaffolding protein for mTORC-1complex, wherein Raptor has been shown to preferentially bind unphosphorylated forms of mTOR targets and recruit the substrates to the mTOR complex for phosphorylation. Role of S6K1 amino terminus in suppressing the inhibitory function of C-terminus remained unclear till identification of an RSPRR motif (a short stretch of 5 amino acids, 433–437) in C-terminus way back in 2005 [47]. This motif has been suggested to negatively regulate S6K1 activation. Evidences, though scant, have propounded that RSPRR motif functions as a docking site for a negative regulator, such as a phosphatase, that is suppressed by mTORC1 [47].R3Amutation of RSPRR motif within the dead ∆NT or TOS motif-mutant (F5A) backbone (NT-R3A or F5A-R3A) has been shown to rescue insulin-stimulated Thr412 phosphorylation and S6K1 activation [15]. These findings though place TOS motif in exhibiting negative control over RSPPR motif inhibition, the exact mechanism behind this regula-

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In 1970s, a bacterial strain, Streptomyces hygroscopicus was isolated from a soil sample from Easter Island ("Rapa Nui" in the native language) [50]. This strain was found to produce an anti-fungal metabolite [51]. Post purifications, the metabolite was characterized as a macrocy-

The strong ability of rapamycin to modulate cellular proliferation besides its promising role in immune suppression for treating various cancers lead to a desperate search in decoding its mode of action and simultaneously initiated hunt for nailing down its targets. The search continued for about two decades and finally culminated in early 1990s with identification of a target protein in yeast, whose mutant conferred resistance to the growth inhibitory effects of rapamycin. The protein was subsequently named as TOR (target of rapamycin) [53–56]. Shortly after, the mammalian counterpart of TOR (mTOR) was identified as physical target of rapamycin by three groups separately in 1994, 1995, respectively [57–59]. However effective mechanism of rapamycin action remained oblivious till intracellular cofactor, the peptidylprolyl cis/trans isomerase, immunophilin FK506-binding protein 12 (FKBP12) was shown to bind rapamycin as a gain-of-function component to mediate its inhibitory effect on TOR [56].

tion still remains largely a mystery.

**3.1. Rapamycin, S6K1 inhibition; a chronology of events**

clic lactone and named "Rapamycin" after its birthplace (**Figure 3**) [52].

**Figure 2.** Stepwise activation of S6K1 via multi-site phosphorylation. Conventional model: the interaction of the C- and N-terminal domains results in auto-inhibition of S6K1. Step 1: mitogens promote C-terminal domain (C) phosphorylation on multiple sites to induce a more relaxed conformation. Step 2: the release of the auto-inhibitory C-terminal domain (CTD) enables mTORC1 access to the HM and thus phosphorylation of Thr412. Step 3: the release of the auto-inhibitory CTD and phosphorylation on Thr412 enables PDK1-mediated phosphorylation of the T-loop on Thr252, resulting in full activation of S6K1. Phospho-Thr412 serves as docking site for PDK1. Owing to insufficient data, the temporal order of TM site phosphorylation (Ser394) is not depicted. Alternative model: Step 1: an unknown kinase phosphorylates the inactive form of S6K on the TM site Ser394. Step 2: mitogens promote C-terminal domain (C) phosphorylation on multiple sites to induce a more relaxed conformation. Step 3: The release of the auto-inhibitory C-terminal domain enables PDK1 access to the T-loop. Step 4: PDK1-mediated phosphorylation of Thr252 promotes mTORC1-mediated phosphorylation on the HM site, Thr412. KD, kinase domain; N,N-terminal domain.

S6K1 amino terminus serves a regulatory role in promoting phosphorylation at HM site and thereby at T-loop site that ultimately brings about its complete activation. The regulation by amino terminus is documented to be two pronged. Firstly, it acts as a receptor of an activating input critical for Thr 412 and Thr 252 phosphorylation; and, secondly, it suppresses an inhibitory function mediated by C-terminus. These observations have strong scientific support as the data generated over a period of time shows that amino terminus truncation of S6K1 (∆NT) abolishes the phosphorylation of rapamycin-sensitive sites Thr 412, Thr 252, Ser 427 and renders it inactive [38, 45] whereas additional deletion of C-terminus (∆NT/∆CT) restores all the rapamycin-sensitive phosphorylations as well as the kinase activity of S6K1. The regulatory function of the amino terminus was mapped to a short stretch of amino acids (comprising of 5–9 amino acids and represented by a signature sequence FDIDL) present at extreme end known as TOS motif [20]. It has been shown that S6K1 truncated of TOS motif or its F5A variant (F5A mutation within the FDIDL sequence) abolishes S6K1 kinase activity as well as Thr412 and Thr252 phosphorylation. However, deletion of C-terminus from the F5A mutant (F5A-∆CT) partially restores kinase activity and Thr 412 phosphorylation [20]. The TOS motif function has been related toward a launch pad required for mTORC1 to engage substrates and to mediate phosphorylation of rapamycin-sensitive sites [46, 47]. The data generated supports the view that mTOR interaction with TOS motif is indirect and mediated by another member of mTORC1 complex, a scaffold protein called Raptor [46, 47]. For phosphorylation of S6K1 and 4EBP-1, the other mTORC1 effector molecule, binding with raptor has been unequivocally stated to be critical. However, the precise mechanism by which raptor mediates efficient phosphorylation of S6K1 and 4EBP-1 remains largely debatable. Two models have however been proposed to explain this mechanism. The first model suggests that raptor and mTOR associate in two states with varying affinities governed by the nutrient availability. During nutrient starved state Raptor binds tightly to mTOR and renders it inactive. While as the loose-binding complex, formed during nutrient sufficiency, activates mTOR and promotes efficient phosphorylation of mTOR targets [48]. Furthermore, overexpression of raptor increases the amount of mTOR found in the tightbinding complex, thereby explaining the observation that overexpression of raptor inhibits mTOR activity. However, it is interesting to note that rapamycin is able to disrupt the raptor-mTOR interaction regardless of nutrient status [49], but it is phosphate dependent. The second model supports the existence of Raptor as a scaffolding protein for mTORC-1complex, wherein Raptor has been shown to preferentially bind unphosphorylated forms of mTOR targets and recruit the substrates to the mTOR complex for phosphorylation. Role of S6K1 amino terminus in suppressing the inhibitory function of C-terminus remained unclear till identification of an RSPRR motif (a short stretch of 5 amino acids, 433–437) in C-terminus way back in 2005 [47]. This motif has been suggested to negatively regulate S6K1 activation. Evidences, though scant, have propounded that RSPRR motif functions as a docking site for a negative regulator, such as a phosphatase, that is suppressed by mTORC1 [47].R3Amutation of RSPRR motif within the dead ∆NT or TOS motif-mutant (F5A) backbone (NT-R3A or F5A-R3A) has been shown to rescue insulin-stimulated Thr412 phosphorylation and S6K1 activation [15]. These findings though place TOS motif in exhibiting negative control over RSPPR motif inhibition, the exact mechanism behind this regulation still remains largely a mystery.

#### **3.1. Rapamycin, S6K1 inhibition; a chronology of events**

S6K1 amino terminus serves a regulatory role in promoting phosphorylation at HM site and thereby at T-loop site that ultimately brings about its complete activation. The regulation by amino terminus is documented to be two pronged. Firstly, it acts as a receptor of an activating input critical for Thr 412 and Thr 252 phosphorylation; and, secondly, it suppresses an inhibitory function mediated by C-terminus. These observations have strong scientific support as the data generated over a period of time shows that amino terminus truncation of S6K1 (∆NT) abolishes the phosphorylation of rapamycin-sensitive sites Thr 412, Thr 252, Ser 427 and renders it inactive [38, 45] whereas additional deletion of C-terminus (∆NT/∆CT) restores all the rapamycin-sensitive phosphorylations as well as the kinase activity of S6K1. The regulatory function of the amino terminus was mapped to a short stretch of amino acids (comprising of 5–9 amino acids and represented by a signature sequence FDIDL) present at extreme end known as TOS motif [20]. It has been shown that S6K1 truncated of TOS motif or its F5A variant (F5A mutation within the FDIDL sequence) abolishes S6K1 kinase activity as well as Thr412 and Thr252 phosphorylation. However, deletion of C-terminus from the F5A mutant (F5A-∆CT) partially restores kinase activity and Thr 412 phosphorylation [20]. The TOS motif function has been related toward a launch pad required for mTORC1 to engage substrates and to mediate phosphorylation of rapamycin-sensitive sites [46, 47]. The data generated supports the view that mTOR interaction with TOS motif is indirect and

HM site, Thr412. KD, kinase domain; N,N-terminal domain.

148 Homeostasis - An Integrated Vision

**Figure 2.** Stepwise activation of S6K1 via multi-site phosphorylation. Conventional model: the interaction of the C- and N-terminal domains results in auto-inhibition of S6K1. Step 1: mitogens promote C-terminal domain (C) phosphorylation on multiple sites to induce a more relaxed conformation. Step 2: the release of the auto-inhibitory C-terminal domain (CTD) enables mTORC1 access to the HM and thus phosphorylation of Thr412. Step 3: the release of the auto-inhibitory CTD and phosphorylation on Thr412 enables PDK1-mediated phosphorylation of the T-loop on Thr252, resulting in full activation of S6K1. Phospho-Thr412 serves as docking site for PDK1. Owing to insufficient data, the temporal order of TM site phosphorylation (Ser394) is not depicted. Alternative model: Step 1: an unknown kinase phosphorylates the inactive form of S6K on the TM site Ser394. Step 2: mitogens promote C-terminal domain (C) phosphorylation on multiple sites to induce a more relaxed conformation. Step 3: The release of the auto-inhibitory C-terminal domain enables PDK1 access to the T-loop. Step 4: PDK1-mediated phosphorylation of Thr252 promotes mTORC1-mediated phosphorylation on the

> In 1970s, a bacterial strain, Streptomyces hygroscopicus was isolated from a soil sample from Easter Island ("Rapa Nui" in the native language) [50]. This strain was found to produce an anti-fungal metabolite [51]. Post purifications, the metabolite was characterized as a macrocyclic lactone and named "Rapamycin" after its birthplace (**Figure 3**) [52].

> The strong ability of rapamycin to modulate cellular proliferation besides its promising role in immune suppression for treating various cancers lead to a desperate search in decoding its mode of action and simultaneously initiated hunt for nailing down its targets. The search continued for about two decades and finally culminated in early 1990s with identification of a target protein in yeast, whose mutant conferred resistance to the growth inhibitory effects of rapamycin. The protein was subsequently named as TOR (target of rapamycin) [53–56]. Shortly after, the mammalian counterpart of TOR (mTOR) was identified as physical target of rapamycin by three groups separately in 1994, 1995, respectively [57–59]. However effective mechanism of rapamycin action remained oblivious till intracellular cofactor, the peptidylprolyl cis/trans isomerase, immunophilin FK506-binding protein 12 (FKBP12) was shown to bind rapamycin as a gain-of-function component to mediate its inhibitory effect on TOR [56].

S6K1 being a downstream effector of mTOR, shows inhibitory response to rapamycin as is

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Early 1990s witnessed a rigorous search for identifying the rapamycin-sensitive regulatory phosphorylation sites in S6K1 and during 1995 three sites, T252 in the activation/T-loop, T412 in hydrophobic motif and S427 in the linker domain, which connects the auto-inhibitory domain to the catalytic domain were identified as principal rapamycin-sensitive sites [65]. These sites were shown to be dephosphorylated by rapamycin in hierarchical fashion T412 > S427 > T252 with T412 dephosphorylation most closely paralleling loss of kinase activity [65]. Besides, these sites were shown to be responsive to mitogenic stimulation as well [66]. Though all these three sites show rapamycin responsiveness, their conversions to either acidic or neutral amino acids reveal that T252 and T412 were critical regulatory sites where as T427 appeared to play a modulatory function [65]. Further T412 was shown to be principal site of rapamycin-induced S6K1 inactivation as T412E showed increased basal activity and was largely rapamycin resistant [65]. In 1995, Weng et al. reported the cooperativity among the two termini for exhibiting their regulatory effects on S6K1 activation by showing that amino- terminus truncated mutant, S6K1-∆NT, was inactive. However, additional deletion of carboxy terminus, generating S6K1-∆NT/∆CT, rescued its phosphorylation and activation state. Surprisingly it was observed that the mutant was rapamycin resistant [45]. However, its responsiveness toward Wortmannin inhibition did not get compromised. These observations combined with the findings that the carboxy-terminal truncated S6K1 (S6K1-∆CT) retains rapamycin sensitivity raised the speculations during latter half of 1990's that the inhibitory effect of the rapamycin–FKBP12 complex on S6K1 was either due to blockade of an upstream activator or activation of some phosphatase which mediate their influence through the two termini. The presence of Thr412 phosphorylation on S6K1-∆NT/∆CT isolated from rapamycintreated cells questioned the idea that mTORC1 represents the sole S6K1 Thr412 kinase. In ∆NT/∆CT, serum and insulin promote Thr412 phosphorylation and kinase activation in a completely rapamycin-resistant manner, suggesting that a rapamycin-insensitive kinase mediates Thr412 phosphorylation [38, 45]. This conundrum was soon resolved in 2005 by observing that rapamycin-insensitive mTORC2 mediates non-physiological S6K1 Thr412 phosphorylation in S6K1 mutants lacking a C-terminus [67]. It was also observed that unlike other AGC kinases S6Ks have atypically an extended C-terminus that imposes additional inhibitory influence on their state of activity [15]. Hence in the dead ∆NT allele, mTORC1 (mTOR/raptor) cannot dock to S6K1 and as such is incapacitated to phosphorylate Thr412, and mTORC2 (mTOR/rictor) cannot phosphorylate Thr412 due to steric hindrance imposed by the extended C-terminus [67]. In the partially rapamycin-resistant ∆CT mutant, both rapamycin-sensitive mTORC1 and rapamycin-insensitive mTORC2 cooperatively mediate Thr412 phosphorylation. In ∆NT/∆CT (and F5A-∆CT), only rapamycin-insensitive mTORC2 mediates Thr412 phosphorylation.

Studies of late propose the involvement of various post translational modifications (PTMs) in regulation of S6K1. Prominent among them are phosphatase-mediated dephosphorylation,

evidenced by loss of its ability to phosphorylate its substrate, ribosomal protein S6.

**4. Other modes of S6K1 regulation**

acetylation and ubiquitination.

**Figure 3.** Electron-density model of a molecule of the immunosuppressant drug rapamycin: It is mainly used to prevent rejection in organ transplantation, and is used in kidney transplants. It also has anti-fungal and anti-cancer properties, and was originally derived from soil bacteria found on Easter Island. The chemical formula is C51.H79.N.O13. The atoms are represented here as color-coded blobs: carbon (green), hydrogen (white), nitrogen (blue) and oxygen (red) (adapted from Dr. Tim Evans Science photo library).

Thus, TOR is also referred to as FKBP12 rapamycin associated protein (FRAP) [60].Rapamycin-FKBP12 complex binds to the FKBP12-rapamycin binding (FRB) domain of TOR (**Figure 4**) [55, 60–62] to inhibit its intrinsic kinase activity, including autophosphorylation, thereby inhibiting access of TOR to its substrates [56]. This finding is however in contradiction with the earlier reports wherein it was shown that rapamycin has little effect on mTOR kinase activity [63]. In mammals, rapamycin in complex with FKBP12 acts as an allosteric inhibitor of mTOR complex1 (mTORC1). Only mTORC1 is acutely sensitive to inhibition of rapamycin. However, long term exposure to rapamycin has been shown to inhibit mTORC2 in certain cell types [64]. It is believed that the rapamycin-FKBP12 complex prevents the association between mTOR and raptor; therefore, downstream targets that depend on raptor binding are specifically inhibited [49].

**Figure 4.** Association of rapamycin with TOR: schematic representation of structural organization of mTOR at domain level and its association with rapamycin–FKBP12 complex.

S6K1 being a downstream effector of mTOR, shows inhibitory response to rapamycin as is evidenced by loss of its ability to phosphorylate its substrate, ribosomal protein S6.

Early 1990s witnessed a rigorous search for identifying the rapamycin-sensitive regulatory phosphorylation sites in S6K1 and during 1995 three sites, T252 in the activation/T-loop, T412 in hydrophobic motif and S427 in the linker domain, which connects the auto-inhibitory domain to the catalytic domain were identified as principal rapamycin-sensitive sites [65]. These sites were shown to be dephosphorylated by rapamycin in hierarchical fashion T412 > S427 > T252 with T412 dephosphorylation most closely paralleling loss of kinase activity [65]. Besides, these sites were shown to be responsive to mitogenic stimulation as well [66]. Though all these three sites show rapamycin responsiveness, their conversions to either acidic or neutral amino acids reveal that T252 and T412 were critical regulatory sites where as T427 appeared to play a modulatory function [65]. Further T412 was shown to be principal site of rapamycin-induced S6K1 inactivation as T412E showed increased basal activity and was largely rapamycin resistant [65]. In 1995, Weng et al. reported the cooperativity among the two termini for exhibiting their regulatory effects on S6K1 activation by showing that amino- terminus truncated mutant, S6K1-∆NT, was inactive. However, additional deletion of carboxy terminus, generating S6K1-∆NT/∆CT, rescued its phosphorylation and activation state. Surprisingly it was observed that the mutant was rapamycin resistant [45]. However, its responsiveness toward Wortmannin inhibition did not get compromised. These observations combined with the findings that the carboxy-terminal truncated S6K1 (S6K1-∆CT) retains rapamycin sensitivity raised the speculations during latter half of 1990's that the inhibitory effect of the rapamycin–FKBP12 complex on S6K1 was either due to blockade of an upstream activator or activation of some phosphatase which mediate their influence through the two termini. The presence of Thr412 phosphorylation on S6K1-∆NT/∆CT isolated from rapamycintreated cells questioned the idea that mTORC1 represents the sole S6K1 Thr412 kinase. In ∆NT/∆CT, serum and insulin promote Thr412 phosphorylation and kinase activation in a completely rapamycin-resistant manner, suggesting that a rapamycin-insensitive kinase mediates Thr412 phosphorylation [38, 45]. This conundrum was soon resolved in 2005 by observing that rapamycin-insensitive mTORC2 mediates non-physiological S6K1 Thr412 phosphorylation in S6K1 mutants lacking a C-terminus [67]. It was also observed that unlike other AGC kinases S6Ks have atypically an extended C-terminus that imposes additional inhibitory influence on their state of activity [15]. Hence in the dead ∆NT allele, mTORC1 (mTOR/raptor) cannot dock to S6K1 and as such is incapacitated to phosphorylate Thr412, and mTORC2 (mTOR/rictor) cannot phosphorylate Thr412 due to steric hindrance imposed by the extended C-terminus [67]. In the partially rapamycin-resistant ∆CT mutant, both rapamycin-sensitive mTORC1 and rapamycin-insensitive mTORC2 cooperatively mediate Thr412 phosphorylation. In ∆NT/∆CT (and F5A-∆CT), only rapamycin-insensitive mTORC2 mediates Thr412 phosphorylation.

#### **4. Other modes of S6K1 regulation**

Thus, TOR is also referred to as FKBP12 rapamycin associated protein (FRAP) [60].Rapamycin-FKBP12 complex binds to the FKBP12-rapamycin binding (FRB) domain of TOR (**Figure 4**) [55, 60–62] to inhibit its intrinsic kinase activity, including autophosphorylation, thereby inhibiting access of TOR to its substrates [56]. This finding is however in contradiction with the earlier reports wherein it was shown that rapamycin has little effect on mTOR kinase activity [63]. In mammals, rapamycin in complex with FKBP12 acts as an allosteric inhibitor of mTOR complex1 (mTORC1). Only mTORC1 is acutely sensitive to inhibition of rapamycin. However, long term exposure to rapamycin has been shown to inhibit mTORC2 in certain cell types [64]. It is believed that the rapamycin-FKBP12 complex prevents the association between mTOR and raptor; therefore, downstream targets that depend on raptor binding are specifically inhibited [49].

**Figure 4.** Association of rapamycin with TOR: schematic representation of structural organization of mTOR at domain

from Dr. Tim Evans Science photo library).

150 Homeostasis - An Integrated Vision

level and its association with rapamycin–FKBP12 complex.

**Figure 3.** Electron-density model of a molecule of the immunosuppressant drug rapamycin: It is mainly used to prevent rejection in organ transplantation, and is used in kidney transplants. It also has anti-fungal and anti-cancer properties, and was originally derived from soil bacteria found on Easter Island. The chemical formula is C51.H79.N.O13. The atoms are represented here as color-coded blobs: carbon (green), hydrogen (white), nitrogen (blue) and oxygen (red) (adapted

> Studies of late propose the involvement of various post translational modifications (PTMs) in regulation of S6K1. Prominent among them are phosphatase-mediated dephosphorylation, acetylation and ubiquitination.

#### **4.1. Dephosphorylation**

S6K1 has been proposed to be regulated by co-ordinated action of phosphatases. This was observed while examining the action of rapamycin on phosphorylation state of S6K1.Whereas as rapamycin could dephosphorylate S6K1 through its inhibitory action on mTORC1 complex; the involvement of certain phosphatases to regulate this event was not ruled out. Indeed, S6Ks have been suspected to represent targets of PP2A (protein phosphatase 2A)-like phosphatases. This is supported by the evidence that regulation of several TOR substrates in *S. cerevisiae* occurs via suppression of PP2A-like phosphatases [68]. Further it has also been shown that PP2A interacts with wild type S6K1 and not ∆NT/∆CT S6K1, a variant truncated of both the termini [69]. It is of late been observed that genetic ablation of PP2A regulatory subunit B (PP2A-B′) in *Drosophila melanogaster* leads to dS6K (Drosophila S6K) deregulation and a variety of metabolic defects [70]. Some further studies report that S6K1 phosphorylation (412) is enhanced in human cells upon knock down of PPP2R5C, the human PP2A-B′ orthologue [70]. All these studies support the role of phosphatases in regulating S6K1 activity. However, it is to be ascertained how mTOR influences these PP2A like phosphatase to modulate S6K1 Thr412 phosphorylation.

extends its influence on various cellular functions, some of its associated functions still appear untenable, reason being the inadequate support data. Some of the S6K1 associated functions

S6K1 has been shown to control cell growth primarily by its ability to phosphorylate S6 protein of 40S ribosomal subunit (rpS6). These phosphorylations are shown to be localized at the C-terminus and happen in sequential order, namely S236 > S235 > S240 > S244 > S247. The dependence of S6 phosphorylation on S6K1 can traced by the observation that rpS6 deficient mice, rps6−/− show phenotypic resemblance to S6K1 deficient mice, S6K1−/−. Although these mice are viable and fertile, however both exhibit cell growth defects. It however is confusing

> / −

significant reduction in rpS6 phosphorylation [77]. The conundrum can partly be resolved by the observation that claims selective recruitment of S6K1 and S6K2 to different cellular compartments and as such enables them to differentially phosphorylate rpS6 or alternatively by the preposition that rpS6 phosphorylation may ensue at a specific developmental stage where S6K1 but not S6K2 is active. Although a discordance seems to appear vis-a-vis S6K1 activation, rpS6 phosphorylation and cell growth, their complete understanding as far as therapeutics is concerned remains but pivotal. Although S6 phosphorylation was initially thought to be required for selectively translating mRNAs characterized by 5′-oligo Pyrimidine tract (5'-TOP). Later studies however confirmed that 5' TOP mRNA translation remains unaffected

S6K1 maintains its influence on cellular translation not only by the ability to phosphorylate ribosomal protein S6 (rpS6) but some studies provide evidences to establish S6K1 as a transcription factor kinase as well. One of the study shows S6K1 to phosphorylate eukaryotic initiation factor 4B (eIF4B), a component of cap-binding complex to control cap dependent translation. While as inactive S6K1 associates with eIF3 (a component of pre-initiation complex) and upon activation by mTORC1, via serum stimulation, dissociates to phosphorylate eIF4B, another component of pre-initiation complex [79, 80]. Besides it also controls translational initiation by phosphorylating PDCD4, a negative regulator of eIF4A (**Figure 5**). Phosphorylated PDCD4 gets ultimately degraded by ubiquitin ligase βTRCP [81]. The importance of S6K1 vis-a-vis translational control can further be demonstrated by the observation which implicates S6K1 in controlling translation elongation as well. The study carried shows S6K1 to phosphorylate and inactivate eEF2k (eukaryotic elongation factor-2 kinase, a negative

S6K1 has been extensively discussed as a kinase that drives cells from G1 to S phase during cell cycle progress. However, the data in support does not seem to match this notion completely

mice display minimal defects in rpS6 phosphorylation, while S6 K2,

mice, which display minimal rpS6 phosphorylation [77], hence suggesting

grow to normal size despite exhibiting a

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**5.1. Cell growth and translational control—what is the connection?**

are briefly described below:

to observe that S6K1−

in *S6K1*<sup>−</sup> */* − */S6K2*<sup>−</sup> */* − / −

these two events to be independent [78].

regulator of translation [82].

**5.2. Cell cycle progression-a reality check**

homolog of S6K1, deficient mice, that is, S6K2−

#### **4.2. Acetylation and ubiquitination**

Acetylation of S6K1 occurs reportedly by the action of two acetyltransferase enzymes, p300/ CBP (cAMP-response-element-binding protein binding protein) and PCAF (p300/CBPassociated factor) [71].While as this PTM has been shown to occur on extreme C-terminus of S6K1 in response to the stimulation by mitogens, its dependence on phosphorylation has not been confirmed [72]. Acetylation has been primarily linked to the stabilization of S6K1 [71]. However, the concrete evidence to support this notion has remained largely elusive till date.

Polyubiquitination is another PTM, S6K1 is responsive to. S6K1 appears to experience the ubiquitination in response to mitogen stimulation [73, 74] through direct action of ubiquitin ligase, ROC1 [75]. RNA interference (RNAi) mediated knock down of ROC1 results in stabilization of S6K. These results are hence suggestive of the notion that polyubiquitination causes destabilization of S6K1 and a resultant attenuation of S6K1 downstream signals. Although regulation of S6K1 through ubiquitination still remains in its infancy; it however has opened a new window toward understanding detailed mechanistic inputs of proteasome mediated S6K1 regulation and function.

## **5. Functions associated with S6K1—a debatable discourse**

S6K1, a downstream effector of mTOR is considered to be a multifaceted effector that regulates cell growth and proliferation by phosphorylating multiple ways. To add to its wide range of functions, S6K1 has much recently been shown to play a pivotal role in cellular senescence through its newly found substrate ZRF1 [76]. S6K1 gets activated in response to various signaling pathways including mTOR, PI3-kinase and MAPK, in a coordinated manner through sequential phosphorylation events directed at multiple sites [62]. While S6K1 extends its influence on various cellular functions, some of its associated functions still appear untenable, reason being the inadequate support data. Some of the S6K1 associated functions are briefly described below:

#### **5.1. Cell growth and translational control—what is the connection?**

**4.1. Dephosphorylation**

152 Homeostasis - An Integrated Vision

Thr412 phosphorylation.

**4.2. Acetylation and ubiquitination**

S6K1 regulation and function.

S6K1 has been proposed to be regulated by co-ordinated action of phosphatases. This was observed while examining the action of rapamycin on phosphorylation state of S6K1.Whereas as rapamycin could dephosphorylate S6K1 through its inhibitory action on mTORC1 complex; the involvement of certain phosphatases to regulate this event was not ruled out. Indeed, S6Ks have been suspected to represent targets of PP2A (protein phosphatase 2A)-like phosphatases. This is supported by the evidence that regulation of several TOR substrates in *S. cerevisiae* occurs via suppression of PP2A-like phosphatases [68]. Further it has also been shown that PP2A interacts with wild type S6K1 and not ∆NT/∆CT S6K1, a variant truncated of both the termini [69]. It is of late been observed that genetic ablation of PP2A regulatory subunit B (PP2A-B′) in *Drosophila melanogaster* leads to dS6K (Drosophila S6K) deregulation and a variety of metabolic defects [70]. Some further studies report that S6K1 phosphorylation (412) is enhanced in human cells upon knock down of PPP2R5C, the human PP2A-B′ orthologue [70]. All these studies support the role of phosphatases in regulating S6K1 activity. However, it is to be ascertained how mTOR influences these PP2A like phosphatase to modulate S6K1

Acetylation of S6K1 occurs reportedly by the action of two acetyltransferase enzymes, p300/ CBP (cAMP-response-element-binding protein binding protein) and PCAF (p300/CBPassociated factor) [71].While as this PTM has been shown to occur on extreme C-terminus of S6K1 in response to the stimulation by mitogens, its dependence on phosphorylation has not been confirmed [72]. Acetylation has been primarily linked to the stabilization of S6K1 [71]. However, the concrete evidence to support this notion has remained largely elusive till date. Polyubiquitination is another PTM, S6K1 is responsive to. S6K1 appears to experience the ubiquitination in response to mitogen stimulation [73, 74] through direct action of ubiquitin ligase, ROC1 [75]. RNA interference (RNAi) mediated knock down of ROC1 results in stabilization of S6K. These results are hence suggestive of the notion that polyubiquitination causes destabilization of S6K1 and a resultant attenuation of S6K1 downstream signals. Although regulation of S6K1 through ubiquitination still remains in its infancy; it however has opened a new window toward understanding detailed mechanistic inputs of proteasome mediated

**5. Functions associated with S6K1—a debatable discourse**

S6K1, a downstream effector of mTOR is considered to be a multifaceted effector that regulates cell growth and proliferation by phosphorylating multiple ways. To add to its wide range of functions, S6K1 has much recently been shown to play a pivotal role in cellular senescence through its newly found substrate ZRF1 [76]. S6K1 gets activated in response to various signaling pathways including mTOR, PI3-kinase and MAPK, in a coordinated manner through sequential phosphorylation events directed at multiple sites [62]. While S6K1 S6K1 has been shown to control cell growth primarily by its ability to phosphorylate S6 protein of 40S ribosomal subunit (rpS6). These phosphorylations are shown to be localized at the C-terminus and happen in sequential order, namely S236 > S235 > S240 > S244 > S247. The dependence of S6 phosphorylation on S6K1 can traced by the observation that rpS6 deficient mice, rps6−/− show phenotypic resemblance to S6K1 deficient mice, S6K1−/−. Although these mice are viable and fertile, however both exhibit cell growth defects. It however is confusing to observe that S6K1− / − mice display minimal defects in rpS6 phosphorylation, while S6 K2, homolog of S6K1, deficient mice, that is, S6K2− / − grow to normal size despite exhibiting a significant reduction in rpS6 phosphorylation [77]. The conundrum can partly be resolved by the observation that claims selective recruitment of S6K1 and S6K2 to different cellular compartments and as such enables them to differentially phosphorylate rpS6 or alternatively by the preposition that rpS6 phosphorylation may ensue at a specific developmental stage where S6K1 but not S6K2 is active. Although a discordance seems to appear vis-a-vis S6K1 activation, rpS6 phosphorylation and cell growth, their complete understanding as far as therapeutics is concerned remains but pivotal. Although S6 phosphorylation was initially thought to be required for selectively translating mRNAs characterized by 5′-oligo Pyrimidine tract (5'-TOP). Later studies however confirmed that 5' TOP mRNA translation remains unaffected in *S6K1*<sup>−</sup> */* − */S6K2*<sup>−</sup> */* − mice, which display minimal rpS6 phosphorylation [77], hence suggesting these two events to be independent [78].

S6K1 maintains its influence on cellular translation not only by the ability to phosphorylate ribosomal protein S6 (rpS6) but some studies provide evidences to establish S6K1 as a transcription factor kinase as well. One of the study shows S6K1 to phosphorylate eukaryotic initiation factor 4B (eIF4B), a component of cap-binding complex to control cap dependent translation. While as inactive S6K1 associates with eIF3 (a component of pre-initiation complex) and upon activation by mTORC1, via serum stimulation, dissociates to phosphorylate eIF4B, another component of pre-initiation complex [79, 80]. Besides it also controls translational initiation by phosphorylating PDCD4, a negative regulator of eIF4A (**Figure 5**). Phosphorylated PDCD4 gets ultimately degraded by ubiquitin ligase βTRCP [81]. The importance of S6K1 vis-a-vis translational control can further be demonstrated by the observation which implicates S6K1 in controlling translation elongation as well. The study carried shows S6K1 to phosphorylate and inactivate eEF2k (eukaryotic elongation factor-2 kinase, a negative regulator of translation [82].

#### **5.2. Cell cycle progression-a reality check**

S6K1 has been extensively discussed as a kinase that drives cells from G1 to S phase during cell cycle progress. However, the data in support does not seem to match this notion completely

already ventured in developing S6K specific inhibitors like ATP-competitive compounds that exhibit selective inhibition of S6K. This remains important in the backdrop of cancer relapses/resistance observed while treating them with mTOR inhibitor rapamycin or its rapalogues [88]. However, until recently it was not clear as to what contributes to the rapamycin resistance in these cells. This conundrum was to a greater extent resolved when MAPK interacting kinase (MNK) was found to be an active player in exerting rapamycin resistance in cancer cells. The study deliberated that cancer cell resistance to the mTORC1 inhibitor rapamycin involves MNK activation via a feedback signaling loop elicited by rapamycin [89, 90]. Much recently a study by Brown et al. reveals that post rapamycin treatment mTORC1 activity is sustained by MNK in cancer cells by way of promoting the association between MNK and mTORC1 to form a sub-complex. This way MNK facilitates binding of mTORC1 with its substrates and with phosphatidyl inositol 3'kinase-related kinase (PIKK) stabilizer, TELO2, while discouraging DEPTOR (endogenous mTOR inhibitor) binding [91]. In addition MNKs are also known for phosphorylating eukaryotic initiation factor 4E, eIF4e [92] and as such may play a role in cellular translation. Further the data generated in our lab hints out at the possible role of eIF4e in regulating S6K1 (unpublished data). We believe that prolonged exposure of cells to rapamycin results in activation of MNKs with the resultant activation of eIF4e. As a result S6K1 gets activated to counter the anti-proliferative/anti-cancerous effects of rapamycin (**Figure 6**). Thus therapeutic interventions envisaging S6K1 as a direct target would help overcome, to a larger extent, the resistance experienced toward mTORC1 inhibitors and as such emerge as more appropriate

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155

**Figure 6.** S6K1, a prudent target for treating cancers: an illustration representing S6K1 as a potential target for the specific inhibitors which could be used to overcome the resistance observed while using mTORC1 inhibitors during

target for treating cancers.

various cancerous situations.

**Figure 5.** S6K1, a component of translation initiation complex: S6K1 is bound to eIF3 in basal (resting) state cells. Upon serum stimulation, mTORC1 is recruited to the complex, wherein it activates S6K1, which dissociates from eIF3 and phosphorylates several of its targets involved in the initiation of translation.

and in fact some of the data available even contests the veracity of these claims. Whereas Embryonic Stem (ES) cells devoid of S6K1 (S6K1−/−) and mouse embryonic fibroblasts (MEFs) obtained from S6K1 deficient mice do not show significant defects in cell proliferation [77], other studies implicate S6K1 in facilitating cell cycle progression through phosphorylation of estrogen receptor leading to the activation of its target genes that ultimately promotes cellular proliferation [83]. Additionally S6K1 exerts its control over cellular transcription through its ability to phosphorylate cAMP response element binding protein (CREB) isoform CREMr and transcription factor UBF-1 [84]. Phosphorylated UBF-1 in turn activates RNA Polymerase 1 driven transcription of genes encoding ribosomal RNAs and as such aid in ribosomal biogenesis. However, contrary to this belief McMullen et al. proposed no absolute requirement of S6K at either transcriptional or translational levels during ribosomal biogenesis. They showed that S6K− / − mice do not exhibit impaired cardiac hypertrophy, a response dependent on ribosomal biogenesis [85]. These observations put a question mark on the actual involvement of S6K1 in controlling cell cycle progression. Howsoever discordant the views with respect to direct involvement of S6K1 may be but the role of S6K1 in accelerating the cell cycle from G1 to S remains undisputed.

#### **5.3. Cellular metastasis—handy option for therapeutic interventions**

S6 kinase, being a downstream effector of mTOR is upregulated in various cancerous situations and as such regulates cellular metastasis. Its involvement in brain tumor pathogenesis was established by the fact that mTOR/S6K axis remains constitutively activated in glioma cells and upon S6K knockdown, the transformed phenotype of these cells is partially rescued [86]. Additionally constitutive activation of S6K1 has been reported in esophageal squamous cell carcinomas (ESCC) [87]. These and several other examples highlight the importance of S6K1 in various cancerous state and as such makes it a potential target for therapeutic interventions to treat cancer. A number of pharmaceutical companies have already ventured in developing S6K specific inhibitors like ATP-competitive compounds that exhibit selective inhibition of S6K. This remains important in the backdrop of cancer relapses/resistance observed while treating them with mTOR inhibitor rapamycin or its rapalogues [88]. However, until recently it was not clear as to what contributes to the rapamycin resistance in these cells. This conundrum was to a greater extent resolved when MAPK interacting kinase (MNK) was found to be an active player in exerting rapamycin resistance in cancer cells. The study deliberated that cancer cell resistance to the mTORC1 inhibitor rapamycin involves MNK activation via a feedback signaling loop elicited by rapamycin [89, 90]. Much recently a study by Brown et al. reveals that post rapamycin treatment mTORC1 activity is sustained by MNK in cancer cells by way of promoting the association between MNK and mTORC1 to form a sub-complex. This way MNK facilitates binding of mTORC1 with its substrates and with phosphatidyl inositol 3'kinase-related kinase (PIKK) stabilizer, TELO2, while discouraging DEPTOR (endogenous mTOR inhibitor) binding [91]. In addition MNKs are also known for phosphorylating eukaryotic initiation factor 4E, eIF4e [92] and as such may play a role in cellular translation. Further the data generated in our lab hints out at the possible role of eIF4e in regulating S6K1 (unpublished data). We believe that prolonged exposure of cells to rapamycin results in activation of MNKs with the resultant activation of eIF4e. As a result S6K1 gets activated to counter the anti-proliferative/anti-cancerous effects of rapamycin (**Figure 6**). Thus therapeutic interventions envisaging S6K1 as a direct target would help overcome, to a larger extent, the resistance experienced toward mTORC1 inhibitors and as such emerge as more appropriate target for treating cancers.

and in fact some of the data available even contests the veracity of these claims. Whereas Embryonic Stem (ES) cells devoid of S6K1 (S6K1−/−) and mouse embryonic fibroblasts (MEFs) obtained from S6K1 deficient mice do not show significant defects in cell proliferation [77], other studies implicate S6K1 in facilitating cell cycle progression through phosphorylation of estrogen receptor leading to the activation of its target genes that ultimately promotes cellular proliferation [83]. Additionally S6K1 exerts its control over cellular transcription through its ability to phosphorylate cAMP response element binding protein (CREB) isoform CREMr and transcription factor UBF-1 [84]. Phosphorylated UBF-1 in turn activates RNA Polymerase 1 driven transcription of genes encoding ribosomal RNAs and as such aid in ribosomal biogenesis. However, contrary to this belief McMullen et al. proposed no absolute requirement of S6K at either transcriptional or translational levels during ribosomal biogenesis. They showed

**Figure 5.** S6K1, a component of translation initiation complex: S6K1 is bound to eIF3 in basal (resting) state cells. Upon serum stimulation, mTORC1 is recruited to the complex, wherein it activates S6K1, which dissociates from eIF3 and

mice do not exhibit impaired cardiac hypertrophy, a response dependent on ribo-

somal biogenesis [85]. These observations put a question mark on the actual involvement of S6K1 in controlling cell cycle progression. Howsoever discordant the views with respect to direct involvement of S6K1 may be but the role of S6K1 in accelerating the cell cycle from G1

S6 kinase, being a downstream effector of mTOR is upregulated in various cancerous situations and as such regulates cellular metastasis. Its involvement in brain tumor pathogenesis was established by the fact that mTOR/S6K axis remains constitutively activated in glioma cells and upon S6K knockdown, the transformed phenotype of these cells is partially rescued [86]. Additionally constitutive activation of S6K1 has been reported in esophageal squamous cell carcinomas (ESCC) [87]. These and several other examples highlight the importance of S6K1 in various cancerous state and as such makes it a potential target for therapeutic interventions to treat cancer. A number of pharmaceutical companies have

**5.3. Cellular metastasis—handy option for therapeutic interventions**

phosphorylates several of its targets involved in the initiation of translation.

that S6K−

/ −

154 Homeostasis - An Integrated Vision

to S remains undisputed.

**Figure 6.** S6K1, a prudent target for treating cancers: an illustration representing S6K1 as a potential target for the specific inhibitors which could be used to overcome the resistance observed while using mTORC1 inhibitors during various cancerous situations.

#### **6. Conclusion**

Understanding S6K1 regulation and its associated functions over the last two decades has seen an upward trend. It has now been possible to establish S6K1 an important regulator of cell growth and proliferation. Further our understanding vis-à-vis S6K1 functions have also improved considerably to dissociate many of its functions from the ones related to phosphorylating rpS6 only. Instead its control over various cellular factors makes it more important candidate for regulating cellular physiology. Of late, S6K1 appears as a prudent target for treating various cancers and as such various trials have been initiated to use specific S6K1 inhibitors with the aim to treat different cancers where the routine practices of using generalized drugs appear less effective. However, achieving a complete success in this endeavor still seems difficult. A major challenge in this regard is to ensure that S6K1 s role is tumor specific regardless of its type or origin. Resolving such issues will of course be a task that would attract more research in the years to come.

[5] Jenö P, Ballou LM, Novak-Hofer I, Thomas G. Identification and characterization of a mitogen-activated S6 kinase. Proceedings of the National Academy of Sciences.

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## **Conflict of interest**

The authors declare that there is no conflict of interest.

#### **Author details**

Sheikh Tahir Majeed<sup>1</sup> , Rabiya Majeed1,2, Ghazia Shah<sup>1</sup> and Khurshid I Andrabi<sup>1</sup> \*

\*Address all correspondence to: andrabik@uok.edu.in

1 Department of Biotechnology, Science block, University of Kashmir, Jammu and Kashmir, India

2 Department of Biochemistry, University of Kashmir, Jammu and Kashmir, India

#### **References**


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**6. Conclusion**

156 Homeostasis - An Integrated Vision

**Conflict of interest**

**Author details**

India

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Sheikh Tahir Majeed<sup>1</sup>

The authors declare that there is no conflict of interest.

\*Address all correspondence to: andrabik@uok.edu.in

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such issues will of course be a task that would attract more research in the years to come.

, Rabiya Majeed1,2, Ghazia Shah<sup>1</sup>

1 Department of Biotechnology, Science block, University of Kashmir, Jammu and Kashmir,

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## *Edited by Fernanda Lasakosvitsch and Sergio Dos Anjos Garnes*

The human body is composed of several systems and organs, consisting of millions of cells that need relatively stable conditions to function and contribute to the survival of the body as a whole. The maintenance of stable conditions for the cells against the variations of the external environment is an essential function of the body and is called homeostasis. As a consequence of the loss of homeostasis, a disease is manifested. This book aims to provide the reader with an up-to-date view of the self-regulatory mechanisms that are activated to achieve homeostasis, the pathways that are altered during the disease process, and how medicine can intervene to restore balance in critical patients.

Published in London, UK © 2019 IntechOpen © leonid\_tit / iStock

Homeostasis - An Integrated Vision

Homeostasis

An Integrated Vision

*Edited by Fernanda Lasakosvitsch* 

*and Sergio Dos Anjos Garnes*