**Meet the editor**

After undergraduate and postgraduate study in the North of England and London respectively, David Gaze works as a Research Biochemist in Clinical Blood Sciences at St George's Hospital and Medical School, London. His area of research interest is the development and clinical utility of cardiovascular biomarkers for assessment of patients with cardiovascular disease and cardiorenal disease. He has

authored and co authored over 115 peer reviewed original papers, editorials and reviews, 145 conference proceedings and has authored two book chapters and a textbook on cardiac troponin. He has delivered over 25 invited international presentations. He received two NACB distinguished abstracts awards (2006/2008) and a diploma for oral presentation from the 17th IFCC–FESCC European Congress of Clinical Chemistry and Laboratory Medicine. He is a member of the American Association of Clinical Biochemistry; Member of the Association for Biochemistry; Associate member of the Royal Institute; Member of the Institute of Biomedical Sciences; Fellow of the Royal Society of Medicine and a member of the Pathological Society of Great Britain.

Contents

**Preface IX** 

**Section 1 Cardiovascular Physiology 1** 

and Sergey Kolmakov

Tadashi Yoshida

Chapter 4 **Control and Coordination** 

Chapter 5 **Hemodynamics 95**  Ali Nasimi

and Xavier F. Figueroa

and Carmen Diniz

Chapter 7 **Endothelial Nitric Oxide Synthase,** 

Grażyna Lutosławska

**in the Vascular System 135** 

Chapter 1 **Control of Cardiovascular System 3**  Mikhail Rudenko, Olga Voronova, Vladimir Zernov, Konstantin Mamberger, Dmitry Makedonsky, Sergey Rudenko

Chapter 2 **Molecular Control of Smooth Muscle Cell** 

Chapter 3 *Trans* **Fatty Acids and Human Health 43**  Sebastjan Filip and Rajko Vidrih

> Mauricio A. Lillo, Francisco R. Pérez, Mariela Puebla, Pablo S. Gaete

**Factor and Its Interacting Proteins 23** 

**Differentiation Marker Genes by Serum Response** 

**of Vasomotor Tone in the Microcirculation 65** 

Chapter 6 **Adenosinergic System in the Mesenteric Vessels 111**  Ana Leitão-Rocha, Joana Beatriz Sousa

**Nitric Oxide and Metabolic Disturbances** 

## Contents

## **Preface XIII**

**Section 1 Cardiovascular Physiology 1**  Chapter 1 **Control of Cardiovascular System 3**  Mikhail Rudenko, Olga Voronova, Vladimir Zernov, Konstantin Mamberger, Dmitry Makedonsky, Sergey Rudenko and Sergey Kolmakov Chapter 2 **Molecular Control of Smooth Muscle Cell Differentiation Marker Genes by Serum Response Factor and Its Interacting Proteins 23**  Tadashi Yoshida Chapter 3 *Trans* **Fatty Acids and Human Health 43**  Sebastjan Filip and Rajko Vidrih Chapter 4 **Control and Coordination of Vasomotor Tone in the Microcirculation 65**  Mauricio A. Lillo, Francisco R. Pérez, Mariela Puebla, Pablo S. Gaete and Xavier F. Figueroa Chapter 5 **Hemodynamics 95**  Ali Nasimi Chapter 6 **Adenosinergic System in the Mesenteric Vessels 111**  Ana Leitão-Rocha, Joana Beatriz Sousa and Carmen Diniz Chapter 7 **Endothelial Nitric Oxide Synthase, Nitric Oxide and Metabolic Disturbances in the Vascular System 135** 

Grażyna Lutosławska


Contents VII

Chapter 17 **Importance of Dermatology in Infective Endocarditis 345** 

**of Intestine, Trophology Status and Systemic** 

**of Hypotension in Premature Infants 419** 

Chapter 21 **Role of Echocardiography in Research into Neglected** 

Francisco Palmero and Cristina Guerrero

**Cardiovascular Diseases in Sub-Saharan Africa 445**

**Functioning in Hostile Defensive Women 465** 

Chapter 18 **Cardiovascular Risk Factors: Implications in Diabetes, Other Disease States and Herbal Drugs 365**

Steve Ogbonnia

Chapter 20 **Evaluation and Treatment** 

Ana Olga Mocumbi

Chapter 22 **Psychophysiological Cardiovascular** 

Chapter 19 **Morphology and Functional Changes** 

G.P. Arutyunov and N.A. Bylova

Shoichi Ezaki and Masanori Tamura

Servy Amandine, Jones Meriem and Valeyrie-Allanore Laurence

**Inflammation in Patients with Chronic Heart Failure 383** 


VI Contents

**Section 2 Cardiovascular Diagnostics 155** 

Chapter 8 **The Diagnostic Performance of Cardiovascular System** 

Mikhail Rudenko, Olga Voronova, Vladimir Zernov, Konstantin Mamberger, Dmitry Makedonsky,

Sergey Rudenko, Yuri Fedossov, Alexander Duyzhikov,

Mikhail Rudenko, Olga Voronova and Vladimir Zernov

**of Phase Functions of Cardiac Muscle Contraction and Biochemical Processes as an Algorithm for** 

Yury Fedosov, Stanislav Zhigalov, Mikhail Rudenko,

**Techniques for Cardiovascular Diagnostics 211** 

Chapter 12 **Analysis of Time Course Changes in the Cardiovascular Response to Head-Up Tilt in Fighter Pilots 241** 

**the Process of Indirect Arterial Pressure Measurement 179**

**Identifying Local Pathologies in Cardiovascular System 195**

**on Heart Cycle Phase Analysis 157** 

Chapter 9 **Biophysical Phenomena in Blood Flow System in** 

Vladimir Zernov and Olga Voronova

C. Nataraj, A. Jalali and P. Ghorbanian

David G. Newman and Robin Callister

**Physiology and Pathophysiology 255** 

Chapter 13 **Physical Activity and Cardiovascular Health 257**

Chapter 14 **Cardiovascular Disease Risk Factors 279** Reza Amani and Nasrin Sharifi

Chapter 15 **Cardiovascular and Cerebrovascular Problems** 

**For Medical Professionals Involved in the Treatment of Atherosclerosis 311** 

**in the Development of Cognitive Impairment:** 

Chapter 16 **French Paradox, Polyphenols and Cardiovascular Protection: The Oestrogenic Receptor-α Implication 319**  Tassadit Benaissa, Thierry Ragot and Angela Tesse

**Section 3 Clinical Impact of Cardiovascular** 

Raul A. Martins

Michihiro Suwa

Chapter 11 **Application of Computational Intelligence** 

Anatoly Orlov and Sergey Sobin

Chapter 10 **Interrelation Between the Changes** 

**and Evaluation of Hemodynamic Parameters Based** 


Preface

main sections:

respiration, metabolism and immunity.

The cardiovascular system includes the heart located centrally in the thorax and the vessels of the body which carry blood. The cardiovascular (or circulatory) system supplies oxygen from inspired air, via the lungs to the tissues around the body. It is also responsible for the removal of the waste product, carbon dioxide via air expired from the lungs. The cardiovascular system also transports nutrients such as electrolytes, amino acids, enzymes, hormones which are integral to cellular

This book is not meant to be an all encompassing text on cardiovascular physiology and pathology rather a selection of chapters from experts in the field who describe recent advances in basic and clinical sciences. As such, the text is divided into three

1. *Cardiovascular Physiology –* In this section, the control of the cardiovascular system is discussed in particular the heaemodynamic mechanisms controlling blood volume, flow and the regulation of systolic blood pressure. The next chapter investigates the molecular control of smooth muscle cell (SMC) differentiation marker genes by serum response factor (SRF) including the interaction of myocardin as a potent cofactor of SRF in SMC differentiation. The chapter also details the interaction of GATA‐6, Klf4, LIM‐only proteins CRP1 and 2 and PIAS‐1 with SRF. The following chapter reports on trans fatty acids (TFA) and human health, detailing the biochemistry of trans fats as well as recommended daily intake. The chapter describes both animal and human studies of TFA. There are details on the analytical determination of TFA as well as their potential antioxidants. There is also a comprehensive overview of TFA and legislative control in food production and consumption. This is followed by a chapter on the control and coordination of vasomotor tone in the microcirculation; concentrating on the cellular membrane potential and potassium channels, the role of prostaglandins, nitric oxide and endothelium‐derived hyperpolarizing factor as paracrine signalling in the wall of the vessel. There is also detail of the role of gap junctions in vascular smooth muscle and endothelium communication processes. The following chapter discusses the concept of hemodynamics, detailing the relationship between physical factors and the effect on blood flow through the vessel in laminar or turbulent flow patterns. The principles of

## Preface

The cardiovascular system includes the heart located centrally in the thorax and the vessels of the body which carry blood. The cardiovascular (or circulatory) system supplies oxygen from inspired air, via the lungs to the tissues around the body. It is also responsible for the removal of the waste product, carbon dioxide via air expired from the lungs. The cardiovascular system also transports nutrients such as electrolytes, amino acids, enzymes, hormones which are integral to cellular respiration, metabolism and immunity.

This book is not meant to be an all encompassing text on cardiovascular physiology and pathology rather a selection of chapters from experts in the field who describe recent advances in basic and clinical sciences. As such, the text is divided into three main sections:

1. *Cardiovascular Physiology –*  In this section, the control of the cardiovascular system is discussed in particular the heaemodynamic mechanisms controlling blood volume, flow and the regulation of systolic blood pressure. The next chapter investigates the molecular control of smooth muscle cell (SMC) differentiation marker genes by serum response factor (SRF) including the interaction of myocardin as a potent cofactor of SRF in SMC differentiation. The chapter also details the interaction of GATA‐6, Klf4, LIM‐only proteins CRP1 and 2 and PIAS‐1 with SRF. The following chapter reports on trans fatty acids (TFA) and human health, detailing the biochemistry of trans fats as well as recommended daily intake. The chapter describes both animal and human studies of TFA. There are details on the analytical determination of TFA as well as their potential antioxidants. There is also a comprehensive overview of TFA and legislative control in food production and consumption. This is followed by a chapter on the control and coordination of vasomotor tone in the microcirculation; concentrating on the cellular membrane potential and potassium channels, the role of prostaglandins, nitric oxide and endothelium‐derived hyperpolarizing factor as paracrine signalling in the wall of the vessel. There is also detail of the role of gap junctions in vascular smooth muscle and endothelium communication processes. The following chapter discusses the concept of hemodynamics, detailing the relationship between physical factors and the effect on blood flow through the vessel in laminar or turbulent flow patterns. The principles of velocity, elasticity and compliance are described. Furthermore the clinical implications such as alteration to blood flow during atherosclerosis and arteriosclerosis are described. The penultimate chapter of this section describes the adenosinergic system in the mesenteric vessels which form the splanchnic circulation. The chapter details the role of adenosine from its production to tissue concentration controlled by nucleoside transporter membrane proteins, namely equilibrative and concentrative nucleoside transporters. The family member subtypes are of these transporter proteins are described thoroughly. The final chapter of section one concentrates on endothelial nitric oxide synthase (eNOS), nitric oxide (NO) and subsequent metabolic disturbances within the vascular system. An overview of vascular dysfunction is given along with the biochemistry of eNOS/NO. The endogenous eNOS and NO inhibitor asymmetric dimethylarginine and its role in the vascular system is also reviewed. The reader is also given the importance of lifestyle on the vascular system, concentrating on dietary habits and physical activity on the eNOS/NO system.

Preface XI

measuring mean arterial pressure, heart rate, stroke volume and total peripheral resistance, the authors compare the cardiovascular responses in fighter pilots

3. *Clinical Impact of Cardiovascular Physiology and Pathophysiology –* The final section of this textbook relates physiology to pathophysiology, clinical presentation and implications of cardiovascular diseases. The first chapter of this section explores the relationship of cardiovascular health and exercise from both the European and North American perspectives, detailing the relationship between physical activity and life expectancy and discusses the pro‐inflammatory state in relation to reduced physical activity and its relationship to cardiovascular disease. The second chapter reviews the global burden of cardiovascular disease and the associated risk factors, including lipid components, inflammatory markers, fibrinogen, smoking and dietary modification to reduce the incidence of cardiac disease. The next chapter details the associations between cardiac and cerebral vascular issues in patients with neurodegenerative diseases such as Alzheimer's disease. Risk factors such as hyperlipidemia, hypertension, diabetes and a history of smoking contribute to deterioration of cognitive function. A reduction of cerebral perfusion following ventricular dysfunction can also contribute to the advancement of cognitive decline. The 'French Paradox' of a low incidence of cardiovascular disease in people who consumed moderate red wine irrespective of the quantity of saturated fatty acids and describes the cardioprotective role of polyphenolic compounds is discussed in the next chapter. The fifth chapter in section 3 discusses the clinical implications of dermatological findings in patients who develop infective endocarditis, in particular the causative microorganisms, risk factors, the clinical signs and symptoms, and the clinical tools to aid diagnosis. The next chapter details the cardiovascular risk factors associated with the development of diabetes mellitus and the role of herbal drugs to control cardiac risk factors. The next chapter reviews the morphology and functional changes of the intestine in patients with heart failure identifying the systemic nature of heart failure. A comprehensive overview of the histological patterns observed and the pro inflammatory state of the gastrointestinal tract is presented. Chapter 20 describes the evaluation and treatment of hypotensive premature infants which is a common phenomenon in the first few weeks of life; describing the interplay between hypovolaemia, tissue hypoxia and myocardial dysfunction. The clinical presentation is described along with diagnostic modalities used to detect hypotensive cardiac problems, followed by the treatment regimens available to correct to the normotensive state. The next interesting chapter discusses the role of echocardiography in Sub‐Saharan Africa. Access to echocardiography is common place in the developed world. In the remoteness of Africa, access to such diagnostic tests are rarely available due to cost, logistical access and the lack of trained sonographers. This chapter reviews the current usage of echocardiography to describe the epidemiology of cardiovascular disease in an otherwise neglected population. The final chapter to

compared to non‐pilots.

2. *Cardiovascular Diagnostics –* Section 2 is concerned with modalities used in the diagnosis and monitoring of parameters associated with the cardiovascular system. The first chapter entitled 'the diagnostic performance of cardiovascular system and evaluation of hemodynamic parameters based on heart cycle phase analysis describes the development and use of the electrocardiogram (ECG) and the rheogram. Furthermore the use of both the ECG and rheogram to assess cardiovascular function in normal and diseased states are described. The second chapter describes the biophysical phenomena of blood flow during indirect arterial pressure measurement. The role of the oscillogram in measuring systolic and diastolic arterial pressure is well described compared to the practice of auscultation of Korotkov sounds. The chapter also notes the peculiarities seen in some oscillogram readings. The third chapter describes the interrelation between changes of phase function of cardiac muscle concentration and the biochemical processes as an algorithm for identifying pathological processes within the cardiovascular system. In this chapter the authors outline their vision of the main biochemical processes determining the clinical meaning of the pathology diagnosed with the aid of the cardiac cycle analysis method. Selection of the therapeutic agents aimed at normalization of the diagnosed functional deviations taking into account the biochemical processes underlying these functions resulted in the recovery of the functions. The next chapter investigates computational intelligence techniques in cardiovascular diagnostics. Continual monitoring of cardiac function in the acute care setting can allow the detection of cardiac arrhythmias. Continuous wavelet transform and principal component analysis are described in detail. The application of these techniques within a multi-layer perceptron neural network is demonstrated. The final chapter of this section analyses the time course changes in the cardiovascular response to head‐up tilt in fighter pilots. In this interesting chapter the authors describe the physiological adaptations that occur following frequent exposure to G‐force acceleration. By

measuring mean arterial pressure, heart rate, stroke volume and total peripheral resistance, the authors compare the cardiovascular responses in fighter pilots compared to non‐pilots.

X Preface

velocity, elasticity and compliance are described. Furthermore the clinical implications such as alteration to blood flow during atherosclerosis and arteriosclerosis are described. The penultimate chapter of this section describes the adenosinergic system in the mesenteric vessels which form the splanchnic circulation. The chapter details the role of adenosine from its production to tissue concentration controlled by nucleoside transporter membrane proteins, namely equilibrative and concentrative nucleoside transporters. The family member subtypes are of these transporter proteins are described thoroughly. The final chapter of section one concentrates on endothelial nitric oxide synthase (eNOS), nitric oxide (NO) and subsequent metabolic disturbances within the vascular system. An overview of vascular dysfunction is given along with the biochemistry of eNOS/NO. The endogenous eNOS and NO inhibitor asymmetric dimethylarginine and its role in the vascular system is also reviewed. The reader is also given the importance of lifestyle on the vascular system, concentrating on

dietary habits and physical activity on the eNOS/NO system.

2. *Cardiovascular Diagnostics –* Section 2 is concerned with modalities used in the diagnosis and monitoring of parameters associated with the cardiovascular system. The first chapter entitled 'the diagnostic performance of cardiovascular system and evaluation of hemodynamic parameters based on heart cycle phase analysis describes the development and use of the electrocardiogram (ECG) and the rheogram. Furthermore the use of both the ECG and rheogram to assess cardiovascular function in normal and diseased states are described. The second chapter describes the biophysical phenomena of blood flow during indirect arterial pressure measurement. The role of the oscillogram in measuring systolic and diastolic arterial pressure is well described compared to the practice of auscultation of Korotkov sounds. The chapter also notes the peculiarities seen in some oscillogram readings. The third chapter describes the interrelation between changes of phase function of cardiac muscle concentration and the biochemical processes as an algorithm for identifying pathological processes within the cardiovascular system. In this chapter the authors outline their vision of the main biochemical processes determining the clinical meaning of the pathology diagnosed with the aid of the cardiac cycle analysis method. Selection of the therapeutic agents aimed at normalization of the diagnosed functional deviations taking into account the biochemical processes underlying these functions resulted in the recovery of the functions. The next chapter investigates computational intelligence techniques in cardiovascular diagnostics. Continual monitoring of cardiac function in the acute care setting can allow the detection of cardiac arrhythmias. Continuous wavelet transform and principal component analysis are described in detail. The application of these techniques within a multi-layer perceptron neural network is demonstrated. The final chapter of this section analyses the time course changes in the cardiovascular response to head‐up tilt in fighter pilots. In this interesting chapter the authors describe the physiological adaptations that occur following frequent exposure to G‐force acceleration. By

3. *Clinical Impact of Cardiovascular Physiology and Pathophysiology –* The final section of this textbook relates physiology to pathophysiology, clinical presentation and implications of cardiovascular diseases. The first chapter of this section explores the relationship of cardiovascular health and exercise from both the European and North American perspectives, detailing the relationship between physical activity and life expectancy and discusses the pro‐inflammatory state in relation to reduced physical activity and its relationship to cardiovascular disease. The second chapter reviews the global burden of cardiovascular disease and the associated risk factors, including lipid components, inflammatory markers, fibrinogen, smoking and dietary modification to reduce the incidence of cardiac disease. The next chapter details the associations between cardiac and cerebral vascular issues in patients with neurodegenerative diseases such as Alzheimer's disease. Risk factors such as hyperlipidemia, hypertension, diabetes and a history of smoking contribute to deterioration of cognitive function. A reduction of cerebral perfusion following ventricular dysfunction can also contribute to the advancement of cognitive decline. The 'French Paradox' of a low incidence of cardiovascular disease in people who consumed moderate red wine irrespective of the quantity of saturated fatty acids and describes the cardioprotective role of polyphenolic compounds is discussed in the next chapter. The fifth chapter in section 3 discusses the clinical implications of dermatological findings in patients who develop infective endocarditis, in particular the causative microorganisms, risk factors, the clinical signs and symptoms, and the clinical tools to aid diagnosis. The next chapter details the cardiovascular risk factors associated with the development of diabetes mellitus and the role of herbal drugs to control cardiac risk factors. The next chapter reviews the morphology and functional changes of the intestine in patients with heart failure identifying the systemic nature of heart failure. A comprehensive overview of the histological patterns observed and the pro inflammatory state of the gastrointestinal tract is presented. Chapter 20 describes the evaluation and treatment of hypotensive premature infants which is a common phenomenon in the first few weeks of life; describing the interplay between hypovolaemia, tissue hypoxia and myocardial dysfunction. The clinical presentation is described along with diagnostic modalities used to detect hypotensive cardiac problems, followed by the treatment regimens available to correct to the normotensive state. The next interesting chapter discusses the role of echocardiography in Sub‐Saharan Africa. Access to echocardiography is common place in the developed world. In the remoteness of Africa, access to such diagnostic tests are rarely available due to cost, logistical access and the lack of trained sonographers. This chapter reviews the current usage of echocardiography to describe the epidemiology of cardiovascular disease in an otherwise neglected population. The final chapter to

#### XII Preface

this section and the whole book describes a study of cardiovascular functional parameters such as heart rate and blood pressure along with a psychophysiological assessment in females displaying defensive hostility demonstrating such women have higher heart rates and blood pressures if they were defensive compared to those with low hostility.

> **David C. Gaze** *Dept of Chemical Pathology Clinical Blood Sciences, St George's Healthcare NHS Trust, London United Kingdom*

#### **Acknowledgements**

I would like to acknowledge the tremendous efforts of the contributing authors to these chapters, especially when writing in English rather than their native tongue. I would also like to thank Ms Iva Simcic of InTECH publishers for keeping the production of this book active and to for steering me to complete the editorial review by the appropriate deadlines.

XII Preface

**Acknowledgements**

by the appropriate deadlines.

this section and the whole book describes a study of cardiovascular functional parameters such as heart rate and blood pressure along with a psychophysiological assessment in females displaying defensive hostility demonstrating such women have higher heart rates and blood pressures if they

I would like to acknowledge the tremendous efforts of the contributing authors to these chapters, especially when writing in English rather than their native tongue. I would also like to thank Ms Iva Simcic of InTECH publishers for keeping the production of this book active and to for steering me to complete the editorial review

*Dept of Chemical Pathology Clinical Blood Sciences,* 

*St George's Healthcare NHS Trust, London* 

**David C. Gaze**

*United Kingdom* 

were defensive compared to those with low hostility.

**Section 1** 

**Cardiovascular Physiology** 

## **Section 1**

**Cardiovascular Physiology** 

**1** 

 *Russia* 

**Control of Cardiovascular System** 

Mikhail Rudenko, Olga Voronova, Vladimir Zernov,

The main method of cognition of the performance of biological systems is their mathematical modeling. The essence of this method should reflect the principle of optimization in biology[9]. Any biosystem cannot function if its energy consumption is

The same is applicable to the blood circulatory system. Its main function is to transport blood throughout the body in order to maintain the proper gaseous exchange, deliver important substances to viscera and tissues in living body and remove decay products. It is impossible to study this function without due consideration of hemodynamic features. But how is the blood circulation provided? It is a question of principle, and so far no

The conventional interpretation of blood circulation is that blood flows through blood vessels under laminar flow conditions to which Poiseuille's law is applicable. But it is a matter of fact that this conventional interpretation concept is inadequate because it is not in compliance with the above principle of optimization in biology, according to which all processes in bio systems show their best performance, i.e., their highest efficiency. It is just the compliance with this principle that is the major criterion to be used for evaluation of adequacy of any theoretical models describing various systems in living body and their

Significant progress in understanding of such phenomena is made after G. Poyedintsev and O.Voronova discovered the so called mode of elevated fluidity, i.e., the third flow conditions that show lesser losses of energy to overcome friction and that is noted for lesser friction

It has been proved that the blood flow through the blood vessels is provided in "the third" flow mode that is the most efficient and therefore fully in compliance with the said principle

The theory of the third mode is a foundation for the development of new mathematical models describing the performance of the blood circulation system. In addition, new methods of quantitative determination of a number of hemodynamic parameters and

**1. Introduction**

inadequately high.

unambiguous answer has been given thereto.

losses and specific pattern of the flow[4].

of optimization.

interactions both with each other and their external environment.

Konstantin Mamberger, Dmitry Makedonsky,

Sergey Rudenko and Sergey Kolmakov

*Russian New University,* 

## **Control of Cardiovascular System**

Mikhail Rudenko, Olga Voronova, Vladimir Zernov, Konstantin Mamberger, Dmitry Makedonsky, Sergey Rudenko and Sergey Kolmakov *Russian New University, Russia* 

## **1. Introduction**

The main method of cognition of the performance of biological systems is their mathematical modeling. The essence of this method should reflect the principle of optimization in biology[9]. Any biosystem cannot function if its energy consumption is inadequately high.

The same is applicable to the blood circulatory system. Its main function is to transport blood throughout the body in order to maintain the proper gaseous exchange, deliver important substances to viscera and tissues in living body and remove decay products. It is impossible to study this function without due consideration of hemodynamic features. But how is the blood circulation provided? It is a question of principle, and so far no unambiguous answer has been given thereto.

The conventional interpretation of blood circulation is that blood flows through blood vessels under laminar flow conditions to which Poiseuille's law is applicable. But it is a matter of fact that this conventional interpretation concept is inadequate because it is not in compliance with the above principle of optimization in biology, according to which all processes in bio systems show their best performance, i.e., their highest efficiency. It is just the compliance with this principle that is the major criterion to be used for evaluation of adequacy of any theoretical models describing various systems in living body and their interactions both with each other and their external environment.

Significant progress in understanding of such phenomena is made after G. Poyedintsev and O.Voronova discovered the so called mode of elevated fluidity, i.e., the third flow conditions that show lesser losses of energy to overcome friction and that is noted for lesser friction losses and specific pattern of the flow[4].

It has been proved that the blood flow through the blood vessels is provided in "the third" flow mode that is the most efficient and therefore fully in compliance with the said principle of optimization.

The theory of the third mode is a foundation for the development of new mathematical models describing the performance of the blood circulation system. In addition, new methods of quantitative determination of a number of hemodynamic parameters and

Control of Cardiovascular System 5

Fig. 1. Formation of concentric waves of friction at initiation of flow in a pipe (according to G.M. Poyedintsev and O.K.Voronova); t1 - moment of pressure difference formation; V1 velocity of plasma in stagnated layers; V2 – velocity of blood elements in accelerated layers

There is another phenomenon typical for the "third" flow mode. If liquid contains suspended particles similar to those in blood, during the development of the above mentioned wave process the particles are concentrated at the wave maxima, and the particle-free liquid is delivered to their minima, correspondingly[3]. When the liquid, patterned in such a way, flows along the pipe axis, the velocity of the concentric particleloaded layers is twice what the liquid pattern-free layers reach. Vectors of velocity are parallel to the axis of the flow. And it is just a prerequisite to elevated fluidity of liquid with reduced friction between the liquid layers and the pipe wall. Figure 2 herein shows the locations of erythrocytes in the blood flow referring to each flow formation stage as mentioned above. At the beginning of the formation of the "third" flow mode, there ringshaped alternating layers of the blood elements and plasma are available, while in the laminar mode all elements are accumulated in the center of the flow. In this case they are located very close to each other forming a thick mass. This process may result in an aggregation of erythrocytes and hemolysis. In order to avoid such pathological consequences, it is a must to manage the blood transportation in the "third" mode of flow,

The theory gives a clue that it can be obtained when transporting liquid in a pulsating mode through an elastic pipe. According to this theory, the pipe clear width and the liquid flow velocity should be changed with every impulse under certain laws[3]. The laws of increasing in the pipe clear width and decreasing in the flow velocity with every impulse

avoiding its transformation into a laminar one.

take the form as follows[4].

qualitative evaluation of some processes occurring in the system have been elaborated. The application of these methods in practice allows filling a lot of gaps in theoretical cardiology and creates at the same time a system of analysis of the functions of the cardiovascular system taking into account the relevant cause-effect relationship.

The detailed description of this theory is given in our book "Theoretical Principles of Heart Cycle Phase Analysis"[3]. Our intention is to outline herein the general principles of the performance of the cardiovascular system only.

## **2. Biophysical processes of formation of hemodynamic mechanism**

#### **2.1 Special features of hemodynamics and its regulation. Hemodynamic volumetric parameters**

There two types of liquid flow conditions described in the classical fluid mechanics: the first type is the laminar flow, and the second one is the turbulent flow mode. In the 80th last century, a new theory of a specific liquid flow mode was developed by G.M. Poyedinstev and O.K. Voronova that was defined by them as the "elevated fluidity mode"[4]. Another name "the third flow mode" was given by the above discoverers to differ it from the two other modes well-known before. Being experts in solving technical problems of fluid mechanics, the authors succeeded in modeling the above elevated fluidity mode in a rigid pipe. For this purpose, hydraulic pulsators of specific design were used. It was established that the energy used to transport liquid in the third flow mode is several times less than it is the case under the laminar flow conditions[3]. Moreover, an efficiency of this process could be considerably increased when liquid is pumped under certain conditions through an elastic piping. The subsequent researches demonstrated that the physical processes producing the elevated fluidity mode and those in the blood circulation are identical. The mathematical tools used to describe "the third" flow mode was applied to describe the hemodynamic processes.

It was established by the authors that there are processes which are always observed in a rigid pipe at the initiation of a liquid flow from a quiescent state, as mentioned below. Whilst particles of liquid are starting their moving in the rigid pipe due to a difference in the static pressure, there a set of concentric waves of friction in the boundary layer is generating, the front of propagation of which is directed towards the pipe axis[3] (Fig. 1). Amplitudes of these waves depend on the diameter of the pipe, acoustic velocity in liquid and an initial difference in pressures at the pipe ends. The length of these traveling waves during this complex process continuously increases. The waves travel towards the axis of the pipe and degenerate. Finally, there a single wave remains only close to the pipe wall, the profile of which becomes parabolic that is typical for the laminar flow (s. Fig. 2 herein).

It should be noted that it is just within this short period of time, i.e., starting from the moment of the motion initiation from a quiescent state till the moment of formation of the laminar flow (s. positions E and F in Fig. 2 herein), when liquid flows in its optimum mode of elevated fluidity, considering it from the point of view of energy consumption (s. positions A, B, C, D in Fig 2 herein). The energy consumption under the laminar flow conditions to transport liquid in the pipe is significantly higher due to increase in the flow resistance.

qualitative evaluation of some processes occurring in the system have been elaborated. The application of these methods in practice allows filling a lot of gaps in theoretical cardiology and creates at the same time a system of analysis of the functions of the cardiovascular

The detailed description of this theory is given in our book "Theoretical Principles of Heart Cycle Phase Analysis"[3]. Our intention is to outline herein the general principles of the

**2.1 Special features of hemodynamics and its regulation. Hemodynamic volumetric** 

There two types of liquid flow conditions described in the classical fluid mechanics: the first type is the laminar flow, and the second one is the turbulent flow mode. In the 80th last century, a new theory of a specific liquid flow mode was developed by G.M. Poyedinstev and O.K. Voronova that was defined by them as the "elevated fluidity mode"[4]. Another name "the third flow mode" was given by the above discoverers to differ it from the two other modes well-known before. Being experts in solving technical problems of fluid mechanics, the authors succeeded in modeling the above elevated fluidity mode in a rigid pipe. For this purpose, hydraulic pulsators of specific design were used. It was established that the energy used to transport liquid in the third flow mode is several times less than it is the case under the laminar flow conditions[3]. Moreover, an efficiency of this process could be considerably increased when liquid is pumped under certain conditions through an elastic piping. The subsequent researches demonstrated that the physical processes producing the elevated fluidity mode and those in the blood circulation are identical. The mathematical tools used to describe "the third" flow mode was applied to describe the

It was established by the authors that there are processes which are always observed in a rigid pipe at the initiation of a liquid flow from a quiescent state, as mentioned below. Whilst particles of liquid are starting their moving in the rigid pipe due to a difference in the static pressure, there a set of concentric waves of friction in the boundary layer is generating, the front of propagation of which is directed towards the pipe axis[3] (Fig. 1). Amplitudes of these waves depend on the diameter of the pipe, acoustic velocity in liquid and an initial difference in pressures at the pipe ends. The length of these traveling waves during this complex process continuously increases. The waves travel towards the axis of the pipe and degenerate. Finally, there a single wave remains only close to the pipe wall, the profile of

It should be noted that it is just within this short period of time, i.e., starting from the moment of the motion initiation from a quiescent state till the moment of formation of the laminar flow (s. positions E and F in Fig. 2 herein), when liquid flows in its optimum mode of elevated fluidity, considering it from the point of view of energy consumption (s. positions A, B, C, D in Fig 2 herein). The energy consumption under the laminar flow conditions to transport liquid in the pipe is significantly higher due to increase in the flow

which becomes parabolic that is typical for the laminar flow (s. Fig. 2 herein).

**2. Biophysical processes of formation of hemodynamic mechanism** 

system taking into account the relevant cause-effect relationship.

performance of the cardiovascular system only.

**parameters** 

hemodynamic processes.

resistance.

Fig. 1. Formation of concentric waves of friction at initiation of flow in a pipe (according to G.M. Poyedintsev and O.K.Voronova); t1 - moment of pressure difference formation; V1 velocity of plasma in stagnated layers; V2 – velocity of blood elements in accelerated layers

There is another phenomenon typical for the "third" flow mode. If liquid contains suspended particles similar to those in blood, during the development of the above mentioned wave process the particles are concentrated at the wave maxima, and the particle-free liquid is delivered to their minima, correspondingly[3]. When the liquid, patterned in such a way, flows along the pipe axis, the velocity of the concentric particleloaded layers is twice what the liquid pattern-free layers reach. Vectors of velocity are parallel to the axis of the flow. And it is just a prerequisite to elevated fluidity of liquid with reduced friction between the liquid layers and the pipe wall. Figure 2 herein shows the locations of erythrocytes in the blood flow referring to each flow formation stage as mentioned above. At the beginning of the formation of the "third" flow mode, there ringshaped alternating layers of the blood elements and plasma are available, while in the laminar mode all elements are accumulated in the center of the flow. In this case they are located very close to each other forming a thick mass. This process may result in an aggregation of erythrocytes and hemolysis. In order to avoid such pathological consequences, it is a must to manage the blood transportation in the "third" mode of flow, avoiding its transformation into a laminar one.

The theory gives a clue that it can be obtained when transporting liquid in a pulsating mode through an elastic pipe. According to this theory, the pipe clear width and the liquid flow velocity should be changed with every impulse under certain laws[3]. The laws of increasing in the pipe clear width and decreasing in the flow velocity with every impulse take the form as follows[4].

Control of Cardiovascular System 7

Fig. 3. Arterial pressure wave shape reography-recorded. ECG recorded simultaneously

the heart changes its shape ten times that corresponds to the heart cycle phases[4].

blood entering or leaving the heart in the respective phase in a cardiac cycle.

α)· [f2(

α)· f4(

ejection, symbolized as PV3 and PV4, respectively, are as follows:

PV3=S· (QR+RS)2· f1(

PV4=S· (QR+RS)2· f1(

where S - cross-section of ascending aorta;

 QR – phase duration according to ECG curve; RS – phase duration according to ECG curve;

> f1(α

10 [ 8

The foundation of hemodynamics is the phase mode of the heart performance. In one beat

The most efficient way is to evaluate the status of hemodynamics not only by values of integral parameters, i.e., stroke and minute volumes, but also phase-related volumes of

So, the final formulae for calculation the volumes of blood in the phase of rapid and slow

α)+f3(

α , β γ ,, δ

)= 243)25(

α

f2(α)= ; <sup>2</sup>

αβδα

]27)25[(5,22072 5

α

−−

1 <sup>5</sup> α−

)= )];(2)(5))(4( <sup>3</sup>

1 <sup>3322</sup> <sup>44</sup> <sup>552</sup>

χδ−−−+−−

α , β γ ,, δ

3

;

αβχαβ

−−

)] (ml); (3)

) (ml), (4)

with Rheogram.

f3(α , β γ ,, δ

Fig. 2. Formation of two-phase pattern at the initiation of the flow from a quiescent state (according to G.M. Poyedintsev and O.K. Voronova), A-F – flow structure in corresponding sections

$$r\_t = r\_0 \left(\frac{t}{t\_0}\right)^{\mu\overline{s}} \tag{1}$$

$$W\_t = W\_0 \left(\frac{t\_0}{t}\right)^{2\xi} \tag{2}$$

where *rt* – current radius of the pipe increasing;

*r0* – initial radius (at *t = t0* );

 *t* - current time (*t ≥ t0)*;

 *t0* – time of acceleration of flow velocity up to maximum velocity in an impulse;

*Wt* – current value of liquid flow velocity;

*W0* – maximum value of velocity in an impulse (at *t = t0*).

It is proved by the authors of this theory that the above conditions are met in the blood circulation system.

This is provided by changing in the clear width of blood vessels in every cardiac cycle and arterial pressure pulsating. The shape of the arterial pressure wave is given herein in Fig. 3 below.

Fig. 2. Formation of two-phase pattern at the initiation of the flow from a quiescent state (according to G.M. Poyedintsev and O.K. Voronova), A-F – flow structure in corresponding

 *t0* – time of acceleration of flow velocity up to maximum velocity in an impulse;

It is proved by the authors of this theory that the above conditions are met in the blood

This is provided by changing in the clear width of blood vessels in every cardiac cycle and arterial pressure pulsating. The shape of the arterial pressure wave is given herein in Fig. 3

51

52 0 <sup>0</sup> <sup>=</sup> *<sup>t</sup> t*

*rrt* (1)

*<sup>t</sup> WW* (2)

0 <sup>0</sup> = *t t*

sections

where *rt* – current radius of the pipe increasing;

*Wt* – current value of liquid flow velocity;

*W0* – maximum value of velocity in an impulse (at *t = t0*).

 *r0* – initial radius (at *t = t0* );  *t* - current time (*t ≥ t0)*;

circulation system.

below.

Fig. 3. Arterial pressure wave shape reography-recorded. ECG recorded simultaneously with Rheogram.

The foundation of hemodynamics is the phase mode of the heart performance. In one beat the heart changes its shape ten times that corresponds to the heart cycle phases[4].

The most efficient way is to evaluate the status of hemodynamics not only by values of integral parameters, i.e., stroke and minute volumes, but also phase-related volumes of blood entering or leaving the heart in the respective phase in a cardiac cycle.

So, the final formulae for calculation the volumes of blood in the phase of rapid and slow ejection, symbolized as PV3 and PV4, respectively, are as follows:

$$\text{PV3} = \text{S} \cdot (\text{QR} + \text{RS})^2 \cdot \text{f}\_{\text{l}}(\mathcal{Q}') \cdot [\text{f}\_2(\mathcal{Q}') + \text{f}\_3(\mathcal{Q}', \mathcal{B}, \mathcal{Y}, \mathcal{S})] \tag{3} \tag{3}$$

$$\text{PV4=S} \cdot (\text{QR+RS})^2 \cdot \text{f}\_l(\mathcal{Q}') \cdot \text{f}\_4(\mathcal{Q}', \mathcal{P}, \mathcal{Y}, \mathcal{S}) \tag{\text{ml}} \\ \tag{4}$$

where S - cross-section of ascending aorta;

QR – phase duration according to ECG curve;

RS – phase duration according to ECG curve;

$$\begin{aligned} \text{f}\_1(\mathcal{O}') &= \frac{22072, 5[(5\alpha - 2)^3 - 27]}{(5\alpha - 2)^5 - 243}; \\\\ \text{f}\_2(\alpha) &= \frac{\alpha^5 - 1}{2}; \end{aligned}$$

$$\mathbb{E}\left(\mathcal{Q}\left(\alpha^{\ast},\beta,\gamma,\delta\right)\right) = \frac{1}{8} [\frac{10}{3}(4\alpha^2 - \delta^2)(\beta^3 - \alpha^3) + 5\,\mathbb{X}\delta(\beta^4 - \alpha^4) - 2\,\mathbb{X}^2(\beta^5 - \alpha^5)]\mathbb{X}$$

Control of Cardiovascular System 9

3. The normal value of interval QT in every specific cardiac cycle is determined from the

 PQсег. = 1 / (10-6 638,44 HR2 + 9,0787) s (9) This equation has been produced according to the method of approximation of normal values PQсег., as known from the sources, considering their dependence on heart rate (HR). These values are used as initial values for calculations of an individual range of normal values of volumetric parameters in hemodynamics considering individual patient cases. In practice, for a better visualization of the data, it should be recommended to present them not only numerically but also graphically, as bar charts, as shown in Figure 4 herein. In the latter case, it is convenient to indicate the deviations from the normal value limits of the

For example. On Figure 4 a), b), c) the result of hemodynamic parameters PV2 measuring volume of blood entering the ventricle in atrial systole- is displayed as follows. Figure 4 a) in column "Blood volumes" shows the result of measuring 18,31 (ml). The second column "% of stroke volume" shows the deviation from the norm. It is 0% here. For quick associative perception of both these values and rapid highlighting of going beyond the bounds of norm parameter, there exists a dark green field with red light indicator to the right of this number in the column "indicators of measurement results". On the left and right sides of the dark green field we see the values of individual range of this hemodynamic parameter, calculated using equation 7, 8, 9. In this case, it is from 15.26 to 35,13 ml. Measured parameter of 18,31 ml is in the middle of the range, which corresponds to the 0% deviation from the norm. And the red light indicator that corresponds to this value is on the dark green background. Light green field - is a bound of "norm - pathology". Sides of this field correspond to excess or deficiency of more than 30% of norm. More than 30% excess requires special attention to the patient. As a rule, such patients needs hospital care. Figure 4 b) shows another patient's result, PV2 = 12,85 ml, and this result goes 15.84% beyond patient's individual norm 15,26 ... 35.13 ml. In this case red light indicates lack of blood volume, rather than redundancy. Lower (upper) than 30% value, but lower (upper) than normal value corridor, denotes further out-patient treatment for this patient. Fig. 4 c) shows a third patient with PV2 = 47,00 ml value, which goes 76.91% beyond his individual norm 10,72 ... 26.13 ml. Red light indicates the redundancy of blood volume. This patient should be examined by cardiac cycle phase analysis to identify the root causes of the disease. It's possible to identify these causes using ECG and RHEO for phase compensation mechanism of the cardio-vascular system

QT = 0.37 RR0.5 , s (male); (7)

QT = 0.4 RR0.5 , s (female) . (8)

QRSmax = 0.1 s. ; QRSmin = 0.08 s.

RSmax = 0.05 s. ; RSmin = 0.035 s.

Bazett formula as follows:

determination.

2. The upper and lower limit of the RS complex values:

4. Normal value PQсег. is calculated from a formula as indicated below:

actually calculated values of hemodynamic parameters as percentage.

$$\begin{aligned} \text{f}\_4(\alpha^\*, \beta^\*, \gamma, \delta) &= \frac{1}{8} [5(\delta^2 - \frac{8}{3}\alpha^2)(\beta^3 - \alpha^3) + 7, 5\chi\delta(\beta^4 - \alpha^4) + 3\chi^2(\beta^3 - \alpha^3)]; \\\\ \alpha &= (1 + \frac{Em}{QR + RS})^{0, 2}; \\\\ \beta &= (1 + \frac{Em + Er}{QR + RS})^{0, 2}; \\\\ \chi &= \frac{2(\alpha - 1)}{\beta - \alpha}; \end{aligned}$$

$$
\delta = \alpha (2 + \mathcal{Z}) \cdot \\
.
$$

Stroke volume SV is calculated by an equation as given below:

$$\text{SV} = \text{PV3} + \text{PV4} = \text{S} \cdot (\text{QR} + \text{RS})^2 \cdot \text{f}\_{\text{l}}(\mathcal{Q}) \cdot [\text{f}\_{\text{2}}(\mathcal{Q}') + \text{f}\_{\text{3}}(\mathcal{Q}, \mathcal{P}, \mathcal{Y}, \mathcal{S}) + \text{f}\_{\text{4}}(\mathcal{Q}', \mathcal{P}, \mathcal{Y}, \mathcal{S})] \quad \text{(ml)} \tag{5}$$

The minute stroke is computed as follows:

$$\mathbf{MV} = \mathbf{SV} \cdot \mathbf{HR} \qquad \text{(l/min)}\tag{6}$$

In similar way calculated are other phase-related volumes of blood as listed below:

PV1 – volume of blood entering the ventricle in premature diastole;

PV2 – volume of blood entering the ventricle in atrial systole;

PV5 – volume of blood pumped by ascending aorta as peristaltic pump.

So, the main parameters in hemodynamics are 7 volumes of blood entering or leaving the heart in different heart cycle phases. They are as follows: stroke volume SV, minute volume MV, two diastolic phase-related volumes PV1 and PV2, two systolic phase-related volumes PV3 and PV4, and PV5 as volume of blood pumped by the aorta.

The authors of this theory in their researches utilized relative phase volumes denoted by RV. Each relative phase volume is that expressed as a percentage of stroke volume SV. These relative parameters demonstrate contributions of each phase process to the formation of the stroke volume in general.

The above hemodynamic parameters should be used mainly in order to evaluate eventual deviations from their normal values, if any. The limits of normal values of hemodynamic parameters are not conditional, and they have their respective calculated values.

With respect to the normal values (the required parameters) in hemodynamics, they have been taken on the basis of the known data on ECG waves, intervals and segments for adults from the literature sources as given below:

1. The upper and lower limit of the QRS complex values:

(1 ) ; 0,2 *QR RS Em* +

(1 ) ; 0,2 *QR RS Em Er* + +

> 2( 1) β α

− <sup>−</sup> <sup>=</sup>

α

α)+f3(

МV = SV· HR (l/min) (6)

So, the main parameters in hemodynamics are 7 volumes of blood entering or leaving the heart in different heart cycle phases. They are as follows: stroke volume SV, minute volume MV, two diastolic phase-related volumes PV1 and PV2, two systolic phase-related volumes

The authors of this theory in their researches utilized relative phase volumes denoted by RV. Each relative phase volume is that expressed as a percentage of stroke volume SV. These relative parameters demonstrate contributions of each phase process to the formation

The above hemodynamic parameters should be used mainly in order to evaluate eventual deviations from their normal values, if any. The limits of normal values of hemodynamic

With respect to the normal values (the required parameters) in hemodynamics, they have been taken on the basis of the known data on ECG waves, intervals and segments for adults

parameters are not conditional, and they have their respective calculated values.

;

α , β,γ ,δ)+f4(

<sup>8</sup> [5( <sup>8</sup>

α

> α= +

β= +

> χ

δ = α(2 + χ).

In similar way calculated are other phase-related volumes of blood as listed below:

α)· [f2(

PV1 – volume of blood entering the ventricle in premature diastole; PV2 – volume of blood entering the ventricle in atrial systole;

PV3 and PV4, and PV5 as volume of blood pumped by the aorta.

PV5 – volume of blood pumped by ascending aorta as peristaltic pump.

Stroke volume SV is calculated by an equation as given below:

SV = PV3+ PV4=S· (QR+RS)2· f1(

of the stroke volume in general.

from the literature sources as given below:

1. The upper and lower limit of the QRS complex values:

The minute stroke is computed as follows:

β −α+

δ−

)= )( ) 7,5 ( ) <sup>3</sup> ( )]; <sup>3</sup>

1 <sup>2</sup> <sup>2</sup> <sup>3</sup> <sup>3</sup> <sup>4</sup> <sup>4</sup> <sup>2</sup> <sup>5</sup> <sup>5</sup>

χδ β −α+ χ β −α

α , β,γ ,δ

)] (ml) (5)

f4(α , β,γ ,δ QRSmax = 0.1 s. ; QRSmin = 0.08 s.


$$\text{QT} = 0.37 \text{ RR} \\ 0.5 \quad \text{s} \quad \text{(male)}; \tag{7}$$

$$\text{'QT=0.4\quadRR0}\text{:}\text{'s} \quad \text{(female)}\,\text{.}\tag{8}$$

4. Normal value PQсег. is calculated from a formula as indicated below:

$$\text{PQ}\_{\text{err.}} = 1 \;/\; \text{(10\;^6\; 638\,44\;\text{ HR2} + 9\,0787\)}\,\text{s} \tag{9}$$

This equation has been produced according to the method of approximation of normal values PQсег., as known from the sources, considering their dependence on heart rate (HR).

These values are used as initial values for calculations of an individual range of normal values of volumetric parameters in hemodynamics considering individual patient cases. In practice, for a better visualization of the data, it should be recommended to present them not only numerically but also graphically, as bar charts, as shown in Figure 4 herein. In the latter case, it is convenient to indicate the deviations from the normal value limits of the actually calculated values of hemodynamic parameters as percentage.

For example. On Figure 4 a), b), c) the result of hemodynamic parameters PV2 measuring volume of blood entering the ventricle in atrial systole- is displayed as follows. Figure 4 a) in column "Blood volumes" shows the result of measuring 18,31 (ml). The second column "% of stroke volume" shows the deviation from the norm. It is 0% here. For quick associative perception of both these values and rapid highlighting of going beyond the bounds of norm parameter, there exists a dark green field with red light indicator to the right of this number in the column "indicators of measurement results". On the left and right sides of the dark green field we see the values of individual range of this hemodynamic parameter, calculated using equation 7, 8, 9. In this case, it is from 15.26 to 35,13 ml. Measured parameter of 18,31 ml is in the middle of the range, which corresponds to the 0% deviation from the norm. And the red light indicator that corresponds to this value is on the dark green background. Light green field - is a bound of "norm - pathology". Sides of this field correspond to excess or deficiency of more than 30% of norm. More than 30% excess requires special attention to the patient. As a rule, such patients needs hospital care. Figure 4 b) shows another patient's result, PV2 = 12,85 ml, and this result goes 15.84% beyond patient's individual norm 15,26 ... 35.13 ml. In this case red light indicates lack of blood volume, rather than redundancy. Lower (upper) than 30% value, but lower (upper) than normal value corridor, denotes further out-patient treatment for this patient. Fig. 4 c) shows a third patient with PV2 = 47,00 ml value, which goes 76.91% beyond his individual norm 10,72 ... 26.13 ml. Red light indicates the redundancy of blood volume. This patient should be examined by cardiac cycle phase analysis to identify the root causes of the disease. It's possible to identify these causes using ECG and RHEO for phase compensation mechanism of the cardio-vascular system determination.

Control of Cardiovascular System 11

reflecting the coordinated operation of the heart and the associated blood vessels. Knowing their ratios and considering the actual anatomic and functional status of the heart and the blood vessels in every phase, we can produce very reliably a diagnosis of the actual status of the blood circulation system, reveal pathology and control the efficiency of therapy, if

The above mentioned evidence is really of fundamental importance. It should be taken into

The above mentioned main volumetric parameters should be complemented by another one: it is arterial pressure (AP). The cardiovascular system has its own mechanism to provide separate regulation of the systolic and diastolic pressures (AP)[8]. A narrowing in sectional areas of the blood vessels in total leads to a displacement of a certain volume of blood that is symbolized by ΔV. The displacement volume enters the ventricles in premature diastole phase T – P. During myocardium contraction phase R – S, the same volume is displaced via the closed aortic valve into the aorta. Actually, before the ejection of stroke volume SV into aorta, the total of displacement volume ΔV enters the aorta. Therefore, it is that the R – S phase, when ΔV can be ejected into the aorta, is preceded by that phase when the motion of the entire mass of blood is actuated, and this preceding phase is the Q – R interval, when the contraction of the septum occurs. It is just the phase when the blood flow becomes its directed vortex motion within the ventricle. Displacement volume ΔV contributes to moving against the total increased resistance of the blood vessels in the next

The blood circulation scheme is shown in Figure 5 herein. The anatomy of the heart is designed in such a way so that the displacement blood can penetrate without hindrance through the closed arteric valve into the aorta. It is determined not only by the configuration of the valves but also the mechanism of the contraction of the heart chambers that consists of three phases. Phase one among them is the contraction of the septum. Phase two provides for the contraction of the ventricle walls. Phase three is the phase of tension. The processes occurring therein are responsible for spinning the blood flows so that the penetration of the displacement blood through the closed valves into the aorta is assisted. Under normal conditions, when there is no displacement volume ΔV available, and, as a consequence, no penetration is required, upon completion of the phase of tension, stroke volume SV residing in the heart is supplied into the aorta. In this case, volume SV added to the volume of blood residing in the aorta creates the systolic pressure that produces a difference in pressures between the aorta and the periphery. Such mechanism required to overcome an increased blood flow resistance operates cyclically till the cause of blood vessel constriction disappears. The processes described above are typical for the mechanism of regulation of the diastolic arterial pressure. Various Rheogram curve shapes reflect this mechanism.

The anatomy design of the heart is determined by the phase mechanism of hemodynamics, i.e., the mechanism of the regulation of the diastolic pressure. This mechanism is responsible for elimination of general vasoconstriction difficulties in blood circulation. Causes of the

said vasoconstriction cannot be diagnostically identified in this case.

required.

account when making diagnosis.

phase which shows rapid blood ejection.

**2.2 Mechanism of regulation of systolic pressure** 


Fig. 4. Displayed measured phase-related values and their qualitative representation as bar charts, with reference to normal values. This figure gives three different measuring cases

The values of phase-related blood volumes are influenced by the mechanism of compensation existing in the cardiovascular system[6]. This mechanism is responsible for the maintenance of the hemodynamic parameters within their respective norms. If any parameter goes far beyond its norm, it means that it is an indication of physiological problems of the respective phase process. In this case, the function in the next phase compensates for the changes in the functioning of the problematic phase[6]. It is the just the case with sportsmen whose cardiovascular system shows the proper performance.

Physical exercise may cause a deficiency in diastolic volumes of blood by more than 500 %.[4] Under the circumstances, the systolic phases undertake to compensate for the above deficiency. For this purpose, the mechanisms may be involved, the manifestations of which cannot be found even in a pathology case. Upon stress relieving, 1 minute later, all phaserelated volumes are normalized again. This kind of the performance of the cardiovascular system hinders an identification of the cause of pathology at early stages for those who are not professional athletes.

As a rule, deviations due to pathology exceed the norm by more than 30 %. Patients, who receive their treatment at cardiology hospital, show sometimes deviations of 50 % and over. The only way to find the primary cause of any pathology, based on the manifestation of the compensation mechanism, can be a thorough analysis of the actual cause-relationship in every individual case.

The phase-related volumetric parameters in hemodynamics are the most informative characteristics of the performance of the cardiovascular system since they are capable of

а) b) c) Fig. 4. Displayed measured phase-related values and their qualitative representation as bar charts, with reference to normal values. This figure gives three different measuring cases

The values of phase-related blood volumes are influenced by the mechanism of compensation existing in the cardiovascular system[6]. This mechanism is responsible for the maintenance of the hemodynamic parameters within their respective norms. If any parameter goes far beyond its norm, it means that it is an indication of physiological problems of the respective phase process. In this case, the function in the next phase compensates for the changes in the functioning of the problematic phase[6]. It is the just the

Physical exercise may cause a deficiency in diastolic volumes of blood by more than 500 %.[4] Under the circumstances, the systolic phases undertake to compensate for the above deficiency. For this purpose, the mechanisms may be involved, the manifestations of which cannot be found even in a pathology case. Upon stress relieving, 1 minute later, all phaserelated volumes are normalized again. This kind of the performance of the cardiovascular system hinders an identification of the cause of pathology at early stages for those who are

As a rule, deviations due to pathology exceed the norm by more than 30 %. Patients, who receive their treatment at cardiology hospital, show sometimes deviations of 50 % and over. The only way to find the primary cause of any pathology, based on the manifestation of the compensation mechanism, can be a thorough analysis of the actual cause-relationship in

The phase-related volumetric parameters in hemodynamics are the most informative characteristics of the performance of the cardiovascular system since they are capable of

case with sportsmen whose cardiovascular system shows the proper performance.

not professional athletes.

every individual case.

reflecting the coordinated operation of the heart and the associated blood vessels. Knowing their ratios and considering the actual anatomic and functional status of the heart and the blood vessels in every phase, we can produce very reliably a diagnosis of the actual status of the blood circulation system, reveal pathology and control the efficiency of therapy, if required.

The above mentioned evidence is really of fundamental importance. It should be taken into account when making diagnosis.

## **2.2 Mechanism of regulation of systolic pressure**

The above mentioned main volumetric parameters should be complemented by another one: it is arterial pressure (AP). The cardiovascular system has its own mechanism to provide separate regulation of the systolic and diastolic pressures (AP)[8]. A narrowing in sectional areas of the blood vessels in total leads to a displacement of a certain volume of blood that is symbolized by ΔV. The displacement volume enters the ventricles in premature diastole phase T – P. During myocardium contraction phase R – S, the same volume is displaced via the closed aortic valve into the aorta. Actually, before the ejection of stroke volume SV into aorta, the total of displacement volume ΔV enters the aorta. Therefore, it is that the R – S phase, when ΔV can be ejected into the aorta, is preceded by that phase when the motion of the entire mass of blood is actuated, and this preceding phase is the Q – R interval, when the contraction of the septum occurs. It is just the phase when the blood flow becomes its directed vortex motion within the ventricle. Displacement volume ΔV contributes to moving against the total increased resistance of the blood vessels in the next phase which shows rapid blood ejection.

The blood circulation scheme is shown in Figure 5 herein. The anatomy of the heart is designed in such a way so that the displacement blood can penetrate without hindrance through the closed arteric valve into the aorta. It is determined not only by the configuration of the valves but also the mechanism of the contraction of the heart chambers that consists of three phases. Phase one among them is the contraction of the septum. Phase two provides for the contraction of the ventricle walls. Phase three is the phase of tension. The processes occurring therein are responsible for spinning the blood flows so that the penetration of the displacement blood through the closed valves into the aorta is assisted. Under normal conditions, when there is no displacement volume ΔV available, and, as a consequence, no penetration is required, upon completion of the phase of tension, stroke volume SV residing in the heart is supplied into the aorta. In this case, volume SV added to the volume of blood residing in the aorta creates the systolic pressure that produces a difference in pressures between the aorta and the periphery. Such mechanism required to overcome an increased blood flow resistance operates cyclically till the cause of blood vessel constriction disappears. The processes described above are typical for the mechanism of regulation of the diastolic arterial pressure. Various Rheogram curve shapes reflect this mechanism.

The anatomy design of the heart is determined by the phase mechanism of hemodynamics, i.e., the mechanism of the regulation of the diastolic pressure. This mechanism is responsible for elimination of general vasoconstriction difficulties in blood circulation. Causes of the said vasoconstriction cannot be diagnostically identified in this case.

Control of Cardiovascular System 13

normal conditions, the pressure increase is provided by the pumping function of the blood

An additional point to emphasize is that there is another biophysical phenomenon connected with hemodynamics. It is cavitation in blood that promotes blood volume expansion[2]. It may spread over very quickly within one heart cycle and is capable of

The cause of the systolic pressure buildup is a reduction in blood supply of some viscera. The pressure buildup is aimed at elimination of hindrances in blood supply in order to maintain the proper blood circulation. The blood supply mechanism of some viscera provides for protection from arterial overpressures. In the first place, the protection of the cerebral blood supply system should be mentioned. The cerebral blood vessels are anatomically connected with veins. During an increase in AP, the venous drainage is hindered, affecting the blood

If for some reason a viscus is not sufficiently supplied with blood, it leads to a systolic AP growth. The venous drainage will be hindered. The first symptoms of this problem could be edema of legs. To solve this problem, required should be elimination of the cause of the improper blood supply to the affected viscus that should decrease the AP and,

vessel constriction and limiting in such a way an excessive AP increase.

**3. Phase structure of heart cycle according to ECG curve** 

The complete phase structure of an ECG is shown in Figure 6 herein.

Phase of buildup of maximum systolic pressure in aorta Tн - Tк,;

Every heart cycle consists of 10 phases. Each phase undertakes its own functions[7].

Each phase serves its purpose. But the phases may be grouped in a manner as follows: **Group of diastol4ic phases which are responsible for blood supply to the ventricles:** 

The phase of premature diastole contains a period of time equal to the duration of wave U which reflects an intensive filling of the coronary vessels with blood. It occurs in

The diastolic phases are described as hemodynamic values PV1 and PV2.

vessels and their increasing resistance.

considerably expanding the blood volume.

subsequently, normalize the venous drainage.

Phase of closing of atrioventricular valve Pк – Q;

Phase of premature diastole of ventricles Uн - P<sup>н</sup> .

Phase of premature diastole of ventricles Uн - Pн;

Phase of closing of atrioventricular valve Pк – Q.

synchronism with filling of the ventricles.

Phase of atrial systole Pн – Pк;

Phase of rapid ejection L – j; Phase of slow ejection j - T<sup>н</sup> ;

Phase of atrial systole Pн – Pк;

Phase of contraction of septum Q – R; Phase of contraction of ventricle walls R – S; Phase of tension of myocardium S – L;

Phase of closing of aortic valve T<sup>к</sup> - Uн;

Fig. 5. а) Blood circulation scheme considering changes in blood vessel resistance. b) AP changes in aorta; c) Changes in AP identifiable on Rheo curve in phase of tension S-L, in proportion to displacement volume ∆V in blood vessel constriction

With synchronous recording of an ECG and a Rheo from the ascending aorta, provided that they are synchronized at wave point S on the ECG curve, the process of the regulation of diastolic pressure may manifest itself as an early AP rise on the respective Rheo curve in phases R – S and S – L.

#### **2.3 Mechanism of regulation of systolic pressure**

The mechanism of regulation of the systolic pressure differs significantly from that responsible for the regulation of the diastolic pressure. It has the function to provide a prerequisite to the blood circulation in the blood vessels due to a difference in pressures between the aorta and veins and manage the transportation of an oxygen quantity as required by tissues and cells. For these purposes, several biophysical processes are engaged.

First and foremost, we should mention the process of myocardium contraction in tension phase S – L. The tension created in this phase presets the velocity of the blood flow during the blood ejection phase. Therefore, the initial velocity of the blood flow in the aorta depends on the degree of the myocardium tension.

The second important process is the phenomenon of an increase in the systolic pressure during the propagation of the AP wave throughout the arteries[1]. The systolic pressure in the aorta and that in the brachial artery may considerably differ from each other. On the

Fig. 5. а) Blood circulation scheme considering changes in blood vessel resistance. b) AP changes in aorta; c) Changes in AP identifiable on Rheo curve in phase of tension S-L, in

With synchronous recording of an ECG and a Rheo from the ascending aorta, provided that they are synchronized at wave point S on the ECG curve, the process of the regulation of diastolic pressure may manifest itself as an early AP rise on the respective Rheo curve in

The mechanism of regulation of the systolic pressure differs significantly from that responsible for the regulation of the diastolic pressure. It has the function to provide a prerequisite to the blood circulation in the blood vessels due to a difference in pressures between the aorta and veins and manage the transportation of an oxygen quantity as required by tissues and cells. For these purposes, several biophysical processes are engaged. First and foremost, we should mention the process of myocardium contraction in tension phase S – L. The tension created in this phase presets the velocity of the blood flow during the blood ejection phase. Therefore, the initial velocity of the blood flow in the aorta

The second important process is the phenomenon of an increase in the systolic pressure during the propagation of the AP wave throughout the arteries[1]. The systolic pressure in the aorta and that in the brachial artery may considerably differ from each other. On the

proportion to displacement volume ∆V in blood vessel constriction

**2.3 Mechanism of regulation of systolic pressure** 

depends on the degree of the myocardium tension.

phases R – S and S – L.

normal conditions, the pressure increase is provided by the pumping function of the blood vessels and their increasing resistance.

An additional point to emphasize is that there is another biophysical phenomenon connected with hemodynamics. It is cavitation in blood that promotes blood volume expansion[2]. It may spread over very quickly within one heart cycle and is capable of considerably expanding the blood volume.

The cause of the systolic pressure buildup is a reduction in blood supply of some viscera. The pressure buildup is aimed at elimination of hindrances in blood supply in order to maintain the proper blood circulation. The blood supply mechanism of some viscera provides for protection from arterial overpressures. In the first place, the protection of the cerebral blood supply system should be mentioned. The cerebral blood vessels are anatomically connected with veins. During an increase in AP, the venous drainage is hindered, affecting the blood vessel constriction and limiting in such a way an excessive AP increase.

If for some reason a viscus is not sufficiently supplied with blood, it leads to a systolic AP growth. The venous drainage will be hindered. The first symptoms of this problem could be edema of legs. To solve this problem, required should be elimination of the cause of the improper blood supply to the affected viscus that should decrease the AP and, subsequently, normalize the venous drainage.

## **3. Phase structure of heart cycle according to ECG curve**

Every heart cycle consists of 10 phases. Each phase undertakes its own functions[7].

The complete phase structure of an ECG is shown in Figure 6 herein.

Phase of atrial systole Pн – Pк; Phase of closing of atrioventricular valve Pк – Q; Phase of contraction of septum Q – R; Phase of contraction of ventricle walls R – S; Phase of tension of myocardium S – L; Phase of rapid ejection L – j; Phase of slow ejection j - T<sup>н</sup> ; Phase of buildup of maximum systolic pressure in aorta Tн - Tк,; Phase of closing of aortic valve T<sup>к</sup> - Uн; Phase of premature diastole of ventricles Uн - P<sup>н</sup> .

Each phase serves its purpose. But the phases may be grouped in a manner as follows:

#### **Group of diastol4ic phases which are responsible for blood supply to the ventricles:**

Phase of premature diastole of ventricles Uн - Pн; Phase of atrial systole Pн – Pк; Phase of closing of atrioventricular valve Pк – Q.

The phase of premature diastole contains a period of time equal to the duration of wave U which reflects an intensive filling of the coronary vessels with blood. It occurs in synchronism with filling of the ventricles.

The diastolic phases are described as hemodynamic values PV1 and PV2.

Control of Cardiovascular System 15

Hemodynamic parameter PV5 shows what share of blood is pumped by the aorta operating

It should be noted that phase of slow ejection j - T<sup>н</sup> is a time when the stroke volume of blood is distributed throughout the large blood vessel, i.e., the time of the aorta expansion. As our investigations demonstrate, in case of improper elasticity of the aorta this period of

An electrocardiogram reflects the most important hemodynamic processes. According to an ECG curve, it is possible to identify an intensity of the contraction of the muscles of the respective segment in the cardiovascular system by analyzing inflection points in the respective heart cycle phase and considering the respective phase amplitudes. However, it is required to understand how the flow of blood changes. For this purpose, rheography should be used. A rheogram shows changes in the arterial pressure. An ECG and a RHEO are produced by using signals of different nature. To record an ECG used is electric potential, and for RHEOgraphy employed are changes in amplitudes of high-frequency AC under the influence of changing blood volumes in blood circulation, which produce changes in the

There is no AP increase in myocardium tension phase S – L. The aortic valve opens at the moment denoted as L. The slope ratio of RHEO in phase of rapid ejection L – j is descriptive of the velocity of stroke volume travel, and, finally, decisive in governing the systolic AP.

When considering an ECG as a complex signal, it should be pointed out that it consists of a number of single-period in-series sinusoidal signals connected. It is referred to a redistribution of energy in bio systems in a not a stepwise, but sinusoidal way, showing halfperiods as follows: energy increase, retardation, attenuation and development. Transition points of these processes should be at the same time the points of inflection of energy functions which are shown by the first derivative at their extrema. Similar processes occur in the cardiovascular system control. Figure 8 represents a schematic model of an ECG

Should an ECG curve be differentiated, 10 extrema on the derivative can be identified which correspond to the boundaries of the respective phases of the heart cycle. It should be mentioned that each phase shall be determined by the same criterion, i.e., by the respective local extremum on the derivative curve. Since a wavefront steepness varies, the respective amplitudes of the derivative extrema differ. The ECG phases are equivalent to those of energy variations responsible for the heart control. For illustration purposes, it is better to

**5. Criteria for recording phases on ECG, Rheo and their derivatives**

The given systolic phases are characterized by hemodynamic values PV3, PV4 and SV.

Phase of closing of aortic valve T<sup>к</sup> - Uн;

time is prolonged.

Hemodynamic value MV is an indication of a blood flow rate.

**4. Phase structure of heart cycle on RHEO curve** 

conductivity within the space between the recording electrodes.

comprising the said in-series single-period sinusoidal waves.

use graphic differentiation.

as a peristaltic pump during the ejection of blood from the ventricles.

Fig. 6. Phase structure of ECG recorded from ascending aorta; Phase of atrial systole Pн – Pк; Phase of closing of atrioventricular valve Pк – Q; Phase of contraction of septum Q – R; Phase of contraction of ventricle walls R – S; Phase of tension of myocardium S – L; Phase of rapid ejection L – j; Phase of slow ejection j - T<sup>н</sup> ; Phase of buildup of maximum systolic pressure in aorta Tн - Tк,; Phase of closing of aortic valve T<sup>к</sup> - Uн; Phase of premature diastole of ventricles Uн - P<sup>н</sup>

**Group of systolic phases which provide** for the conditions for the proper blood circulation. They can be divided into subgroups undertaking certain functions as given below:

#### **Subgroup responsible for diastolic AP regulation:**

Phase of contraction of septum Q – R; Phase of contraction of ventricle walls R – S; Phase of tension of myocardium S – L (partially).

#### **Subgroup responsible for systolic AP regulation:**

Phase of tension of myocardium S – L, Phase of rapid ejection L – j.

#### **Subgroup responsible for aorta pumping function control:**

Phase of slow ejection j - T<sup>н</sup> ; Phase of buildup of maximum systolic pressure in aorta Tн - Tк,;

Fig. 6. Phase structure of ECG recorded from ascending aorta; Phase of atrial systole Pн – Pк; Phase of closing of atrioventricular valve Pк – Q; Phase of contraction of septum Q – R; Phase of contraction of ventricle walls R – S; Phase of tension of myocardium S – L; Phase of rapid ejection L – j; Phase of slow ejection j - T<sup>н</sup> ; Phase of buildup of maximum systolic pressure in aorta Tн - Tк,; Phase of closing of aortic valve T<sup>к</sup> - Uн; Phase of premature diastole

**Group of systolic phases which provide** for the conditions for the proper blood circulation.

They can be divided into subgroups undertaking certain functions as given below:

**Subgroup responsible for diastolic AP regulation:** 

**Subgroup responsible for aorta pumping function control:** 

Phase of buildup of maximum systolic pressure in aorta Tн - Tк,;

Phase of contraction of septum Q – R; Phase of contraction of ventricle walls R – S; Phase of tension of myocardium S – L (partially). **Subgroup responsible for systolic AP regulation:** 

Phase of tension of myocardium S – L,

Phase of rapid ejection L – j.

Phase of slow ejection j - T<sup>н</sup> ;

of ventricles Uн - P<sup>н</sup>

Phase of closing of aortic valve T<sup>к</sup> - Uн;

The given systolic phases are characterized by hemodynamic values PV3, PV4 and SV.

Hemodynamic value MV is an indication of a blood flow rate.

Hemodynamic parameter PV5 shows what share of blood is pumped by the aorta operating as a peristaltic pump during the ejection of blood from the ventricles.

It should be noted that phase of slow ejection j - T<sup>н</sup> is a time when the stroke volume of blood is distributed throughout the large blood vessel, i.e., the time of the aorta expansion. As our investigations demonstrate, in case of improper elasticity of the aorta this period of time is prolonged.

## **4. Phase structure of heart cycle on RHEO curve**

An electrocardiogram reflects the most important hemodynamic processes. According to an ECG curve, it is possible to identify an intensity of the contraction of the muscles of the respective segment in the cardiovascular system by analyzing inflection points in the respective heart cycle phase and considering the respective phase amplitudes. However, it is required to understand how the flow of blood changes. For this purpose, rheography should be used. A rheogram shows changes in the arterial pressure. An ECG and a RHEO are produced by using signals of different nature. To record an ECG used is electric potential, and for RHEOgraphy employed are changes in amplitudes of high-frequency AC under the influence of changing blood volumes in blood circulation, which produce changes in the conductivity within the space between the recording electrodes.

There is no AP increase in myocardium tension phase S – L. The aortic valve opens at the moment denoted as L. The slope ratio of RHEO in phase of rapid ejection L – j is descriptive of the velocity of stroke volume travel, and, finally, decisive in governing the systolic AP.

## **5. Criteria for recording phases on ECG, Rheo and their derivatives**

When considering an ECG as a complex signal, it should be pointed out that it consists of a number of single-period in-series sinusoidal signals connected. It is referred to a redistribution of energy in bio systems in a not a stepwise, but sinusoidal way, showing halfperiods as follows: energy increase, retardation, attenuation and development. Transition points of these processes should be at the same time the points of inflection of energy functions which are shown by the first derivative at their extrema. Similar processes occur in the cardiovascular system control. Figure 8 represents a schematic model of an ECG comprising the said in-series single-period sinusoidal waves.

Should an ECG curve be differentiated, 10 extrema on the derivative can be identified which correspond to the boundaries of the respective phases of the heart cycle. It should be mentioned that each phase shall be determined by the same criterion, i.e., by the respective local extremum on the derivative curve. Since a wavefront steepness varies, the respective amplitudes of the derivative extrema differ. The ECG phases are equivalent to those of energy variations responsible for the heart control. For illustration purposes, it is better to use graphic differentiation.

Control of Cardiovascular System 17

Fig. 8. Schematic model of ECG comprising in-series single-period sinusoidal variations

Fig. 9. Graphic differentiation of ECG curve. Shown are an ECG and its first derivative. Wave points on the ECG curve are its inflection points that correspond to the local extrema

on the derivative

It is just the graphic differentiation that is capable of clearly illustrating all specific points of such complex signal like an ECG signal. Whereas it is practically impossible to detect visually on an ECG curve the inflection points, they can be easy identified on the derivative by local extrema without error. Figure 9 gives an ECG curve and its first derivative. It is evident that point P on the ECG curve corresponds to point Р on the derivative that is found by the respective local extremum. In the same way point T should be identified. It is of great importance to localize point S. There are no other methods capable of identifying this point.

Fig. 7. Phase structure of RHEO recorded from ascending aorta

Fig. 7. Phase structure of RHEO recorded from ascending aorta

Fig. 8. Schematic model of ECG comprising in-series single-period sinusoidal variations

It is just the graphic differentiation that is capable of clearly illustrating all specific points of such complex signal like an ECG signal. Whereas it is practically impossible to detect visually on an ECG curve the inflection points, they can be easy identified on the derivative by local extrema without error. Figure 9 gives an ECG curve and its first derivative. It is evident that point P on the ECG curve corresponds to point Р on the derivative that is found by the respective local extremum. In the same way point T should be identified. It is of great importance to localize point S. There are no other methods capable of identifying this point.

Fig. 9. Graphic differentiation of ECG curve. Shown are an ECG and its first derivative. Wave points on the ECG curve are its inflection points that correspond to the local extrema on the derivative

Control of Cardiovascular System 19

Fig. 11. Identification of phases on an ECG curve with use of the first derivative graph

**cycle phase analysis** 

mentioned:

to clearly identifying such imbalances.

5 condition of venous flow

6 condition of pulmonary function <sup>7</sup>whether pre-stroke conditions are available or not

<sup>8</sup>problems with coronary blood

Table 1. Main functions and regulated parameters of cardiovascular system

flow

**6. Functions of cardiovascular system to be evaluated on the basis of heart** 

The complex of the functions of the cardiovascular system is a combination of the functions in every individual heart cycle phase. There is a certain logic design available explaining this. Every phase has its own significance but the basis of all phases is the mechanism of contraction or relaxation of muscles. Should metabolic disturbance in a muscle occur, its contraction or relaxation will be diminished. In this case, every next phase will undertake to compensate for this malfunction by enhancing its activity. The phase analysis gives us a clue

In this connection, the following functions of the cardiovascular system should be

№ Function Regulated parameter 1 Contraction of septum diastolic AP in the aorta 2 Contraction of myocardium; diastolic AP in the aorta 3 Tension of myocardium muscles systolic AP in the aorta 4 Elasticity of aorta Maintain blood flow structure

It is just the derivative that is capable of recognizing point S very clearly by the respective local positive extremum. The proposed procedure of identifying the above mentioned key points makes possible to develop a computer-assisted technology for measuring durations of every heart cycle phase.

For the same purpose, the second derivative may be used, too, but in this case there is no need to do it since the informative content of the heart cycle phase identifiable criteria with utilization of the first derivative is quite sufficient.

Some real ECG curves recorded from the aorta are given in Figure 10 herein. Wave points P, Q, S and T are marked on the curves which are reliably found according to the first derivative.

Figure 11 herein illustrates real ECG signals and the first derivative of this ECG. The ECG shape shown in this Figure is close to an ideal one. It is the matter of fact that in practice we deal with such ECG curves that significantly differ from the ideal ECG type represented herein. Therefore, it is the differentiation only that can very reliably identify the boundaries of every phase in every heart cycle.

Fig. 10. Key points P, Q, S and T on ECG curve, characterizing the respective phases of the heart cycle and corresponding to the respective local extrema on the derivative

It is just the derivative that is capable of recognizing point S very clearly by the respective local positive extremum. The proposed procedure of identifying the above mentioned key points makes possible to develop a computer-assisted technology for measuring durations

For the same purpose, the second derivative may be used, too, but in this case there is no need to do it since the informative content of the heart cycle phase identifiable criteria with

Some real ECG curves recorded from the aorta are given in Figure 10 herein. Wave points P, Q, S and T are marked on the curves which are reliably found according to the first

Figure 11 herein illustrates real ECG signals and the first derivative of this ECG. The ECG shape shown in this Figure is close to an ideal one. It is the matter of fact that in practice we deal with such ECG curves that significantly differ from the ideal ECG type represented herein. Therefore, it is the differentiation only that can very reliably identify the boundaries

heart cycle and corresponding to the respective local extrema on the derivative

 point S point T Fig. 10. Key points P, Q, S and T on ECG curve, characterizing the respective phases of the

point P point Q

of every heart cycle phase.

of every phase in every heart cycle.

derivative.

utilization of the first derivative is quite sufficient.

Fig. 11. Identification of phases on an ECG curve with use of the first derivative graph

## **6. Functions of cardiovascular system to be evaluated on the basis of heart cycle phase analysis**

The complex of the functions of the cardiovascular system is a combination of the functions in every individual heart cycle phase. There is a certain logic design available explaining this. Every phase has its own significance but the basis of all phases is the mechanism of contraction or relaxation of muscles. Should metabolic disturbance in a muscle occur, its contraction or relaxation will be diminished. In this case, every next phase will undertake to compensate for this malfunction by enhancing its activity. The phase analysis gives us a clue to clearly identifying such imbalances.

In this connection, the following functions of the cardiovascular system should be mentioned:


Table 1. Main functions and regulated parameters of cardiovascular system

Control of Cardiovascular System 21

Fig. 13. Anatomical design of the heart predetermined by the required functions in every

developing and validating of new high-efficient therapy methods.

mathematical modeling and instrumentation technology.

CARDIOCODE-Finland. She remains in our memory for ever.

Our clinical studies offer a clearer view of how many difficult issues associated with biochemical reactions responsible for the stable maintenance of the hemodynamic and the entire performance of the cardiovascular system can be answered. This is a pre-requisite to

Hereby the authors would like to express their hope that within the nearest future we shall deal with a new research field, which is cardiometry. The basis of this science should create

The well-known recipe for success in any work is to create a team of like-minded researches working for the same cause. If the concept of their work is that the point of life is work, and if the work results encourage and motivate them, then success is assured. But our life is able to make its corrections. We regret to say that, one of the authors of our discovery, who originated the idea of the "third" mode of flow, died. We speak about Gustav M. Poyedintsev, a great mathematician and scientist. Our last book *Theoretical Principles of Heart Cycle Phase Analysis* published in 2007 was devoted to the memory of him and his work.

The other sad news has been received by us when we were working on this Chapter: Jaana Koponen-Kolmakova, another member of our R & D team, has departed this life. She was really an outstanding person! She was the General Manager of the Company

heart cycle

**8. Acknowledgements** 

Figure 12 given below demonstrates the relations between the heart cycle phases on an ECG & RHEO and the respective functions of the cardiovascular system. Although it seems that the hemodynamic mechanism as a whole and the performance of the cardiovascular system are very complicated, the heart cycle phase analysis allows establishing of cause-effect relationship of any pathology in every individual case within the shortest time. It is very important that it makes possible to detect the primary cause of a cardiac disease.

Figure 13 displays anatomic segments of the heart and their respective functions in every heart cycle phase.

Fig. 12. Diagnosable heart segments with their functions and their relations to heart cycle phases on ECG and RHEO

## **7. Conclusion**

Making progress in research of biophysical processes of the formation of the hemodynamic mechanism is possible only when theoretical models are tested for their compliance in practice, i.e., a model to be validated should show in practice its compliance with the requirements for all simulated functions. The results of many years' researches accumulated by our R & D team made it possible not only to develop an innovative, radically new theory of the heart cycle phase analysis but also provide metrology for such field of medical science as cardiology[4]. We have succeeded in solving the problem of indirect measuring technologies for hemodynamic parameters, including phase-related volumes, by the mathematical modeling.

Figure 12 given below demonstrates the relations between the heart cycle phases on an ECG & RHEO and the respective functions of the cardiovascular system. Although it seems that the hemodynamic mechanism as a whole and the performance of the cardiovascular system are very complicated, the heart cycle phase analysis allows establishing of cause-effect relationship of any pathology in every individual case within the shortest time. It is very

Figure 13 displays anatomic segments of the heart and their respective functions in every

Fig. 12. Diagnosable heart segments with their functions and their relations to heart cycle

Making progress in research of biophysical processes of the formation of the hemodynamic mechanism is possible only when theoretical models are tested for their compliance in practice, i.e., a model to be validated should show in practice its compliance with the requirements for all simulated functions. The results of many years' researches accumulated by our R & D team made it possible not only to develop an innovative, radically new theory of the heart cycle phase analysis but also provide metrology for such field of medical science as cardiology[4]. We have succeeded in solving the problem of indirect measuring technologies for hemodynamic parameters, including phase-related volumes, by the

important that it makes possible to detect the primary cause of a cardiac disease.

heart cycle phase.

phases on ECG and RHEO

mathematical modeling.

**7. Conclusion** 

Fig. 13. Anatomical design of the heart predetermined by the required functions in every heart cycle

Our clinical studies offer a clearer view of how many difficult issues associated with biochemical reactions responsible for the stable maintenance of the hemodynamic and the entire performance of the cardiovascular system can be answered. This is a pre-requisite to developing and validating of new high-efficient therapy methods.

Hereby the authors would like to express their hope that within the nearest future we shall deal with a new research field, which is cardiometry. The basis of this science should create mathematical modeling and instrumentation technology.

## **8. Acknowledgements**

The well-known recipe for success in any work is to create a team of like-minded researches working for the same cause. If the concept of their work is that the point of life is work, and if the work results encourage and motivate them, then success is assured. But our life is able to make its corrections. We regret to say that, one of the authors of our discovery, who originated the idea of the "third" mode of flow, died. We speak about Gustav M. Poyedintsev, a great mathematician and scientist. Our last book *Theoretical Principles of Heart Cycle Phase Analysis* published in 2007 was devoted to the memory of him and his work.

The other sad news has been received by us when we were working on this Chapter: Jaana Koponen-Kolmakova, another member of our R & D team, has departed this life. She was really an outstanding person! She was the General Manager of the Company CARDIOCODE-Finland. She remains in our memory for ever.

**2** 

*Japan* 

Tadashi Yoshida

*Apheresis and Dialysis Center School of Medicine, Keio University* 

**Molecular Control of Smooth Muscle Cell** 

**Response Factor and Its Interacting Proteins** 

Vascular smooth muscle cells (SMCs) exhibit a wide range of different phenotypes at different stages of development (Owens, 1995; Owens et al., 2004; Yoshida & Owens, 2005). Even in mature animals, SMCs retain the capability to change their phenotype in response to multiple local environmental cues. The plasticity of SMCs enables them to play a critical role in physiological processes in the vasculature, as well as the pathogenesis of numerous vascular diseases including atherosclerosis, re-stenosis after percutaneous coronary intervention, aortic aneurysm, and hypertension. Thus, it is important to understand the precise mechanisms whereby SMCs exhibit different phenotypes under distinct conditions. Because one of the most remarkable differences among SMC subtypes is the difference in expression levels of SMC-specific/-selective genes, elucidation of the molecular mechanisms

controlling SMC differentiation marker gene expression may shed light on this issue.

Most of SMC differentiation marker genes characterized to date, including *smooth muscle* 

kb to recapitulate the expression patterns of the endogenous gene, and mutation of any one of three conserved CArG elements within the regions abolishes the expression (Mack & Owens, 1999). Likewise, SMC-specific expression of the *SM-MHC* gene requires 4.2 kb of the 5'-flanking region, the entire first exon, and 11.5 kb of the first intronic sequence, and mutation of CArG elements in the 5'-flanking region abolishes the expression (Manabe & Owens, 2001a). These results indicate the critical roles of CArG elements in the regulation of SMC differentiation marker gene expression. Currently, it is reported that over 60 of SMCspecific/-selective genes possess CArG elements in the promoter-enhancer regions by insilico analysis (Miano, 2003), although it is not fully determined how many CArG elements

*-actin* (Mack & Owens, 1999), *SM-myosin heavy chain* (*SM-MHC*) (Madsen et al., 1998),

 (Li et al., 1996), and *h1-calponin* (Miano et al., 2000), have multiple highly conserved CC(A/T-rich)6GG (CArG) elements in their promoter-enhancer regions. Results of studies *in vivo* have shown that expression of these genes is dependent on the presence of CArG elements (Li et al., 1997; Mack & Owens, 1999; Manabe & Owens, 2001a). For example,

*-actin* gene requires a promoter-enhancer region from -2.6 kb to +2.8

**1. Introduction** 

*(SM)* α

*SM22*α

expression of the *SM* 

of them are functional.

α

**Differentiation Marker Genes by Serum** 

During our work we meet a lot of people who dedicate their life to science. We always enjoy communicating with them. This is a sort of people who deserve our special recognition and respect.

#### **9. References**


## **Molecular Control of Smooth Muscle Cell Differentiation Marker Genes by Serum Response Factor and Its Interacting Proteins**

Tadashi Yoshida *Apheresis and Dialysis Center School of Medicine, Keio University Japan* 

## **1. Introduction**

22 The Cardiovascular System – Physiology, Diagnostics and Clinical Implications

During our work we meet a lot of people who dedicate their life to science. We always enjoy communicating with them. This is a sort of people who deserve our special recognition and

[1] Caro, C.; Padley, T.; Shroter, R. & Sid, W. (1981) *Blood Circulation Mechanics.* Mir. M.

[2] Goncharenko, A. & Goncharenko, S. (2005) Extrasensory Capabilities of Heart. Magazin

[3] М. Rudenko, M.; Voronova, O. & Zernov. V. (2009). *Theoretical Principles of Heart Cycle* 

[4] Voronova, O. (1995). *Development of Models & Algorithms of Automated Transport Function* 

[5] Voronova, O. & Poyedintsev, G. Patent № 94031904 (RF). *Method of Determination of the Functional Status of the Left Sections of the Heart & their Associated Large Blood Vessels*. [6] Rudenko, M.; Voronova, O. & Zernov. V. Innovation in cardiology. A new diagnostic

"Technika Molodyozhi", No. 5, ISSN 0320 – 331 Х Rosen, P. (1969) *The Principle of* 

*Phase Analysis*. Fouqué Literaturverlag. ISBN 978-3-937909-57-8, Frankfurt a/M.

*of The Cardiovascular System*. Doctorate Thesis. Prepared by Mrs. O.K. Voronova,

standard establishing criteria of quantitative & qualitative evaluation of main parameters of the cardiac & cardiovascular system according to ECG and Rheo based on cardiac cycle phase analysis (for concurrent single-channel recording of cardiac signals from ascending aorta). (npre.2009.3667.1). *Nature Precedings.* Available from: http://precedings.nature.com/documents/3667/version/1/html [7] Rudenko, M.; Voronova, O. & Zernov. V. (2009) Study of Hemodynamic Parameters

Using Phase Analysis of the Cardiac Cycle. *Biomedical Engineering. Springer New York.* ISSN 0006-3398 (Print) 1573-8256 (Online). Volume 43, Number 4 / July, 2009.

Phase mechanism of regulation of diastolic pressure. Arrhythmology Bulletin

[8] Rudenko, M.; Voronova, O. & Zernov. V. (2010) Innovation in theoretical cardiology.

respect.

**9. References** 

(fig.12.14).

Р. 151 -155.

(Appendix B) – М. - P. 133.

[9] Rosen, P. (1969) *The Principle of Optimization in Biology.* Mir. M.

*Optimization in Biology.* Mir.

München London - New York.

Ph.D., VGTU, Voronezh.

Vascular smooth muscle cells (SMCs) exhibit a wide range of different phenotypes at different stages of development (Owens, 1995; Owens et al., 2004; Yoshida & Owens, 2005). Even in mature animals, SMCs retain the capability to change their phenotype in response to multiple local environmental cues. The plasticity of SMCs enables them to play a critical role in physiological processes in the vasculature, as well as the pathogenesis of numerous vascular diseases including atherosclerosis, re-stenosis after percutaneous coronary intervention, aortic aneurysm, and hypertension. Thus, it is important to understand the precise mechanisms whereby SMCs exhibit different phenotypes under distinct conditions. Because one of the most remarkable differences among SMC subtypes is the difference in expression levels of SMC-specific/-selective genes, elucidation of the molecular mechanisms controlling SMC differentiation marker gene expression may shed light on this issue.

Most of SMC differentiation marker genes characterized to date, including *smooth muscle (SM)* α*-actin* (Mack & Owens, 1999), *SM-myosin heavy chain* (*SM-MHC*) (Madsen et al., 1998), *SM22*α (Li et al., 1996), and *h1-calponin* (Miano et al., 2000), have multiple highly conserved CC(A/T-rich)6GG (CArG) elements in their promoter-enhancer regions. Results of studies *in vivo* have shown that expression of these genes is dependent on the presence of CArG elements (Li et al., 1997; Mack & Owens, 1999; Manabe & Owens, 2001a). For example, expression of the *SM* α*-actin* gene requires a promoter-enhancer region from -2.6 kb to +2.8 kb to recapitulate the expression patterns of the endogenous gene, and mutation of any one of three conserved CArG elements within the regions abolishes the expression (Mack & Owens, 1999). Likewise, SMC-specific expression of the *SM-MHC* gene requires 4.2 kb of the 5'-flanking region, the entire first exon, and 11.5 kb of the first intronic sequence, and mutation of CArG elements in the 5'-flanking region abolishes the expression (Manabe & Owens, 2001a). These results indicate the critical roles of CArG elements in the regulation of SMC differentiation marker gene expression. Currently, it is reported that over 60 of SMCspecific/-selective genes possess CArG elements in the promoter-enhancer regions by insilico analysis (Miano, 2003), although it is not fully determined how many CArG elements of them are functional.

Molecular Control of Smooth Muscle Cell Differentiation

differentiation marker genes.

promoter.

Marker Genes by Serum Response Factor and Its Interacting Proteins 25

the responsiveness to myocardin, suggesting that myocardin activates the transcription in a CArG-dependent manner. However, myocardin showed no DNA binding activity, but showed interaction with SRF. In addition, myocardin failed to activate the transcription of CArG-dependent genes in the absence of SRF (Du et al., 2003), demonstrating that myocardin is a co-activator of SRF. Over-expression of myocardin also induced the endogenous expression of SMC differentiation marker genes in cultured SMCs and non-SMCs, including 3T3 fibroblasts, L6 myoblasts, 3T3-L1 preadipocytes, COS cells, and undifferentiated embryonic stem cells (Chen et al., 2002; Du et al., 2003; Du et al., 2004; Wang et al., 2001; Wang et al., 2003; Yoshida et al., 2003; Yoshida et al., 2004b). However, forced expression of myocardin in non-SMCs was not sufficient to induce the full SMC differentiation program, because some SMC-enriched genes, which do not contain CArG elements in their promoter-enhancer region, were not induced (Yoshida et al., 2004b). Nevertheless, it was sufficient to establish a SMC-like contractile phenotype (Long et al., 2008). Either dominant-negative forms of myocardin or siRNA-induced suppression of myocardin decreased the transcription of SMC differentiation marker genes in cultured SMCs (Du et al., 2003; Wang et al., 2003; Yoshida et al., 2003). In addition, *myocardin*deficient mice exhibited no vascular SMC differentiation and died by embryonic day 10.5 (Li et al., 2003), although this may have been secondary to the defect in the extra-embryonic circulation. Moreover, mice lacking the *myocardin* gene in neural crest-derived cells died prior to postnatal day 3 from patent ductus arteriosus, and neural crest-derived SMCs in these mice exhibited a cell-autonomous block in expression of SMC differentiation marker genes (Huang et al., 2008). Taken together, the preceding results provide compelling evidence that myocardin plays a key role in the regulation of expression of SMC

Fig. 1. Myocardin potently induces the transcription of CArG-element containing SMC differentiation marker genes. Myocardin preferentially activates SMC differentiation marker

genes which contain multiple CArG elements in their promoter-enhancer regions. Homodimerization of myocardin through the leucine zipper-like domain efficiently activates the transcription. In contrast, myocardin does not induce the transcription of the growth factor-inducible gene, *c-fos*, because it only contains a single CArG element in the

The binding factor for CArG elements is the ubiquitously expressed transcription factor, serum response factor (SRF) (Norman et al., 1988). Knockout of the *SRF* gene in mice resulted in early embryonic lethality due to abnormal gastrulation and loss in key mesodermal markers (Arsenian et al., 1998), precluding the evaluation of requirement of SRF for SMC differentiation. Instead, conditional knockout of the *SRF* gene in the heart and SMCs exhibited the attenuation in cardiac trabeculation and the compact layer expansion, as well as decreases in SMC-specific/-selective genes including *SM* α*-actin* in aortic SMCs (Miano et al., 2004). Moreover, SRF has been shown to be required for differentiation of SMCs in an *in vitro* model of coronary SMC differentiation (Landerholm et al., 1999). Indeed, over-expression of dominant-negative forms of SRF inhibited the induction of SMC differentiation marker genes including *SM22*α, *h1-calponin*, and *SM* α*-actin* in proepicardial cells excised from quail embryos. As such, the preceding studies provide evidence indicating that the CArG-SRF complex plays an important role in the regulation of SMC differentiation marker gene expression. However, SRF was first cloned as a binding factor for the core sequences of serum response element (SRE) in the *c-fos* gene (Norman et al., 1988). Because the *c-fos* gene is known as one of the growth factor-inducible genes, major unresolved issues in the field are to identify the mechanisms whereby: (1) the CArG-SRF complex can simultaneously contribute to two disparate processes: induction of SMC differentiation marker gene expression versus activation of growth-regulated genes; and (2) the ubiquitously expressed SRF can contribute to SMC-specific/-selective expression of target genes.

To date, a number of factors have been reported to interact with SRF. Several recent studies suggest that these interactions are responsible for multiple actions of SRF. Therefore, this review article will summarize recent progress in our understanding of the transcriptional mechanisms involved in controlling expression of SMC differentiation marker genes by focusing on SRF and its interacting factors.

## **2. Myocardin is a potent co-factor of SRF for SMC differentiation marker gene expression**

One of the major breakthroughs in the SMC field was the discovery of myocardin (Wang et al., 2001). Myocardin was cloned as a co-factor of SRF by a bioinformatics-based screen and found to be exclusively expressed in SMCs and cardiomyocytes (Chen et al., 2002; Du et al., 2003; Wang et al., 2001; Yoshida et al., 2003). It has two isoforms, and smooth muscleenriched isoform consists of 856 amino acids (Creemers et al., 2006). Myocardin has several domains including three RPEL domains, a basic domain, a glutamine-rich domain, a SAP (Scaffold attachment factors A and B, Acinus, Protein inhibitor of activated STAT) domain, and a leucine zipper-like domain. It has been shown that leucine zipper-like domain is required for homodimerization of myocardin (Figure 1) (Wang et al., 2003), but the function of the other domains is not well understood. Transcriptional activation domain, TAD, is localized at the carboxy-terminal region, and deletion mutants that lack TAD behaved as dominant-negative forms (Wang et al., 2001; Yoshida et al., 2003). Over-expression of myocardin potently induces transcription of virtually all CArG-dependent SMC differentiation marker genes, including *SM* α*-actin*, *SM-MHC*, *SM22*α, *h1-calponin*, and *myosin light chain kinase* (*MLCK*) (Chen et al., 2002; Du et al., 2003; Wang et al., 2001; Wang et al., 2003; Yoshida et al., 2003). Mutation of CArG elements in the SMC promoters abolished

The binding factor for CArG elements is the ubiquitously expressed transcription factor, serum response factor (SRF) (Norman et al., 1988). Knockout of the *SRF* gene in mice resulted in early embryonic lethality due to abnormal gastrulation and loss in key mesodermal markers (Arsenian et al., 1998), precluding the evaluation of requirement of SRF for SMC differentiation. Instead, conditional knockout of the *SRF* gene in the heart and SMCs exhibited the attenuation in cardiac trabeculation and the compact layer expansion, as

(Miano et al., 2004). Moreover, SRF has been shown to be required for differentiation of SMCs in an *in vitro* model of coronary SMC differentiation (Landerholm et al., 1999). Indeed, over-expression of dominant-negative forms of SRF inhibited the induction of SMC

α

cells excised from quail embryos. As such, the preceding studies provide evidence indicating that the CArG-SRF complex plays an important role in the regulation of SMC differentiation marker gene expression. However, SRF was first cloned as a binding factor for the core sequences of serum response element (SRE) in the *c-fos* gene (Norman et al., 1988). Because the *c-fos* gene is known as one of the growth factor-inducible genes, major unresolved issues in the field are to identify the mechanisms whereby: (1) the CArG-SRF complex can simultaneously contribute to two disparate processes: induction of SMC differentiation marker gene expression versus activation of growth-regulated genes; and (2) the ubiquitously expressed SRF can contribute to SMC-specific/-selective expression of

To date, a number of factors have been reported to interact with SRF. Several recent studies suggest that these interactions are responsible for multiple actions of SRF. Therefore, this review article will summarize recent progress in our understanding of the transcriptional mechanisms involved in controlling expression of SMC differentiation marker genes by

**2. Myocardin is a potent co-factor of SRF for SMC differentiation marker gene** 

One of the major breakthroughs in the SMC field was the discovery of myocardin (Wang et al., 2001). Myocardin was cloned as a co-factor of SRF by a bioinformatics-based screen and found to be exclusively expressed in SMCs and cardiomyocytes (Chen et al., 2002; Du et al., 2003; Wang et al., 2001; Yoshida et al., 2003). It has two isoforms, and smooth muscleenriched isoform consists of 856 amino acids (Creemers et al., 2006). Myocardin has several domains including three RPEL domains, a basic domain, a glutamine-rich domain, a SAP (Scaffold attachment factors A and B, Acinus, Protein inhibitor of activated STAT) domain, and a leucine zipper-like domain. It has been shown that leucine zipper-like domain is required for homodimerization of myocardin (Figure 1) (Wang et al., 2003), but the function of the other domains is not well understood. Transcriptional activation domain, TAD, is localized at the carboxy-terminal region, and deletion mutants that lack TAD behaved as dominant-negative forms (Wang et al., 2001; Yoshida et al., 2003). Over-expression of myocardin potently induces transcription of virtually all CArG-dependent SMC

α

*myosin light chain kinase* (*MLCK*) (Chen et al., 2002; Du et al., 2003; Wang et al., 2001; Wang et al., 2003; Yoshida et al., 2003). Mutation of CArG elements in the SMC promoters abolished

*-actin*, *SM-MHC*, *SM22*

α

, *h1-calponin*, and

, *h1-calponin*, and *SM* 

α

α

*-actin* in aortic SMCs

*-actin* in proepicardial

well as decreases in SMC-specific/-selective genes including *SM* 

differentiation marker genes including *SM22*

focusing on SRF and its interacting factors.

differentiation marker genes, including *SM* 

target genes.

**expression** 

the responsiveness to myocardin, suggesting that myocardin activates the transcription in a CArG-dependent manner. However, myocardin showed no DNA binding activity, but showed interaction with SRF. In addition, myocardin failed to activate the transcription of CArG-dependent genes in the absence of SRF (Du et al., 2003), demonstrating that myocardin is a co-activator of SRF. Over-expression of myocardin also induced the endogenous expression of SMC differentiation marker genes in cultured SMCs and non-SMCs, including 3T3 fibroblasts, L6 myoblasts, 3T3-L1 preadipocytes, COS cells, and undifferentiated embryonic stem cells (Chen et al., 2002; Du et al., 2003; Du et al., 2004; Wang et al., 2001; Wang et al., 2003; Yoshida et al., 2003; Yoshida et al., 2004b). However, forced expression of myocardin in non-SMCs was not sufficient to induce the full SMC differentiation program, because some SMC-enriched genes, which do not contain CArG elements in their promoter-enhancer region, were not induced (Yoshida et al., 2004b). Nevertheless, it was sufficient to establish a SMC-like contractile phenotype (Long et al., 2008). Either dominant-negative forms of myocardin or siRNA-induced suppression of myocardin decreased the transcription of SMC differentiation marker genes in cultured SMCs (Du et al., 2003; Wang et al., 2003; Yoshida et al., 2003). In addition, *myocardin*deficient mice exhibited no vascular SMC differentiation and died by embryonic day 10.5 (Li et al., 2003), although this may have been secondary to the defect in the extra-embryonic circulation. Moreover, mice lacking the *myocardin* gene in neural crest-derived cells died prior to postnatal day 3 from patent ductus arteriosus, and neural crest-derived SMCs in these mice exhibited a cell-autonomous block in expression of SMC differentiation marker genes (Huang et al., 2008). Taken together, the preceding results provide compelling evidence that myocardin plays a key role in the regulation of expression of SMC differentiation marker genes.

Fig. 1. Myocardin potently induces the transcription of CArG-element containing SMC differentiation marker genes. Myocardin preferentially activates SMC differentiation marker genes which contain multiple CArG elements in their promoter-enhancer regions. Homodimerization of myocardin through the leucine zipper-like domain efficiently activates the transcription. In contrast, myocardin does not induce the transcription of the growth factor-inducible gene, *c-fos*, because it only contains a single CArG element in the promoter.

Molecular Control of Smooth Muscle Cell Differentiation

the vicinity of CArG elements at the *SM* 

containing regions of the *SM* 

transcriptional activity of the *SM22*

and these chromatin modifying enzymes.

myocardin on the transcription of the *SM22*

the determinants of the transcriptional activity of myocardin.

α

**2.2 Role of the myocardin-related family in SMC differentiation** 

selectively enhanced SRF binding to CArG-containing region of the *SM* 

α

Marker Genes by Serum Response Factor and Its Interacting Proteins 27

consensus CArG element. If this is the case, it is likely that reduced SRF binding to CArG elements, which does not necessarily have G or C substitutions, is one of the mechanisms for target gene selectivity of myocardin. If this is not the case, it is still possible that the degeneracy in CArG elements may explain a part of the promoter selectivity of myocardin, but this mechanism cannot be applicable to all of the SMC differentiation marker genes.

Regarding the mechanism of myocardin-induced transcription of SMC differentiation marker genes, the physical interaction of myocardin with histone acetyltransferase, p300, and class II histone deacetylases, HDAC4 and HDAC5, has been reported (Cao et al., 2005). Indeed, results showed that over-expression of myocardin induced histone H3 acetylation in

et al., 2005). In addition, they showed that p300 augmented the stimulatory effect of

repressed the effect of myocardin by co-transfection/reporter assays. Moreover, they demonstrated that p300 and HDACs, respectively, bound to distinct domains of myocardin simultaneously, suggesting that the balance between p300 and HDACs is likely to be one of

These results are of significant interest in that they provided evidence that transcription of SMC differentiation marker genes is regulated by the recruitment of chromatin modifying enzymes by myocardin. Previous studies showed that SMC differentiation was associated with increased binding of SRF and hyperacetylation of histones H3 and H4 at CArG-

(Manabe & Owens, 2001b). In addition, we showed that over-expression of myocardin

to that of the *c-fos* gene in the context of intact chromatin in SMCs (Hendrix et al., 2005). Results of studies by another group (Qiu & Li, 2002) also showed that HDACs reduced the

findings are consistent with the results showing the association of myocardin with p300 or HDACs (Cao et al., 2005). However, it remains unknown how the association between myocardin and p300 or HDACs regulates the accessibility of SRF to CArG elements, as has been observed during the induction of SMC differentiation in A404 cells (Manabe & Owens, 2001b). It is possible that particular histone modifications by the myocardin-p300 complex enable SRF to bind to CArG-elements within the SMC promoters. It is also possible that the association between myocardin and chromatin modifying enzymes including p300 may alter the binding affinity of myocardin to SRF. Because regulation of SMC differentiation marker genes by platelet-derived growth factor-BB (PDGF-BB) or oxidized phospholipids has been shown to be accompanied by the recruitment of HDACs and thereby changes in acetylation levels at the SMC promoters (Yoshida et al., 2007, 2008a), it is interesting to determine if these changes are caused by the modulation of association between myocardin

Two factors were identified as members of the myocardin-related transcription factors: MKL1 (also referred to as MAL, BSAC, and MRTF-A) (Cen et al., 2003; Miralles et al., 2003; Sasazuki et al., 2002; Wang et al., 2002) and MKL2 (also referred to as MRTF-B) (Selvaraj & Prywes, 2003; Wang et al., 2002). It has been shown that expression of *MKL1* mRNA is

α

*-actin* and *SM22*

α

*-actin* and *SM-MHC* genes in A404 SMC precursor cells

gene in a CArG-element dependent manner. These

promoters in 10T1/2 cells (Cao

gene, whereas either HDAC4 or HDAC5

α

*-actin* gene, but not

α

#### **2.1 Transcriptional mechanism for myocardin-dependent SMC differentiation marker genes**

Although myocardin is a powerful transcriptional co-activator of SRF, there are still some questions for the mechanisms whereby myocardin induces SMC differentiation marker genes. One of these questions is: "what *cis*-elements and transcriptional co-activators other than SRF are required for the function of myocardin?" Initial studies (Wang et al., 2001) suggested that myocardin activated the transcription through the formation of complex with SRF and multiple CArG elements, based on the findings that: (1) the single CArG-containing *c-fos* gene had no responsiveness to myocardin; and (2) myocardin could activate an artificial promoter consisting of 4x *c-fos* SREs coupled to the basal promoter. Such a "2- CArG" model, in which multiple CArG elements are required for myocardin-induced transactivation, is strengthened by the results showing that homodimerization of myocardin extraordinary augmented the transcriptional activity of SMC differentiation marker genes (Figure 1) (Wang et al., 2003). However, several SMC-specific genes that only contain single CArG element in their promoter, such as the *telokin* gene and the *cysteine-rich protein-1* (*CRP-1*) gene, have also been shown to be activated by myocardin (Wang et al., 2003; Yoshida et al., 2004b). These results raised a question as to how myocardin distinguishes these single CArG-containing SMC differentiation marker genes from the *c-fos* gene. One hypothesis is that the presence of a ternary complex factor (TCF)-binding site in the *c-fos* promoter regulates the binding of myocardin to SRF. In support of this, it has been shown that one of the TCFs, Elk-1, could compete for SRF binding with myocardin on the SMC promoters (Wang et al., 2004; Yoshida et al., 2007; Zhou et al., 2005). Such a possibility will be discussed in detail in a later section.

An additional possibility is that degeneracy within CArG elements, i.e. conserved base pair substitutions that reduce SRF binding affinity, contributes to the promoter selectivity of myocardin. Consistent with this idea, the majority of SMC differentiation marker genes including *SM* α*-actin* and *SM-MHC* have degenerate CArG elements in their promoterenhancer regions (Miano, 2003). For example, both of CArG elements located within 5' flanking region of the *SM* α*-actin* gene contain a single G or C substitution within their A/Trich cores that is 100% conserved between species as divergent as humans and chickens (Shimizu et al., 1995). Results of our previous studies showed that substitution of *SM* α*-actin* 5' CArGs with the *c-fos* consensus CArGs significantly attenuated injury-induced downregulation of *SM* α*-actin* expression (Hendrix et al., 2005). In addition, of interest, overexpression of myocardin selectively enhanced SRF binding to degenerate *SM* α*-actin* CArG elements compared to *c-fos* consensus CArG element in SMCs, as determined by quantitative chromatin immunoprecipitation assays. These results raise a possibility that the degeneracy in the CArG elements is one of the determinants of promoter selectivity of myocardin. However, it should be noted that there is a difference not only in the sequence context of CArG elements, but also in the number of CArG elements between the *SM* α*-actin* gene versus the *c-fos* gene. Moreover, there is no G or C substitution in the CArG elements of several SMC differentiation marker genes including the *SM22*α, *telokin*, and *CRP-1* genes (Miano, 2003), although previous studies showed that the binding affinity of SRF to *SM22*α CArG-near element was lower than that to the *c-fos* CArG element by electromobility shift assays (EMSA) (Chang et al., 2001). It is interesting to determine whether CArG elements in the *telokin* gene and the *CRP-1* genes also exhibit lower binding affinity to SRF than the *c-fos*

**2.1 Transcriptional mechanism for myocardin-dependent SMC differentiation marker** 

Although myocardin is a powerful transcriptional co-activator of SRF, there are still some questions for the mechanisms whereby myocardin induces SMC differentiation marker genes. One of these questions is: "what *cis*-elements and transcriptional co-activators other than SRF are required for the function of myocardin?" Initial studies (Wang et al., 2001) suggested that myocardin activated the transcription through the formation of complex with SRF and multiple CArG elements, based on the findings that: (1) the single CArG-containing *c-fos* gene had no responsiveness to myocardin; and (2) myocardin could activate an artificial promoter consisting of 4x *c-fos* SREs coupled to the basal promoter. Such a "2- CArG" model, in which multiple CArG elements are required for myocardin-induced transactivation, is strengthened by the results showing that homodimerization of myocardin extraordinary augmented the transcriptional activity of SMC differentiation marker genes (Figure 1) (Wang et al., 2003). However, several SMC-specific genes that only contain single CArG element in their promoter, such as the *telokin* gene and the *cysteine-rich protein-1* (*CRP-1*) gene, have also been shown to be activated by myocardin (Wang et al., 2003; Yoshida et al., 2004b). These results raised a question as to how myocardin distinguishes these single CArG-containing SMC differentiation marker genes from the *c-fos* gene. One hypothesis is that the presence of a ternary complex factor (TCF)-binding site in the *c-fos* promoter regulates the binding of myocardin to SRF. In support of this, it has been shown that one of the TCFs, Elk-1, could compete for SRF binding with myocardin on the SMC promoters (Wang et al., 2004; Yoshida et al., 2007; Zhou et al., 2005). Such a possibility will be discussed

An additional possibility is that degeneracy within CArG elements, i.e. conserved base pair substitutions that reduce SRF binding affinity, contributes to the promoter selectivity of myocardin. Consistent with this idea, the majority of SMC differentiation marker genes

enhancer regions (Miano, 2003). For example, both of CArG elements located within 5'-

rich cores that is 100% conserved between species as divergent as humans and chickens

5' CArGs with the *c-fos* consensus CArGs significantly attenuated injury-induced

elements compared to *c-fos* consensus CArG element in SMCs, as determined by quantitative chromatin immunoprecipitation assays. These results raise a possibility that the degeneracy in the CArG elements is one of the determinants of promoter selectivity of myocardin. However, it should be noted that there is a difference not only in the sequence

gene versus the *c-fos* gene. Moreover, there is no G or C substitution in the CArG elements

(Miano, 2003), although previous studies showed that the binding affinity of SRF to *SM22*

CArG-near element was lower than that to the *c-fos* CArG element by electromobility shift assays (EMSA) (Chang et al., 2001). It is interesting to determine whether CArG elements in the *telokin* gene and the *CRP-1* genes also exhibit lower binding affinity to SRF than the *c-fos*

(Shimizu et al., 1995). Results of our previous studies showed that substitution of *SM* 

context of CArG elements, but also in the number of CArG elements between the *SM* 

expression of myocardin selectively enhanced SRF binding to degenerate *SM* 

of several SMC differentiation marker genes including the *SM22*

*-actin* and *SM-MHC* have degenerate CArG elements in their promoter-

*-actin* gene contain a single G or C substitution within their A/T-

*-actin* expression (Hendrix et al., 2005). In addition, of interest, over-

α

α*-actin*

*-actin* CArG

α*-actin*

α

α

, *telokin*, and *CRP-1* genes

**genes** 

in detail in a later section.

flanking region of the *SM* 

downregulation of *SM* 

α

α

α

including *SM* 

consensus CArG element. If this is the case, it is likely that reduced SRF binding to CArG elements, which does not necessarily have G or C substitutions, is one of the mechanisms for target gene selectivity of myocardin. If this is not the case, it is still possible that the degeneracy in CArG elements may explain a part of the promoter selectivity of myocardin, but this mechanism cannot be applicable to all of the SMC differentiation marker genes.

Regarding the mechanism of myocardin-induced transcription of SMC differentiation marker genes, the physical interaction of myocardin with histone acetyltransferase, p300, and class II histone deacetylases, HDAC4 and HDAC5, has been reported (Cao et al., 2005). Indeed, results showed that over-expression of myocardin induced histone H3 acetylation in the vicinity of CArG elements at the *SM* α*-actin* and *SM22*α promoters in 10T1/2 cells (Cao et al., 2005). In addition, they showed that p300 augmented the stimulatory effect of myocardin on the transcription of the *SM22*α gene, whereas either HDAC4 or HDAC5 repressed the effect of myocardin by co-transfection/reporter assays. Moreover, they demonstrated that p300 and HDACs, respectively, bound to distinct domains of myocardin simultaneously, suggesting that the balance between p300 and HDACs is likely to be one of the determinants of the transcriptional activity of myocardin.

These results are of significant interest in that they provided evidence that transcription of SMC differentiation marker genes is regulated by the recruitment of chromatin modifying enzymes by myocardin. Previous studies showed that SMC differentiation was associated with increased binding of SRF and hyperacetylation of histones H3 and H4 at CArGcontaining regions of the *SM* α*-actin* and *SM-MHC* genes in A404 SMC precursor cells (Manabe & Owens, 2001b). In addition, we showed that over-expression of myocardin selectively enhanced SRF binding to CArG-containing region of the *SM* α*-actin* gene, but not to that of the *c-fos* gene in the context of intact chromatin in SMCs (Hendrix et al., 2005). Results of studies by another group (Qiu & Li, 2002) also showed that HDACs reduced the transcriptional activity of the *SM22*α gene in a CArG-element dependent manner. These findings are consistent with the results showing the association of myocardin with p300 or HDACs (Cao et al., 2005). However, it remains unknown how the association between myocardin and p300 or HDACs regulates the accessibility of SRF to CArG elements, as has been observed during the induction of SMC differentiation in A404 cells (Manabe & Owens, 2001b). It is possible that particular histone modifications by the myocardin-p300 complex enable SRF to bind to CArG-elements within the SMC promoters. It is also possible that the association between myocardin and chromatin modifying enzymes including p300 may alter the binding affinity of myocardin to SRF. Because regulation of SMC differentiation marker genes by platelet-derived growth factor-BB (PDGF-BB) or oxidized phospholipids has been shown to be accompanied by the recruitment of HDACs and thereby changes in acetylation levels at the SMC promoters (Yoshida et al., 2007, 2008a), it is interesting to determine if these changes are caused by the modulation of association between myocardin and these chromatin modifying enzymes.

#### **2.2 Role of the myocardin-related family in SMC differentiation**

Two factors were identified as members of the myocardin-related transcription factors: MKL1 (also referred to as MAL, BSAC, and MRTF-A) (Cen et al., 2003; Miralles et al., 2003; Sasazuki et al., 2002; Wang et al., 2002) and MKL2 (also referred to as MRTF-B) (Selvaraj & Prywes, 2003; Wang et al., 2002). It has been shown that expression of *MKL1* mRNA is

Molecular Control of Smooth Muscle Cell Differentiation

transcription of the *c-fos* gene (Buchwalter et al., 2004).

*-actin* and *SM22*

to the TCF-binding site within the *SM22*

genes.

α

including *SM* 

α

Marker Genes by Serum Response Factor and Its Interacting Proteins 29

of the *c-fos* gene with SRF dimers both before and after growth factor stimulation, and that after the stimulation with growth factors, TCFs are phosphorylated and activate

Although it has been believed, for a long time, that most of SMC differentiation marker genes lack the TCF-binding site in their promoter regions (Miano 2003), results of recent studies by multiple laboratories including our own (Wang et al., 2004; Yoshida et al., 2007; Zhou et al., 2005) suggest the involvement of Elk-1 in the regulation of SMC differentiation marker genes. They presented evidence that repression of SMC differentiation marker genes

from SRF by phosphorylated Elk-1 in cultured SMCs (Figure 2). Indeed, they showed that treatment with PDGF-BB induced phosphorylation of Elk-1 through the activation of the MEK1/2-Erk1/2 pathway and increased the association between Elk-1 and SRF, whereas the association between myocardin and SRF was decreased at the same time. By extensively mapping the domain of myocardin and Elk-1, they found that both factors have a structurally related SRF-binding motif and thereby compete for the common docking region of SRF. These results are very interesting in that phosphorylation of Elk-1 simultaneously exhibits the dual roles in the regulation of CArG-dependent genes: transcriptional activation of the *c-fos* gene versus transcriptional repression of SMC differentiation marker genes.

Fig. 2. Phosphorylation of Elk-1 competes for SRF binding with myocardin. The myocardin-SRF-CArG complex activates the transcription of SMC differentiation marker genes in the absence of growth factors as shown in Fig. 1. Activation of the Erk1/2 pathway by growth factors such as PDGF-BB induces phosphorylation of Elk-1. Phosphorylated Elk-1 displaces myocardin from SRF and binds to SRF, thereby suppressing the transcription of SMC differentiation marker genes. It has been reported that phosphorylated Elk-1 is able to bind

binding site is not present within the promoter region of most SMC differentiation marker

However, the mechanisms responsible for these dual effects have not been clearly understood yet. That is, although the binding of Elk-1 on the putative TCF-binding site (5'-

α

TTCCCG-3') adjacent to the CArG-far element at the *SM22*

by PDGF-BB was due to the displacement of myocardin

promoter (Wang et al., 2004), although the TCF-

α

promoter was detected by

ubiquitous, whereas expression of *MKL2* mRNA is restricted to several tissues including the brain and the heart (Cen et al., 2003; Selvaraj & Prywes, 2003; Wang et al., 2002). Cotransfection studies revealed that both MKL1 and MKL2 were capable of inducing the transcription of multiple CArG-containing promoters including *atrial natriuretic factor* (*ANF*), *SM22*α, *SM* α*-actin*, and *cardiac* α*-actin*. A truncated MKL2 protein that lacks both aminoterminal region and carboxy-terminal region (MKL2ΔNΔC700) behaved as a dominantnegative manner for both MKL1 and MKL2, and over-expression of MKL2ΔNΔC700 inhibited skeletal muscle differentiation in C2C12 skeletal myoblasts (Selvaraj & Prywes, 2003). In addition, MKL1 strongly induced SMC differentiation marker gene expression in undifferentiated embryonic stem cells, even in the absence of myocardin (Du et al., 2004). Moreover, a truncated form of MKL1, which behaved as a dominant-negative form of MKL1 and myocardin, inhibited MKL1-induced transcription of the *SM22*α gene (Du et al., 2004). Taken together, MKL factors appear to be important regulators of SMC differentiation marker gene expression as well as myocardin, and they appear to exhibit the redundant function with myocardin as SRF co-factors. However, the precise roles of MKL factors in SMC differentiation marker gene expression in SMCs are still unclear, because most of these studies analyzing the function of MKL factors have been performed by over-expression experiments. Regarding this point, there are several interesting studies as described below. First, *MKL1* knockout mice were viable, but were unable to effectively nurse their offspring due to a failure in maintenance of the differentiated state of mammary myoepithelial cells during lactation (Li et al., 2006; Sun et al., 2006). Second, conditional knockout of the *MKL2* gene in neural crestderived cells exhibited a spectrum of cardiovascular defects including abnormal patterning of the branchial arch arteries (Li et al., 2005; Oh et al., 2005). The abnormalities in *MKL2* knockout mice were accompanied by a decrease in SM α-actin expression in SMCs within the branchial arch arteries. Based on the results of these studies, MKL1 is unlikely to play an important role in expression of SMC differentiation marker genes *in vivo*. In addition, role of MKL2 for SMC differentiation in SMCs derived from other origins is still unknown. A biggest issue is how broadly expressed MKL factors regulate SMC-specific/-selective CArG-dependent genes. Recently, several studies suggest the importance of intracellular localization of MKL factors in SMCs and non-SMCs (Hinson et al., 2007; Nakamura et al., 2010; Yoshida et al., 2007). Further studies are required to address this issue.

In summary, it is clear that myocardin plays a critical role in SMC differentiation in concert with the CArG-SRF complex. However, myocardin is not a SMC-specific gene in that it is also expressed in cardiomyocytes, suggesting that myocardin alone is not enough to coordinate expression of SMC differentiation marker genes. It is highly likely that cooperative interaction of the SRF-myocardin complex with other transcription factors is necessary for expression of SMC differentiation marker genes in SMCs. Further studies are needed to clarify these combinatorial mechanisms.

#### **3. Ternary complex factors exhibit dual roles in the transcription of SRFdependent CArG-Containing genes**

TCFs are a subfamily of the Ets domain transcription factors (Buchwalter et al., 2004). TCF was first described as 62 kD nuclear fractions (p62) that form a ternary complex with SRF on the *c-fos* SRE (Shaw et al., 1989). Three members, Elk-1, Sap-1/Elk-4, and Net/Sap-2/Elk-3, have been identified as TCFs. Previous studies demonstrated that TCFs are present on SREs

ubiquitous, whereas expression of *MKL2* mRNA is restricted to several tissues including the brain and the heart (Cen et al., 2003; Selvaraj & Prywes, 2003; Wang et al., 2002). Cotransfection studies revealed that both MKL1 and MKL2 were capable of inducing the transcription of multiple CArG-containing promoters including *atrial natriuretic factor* (*ANF*),

terminal region and carboxy-terminal region (MKL2ΔNΔC700) behaved as a dominantnegative manner for both MKL1 and MKL2, and over-expression of MKL2ΔNΔC700 inhibited skeletal muscle differentiation in C2C12 skeletal myoblasts (Selvaraj & Prywes, 2003). In addition, MKL1 strongly induced SMC differentiation marker gene expression in undifferentiated embryonic stem cells, even in the absence of myocardin (Du et al., 2004). Moreover, a truncated form of MKL1, which behaved as a dominant-negative form of MKL1

Taken together, MKL factors appear to be important regulators of SMC differentiation marker gene expression as well as myocardin, and they appear to exhibit the redundant function with myocardin as SRF co-factors. However, the precise roles of MKL factors in SMC differentiation marker gene expression in SMCs are still unclear, because most of these studies analyzing the function of MKL factors have been performed by over-expression experiments. Regarding this point, there are several interesting studies as described below. First, *MKL1* knockout mice were viable, but were unable to effectively nurse their offspring due to a failure in maintenance of the differentiated state of mammary myoepithelial cells during lactation (Li et al., 2006; Sun et al., 2006). Second, conditional knockout of the *MKL2* gene in neural crestderived cells exhibited a spectrum of cardiovascular defects including abnormal patterning of the branchial arch arteries (Li et al., 2005; Oh et al., 2005). The abnormalities in *MKL2* knockout mice were accompanied by a decrease in SM α-actin expression in SMCs within the branchial arch arteries. Based on the results of these studies, MKL1 is unlikely to play an important role in expression of SMC differentiation marker genes *in vivo*. In addition, role of MKL2 for SMC differentiation in SMCs derived from other origins is still unknown. A biggest issue is how broadly expressed MKL factors regulate SMC-specific/-selective CArG-dependent genes. Recently, several studies suggest the importance of intracellular localization of MKL factors in SMCs and non-SMCs (Hinson et al., 2007; Nakamura et al., 2010; Yoshida et al., 2007). Further

In summary, it is clear that myocardin plays a critical role in SMC differentiation in concert with the CArG-SRF complex. However, myocardin is not a SMC-specific gene in that it is also expressed in cardiomyocytes, suggesting that myocardin alone is not enough to coordinate expression of SMC differentiation marker genes. It is highly likely that cooperative interaction of the SRF-myocardin complex with other transcription factors is necessary for expression of SMC differentiation marker genes in SMCs. Further studies are

**3. Ternary complex factors exhibit dual roles in the transcription of SRF-**

TCFs are a subfamily of the Ets domain transcription factors (Buchwalter et al., 2004). TCF was first described as 62 kD nuclear fractions (p62) that form a ternary complex with SRF on the *c-fos* SRE (Shaw et al., 1989). Three members, Elk-1, Sap-1/Elk-4, and Net/Sap-2/Elk-3, have been identified as TCFs. Previous studies demonstrated that TCFs are present on SREs

*-actin*. A truncated MKL2 protein that lacks both amino-

α

gene (Du et al., 2004).

*SM22*α, *SM* α

*-actin*, and *cardiac* 

studies are required to address this issue.

needed to clarify these combinatorial mechanisms.

**dependent CArG-Containing genes** 

α

and myocardin, inhibited MKL1-induced transcription of the *SM22*

of the *c-fos* gene with SRF dimers both before and after growth factor stimulation, and that after the stimulation with growth factors, TCFs are phosphorylated and activate transcription of the *c-fos* gene (Buchwalter et al., 2004).

Although it has been believed, for a long time, that most of SMC differentiation marker genes lack the TCF-binding site in their promoter regions (Miano 2003), results of recent studies by multiple laboratories including our own (Wang et al., 2004; Yoshida et al., 2007; Zhou et al., 2005) suggest the involvement of Elk-1 in the regulation of SMC differentiation marker genes. They presented evidence that repression of SMC differentiation marker genes including *SM* α*-actin* and *SM22*α by PDGF-BB was due to the displacement of myocardin from SRF by phosphorylated Elk-1 in cultured SMCs (Figure 2). Indeed, they showed that treatment with PDGF-BB induced phosphorylation of Elk-1 through the activation of the MEK1/2-Erk1/2 pathway and increased the association between Elk-1 and SRF, whereas the association between myocardin and SRF was decreased at the same time. By extensively mapping the domain of myocardin and Elk-1, they found that both factors have a structurally related SRF-binding motif and thereby compete for the common docking region of SRF. These results are very interesting in that phosphorylation of Elk-1 simultaneously exhibits the dual roles in the regulation of CArG-dependent genes: transcriptional activation of the *c-fos* gene versus transcriptional repression of SMC differentiation marker genes.

Fig. 2. Phosphorylation of Elk-1 competes for SRF binding with myocardin. The myocardin-SRF-CArG complex activates the transcription of SMC differentiation marker genes in the absence of growth factors as shown in Fig. 1. Activation of the Erk1/2 pathway by growth factors such as PDGF-BB induces phosphorylation of Elk-1. Phosphorylated Elk-1 displaces myocardin from SRF and binds to SRF, thereby suppressing the transcription of SMC differentiation marker genes. It has been reported that phosphorylated Elk-1 is able to bind to the TCF-binding site within the *SM22*α promoter (Wang et al., 2004), although the TCFbinding site is not present within the promoter region of most SMC differentiation marker genes.

However, the mechanisms responsible for these dual effects have not been clearly understood yet. That is, although the binding of Elk-1 on the putative TCF-binding site (5'- TTCCCG-3') adjacent to the CArG-far element at the *SM22*αpromoter was detected by

Molecular Control of Smooth Muscle Cell Differentiation

differentiation of SMCs by interacting with the CArG-SRF complex.

multiple SMC differentiation marker genes including *SM* 

regulating the binding of SRF to CArG elements.

*SM* α

both basal and angiotensin II-induced transcription of the *SM* 

dependent SMC differentiation marker gene expression in these mice.

Marker Genes by Serum Response Factor and Its Interacting Proteins 31

types during embryogenesis (Gorski & Walsh, 2003). This family is comprised of over 160 genes, and it has been reported that several homeodomain proteins are able to regulate

One of these factors is Prx-1 (Paired-related homeobox gene-1), which is also known as MHox and Phox (Cserjesi et al., 1992; Grueneberg et al., 1992). Expression of Prx-1 is completely restricted to mesodermally derived cell types during embryogenesis and to cell lines of mesodermal origin including cultured aortic SMCs (Blank et al., 1995; Cserjesi et al., 1992). Previous studies from our laboratory and others showed that Prx-1 was capable of inducing the transcription of the CArG-SRF dependent genes (Grueneberg et al., 1992; Hautmann et al., 1997; Yoshida et al., 2004a). Indeed, we found that angiotensin II increased expression of

cultured SMCs (Hautmann et al., 1997; Turla et al., 1991; Yoshida et al., 2004a). Of major interest, we provided evidence that siRNA-induced suppression of Prx-1 dramatically reduced

2004a). In addition, Prx-1 increased the SRF binding to degenerate CArG B element within the

Although the preceding results suggest that Prx-1 is involved in the regulation of SMC differentiation marker gene expression (Hautmann et al., 1997; Yoshida et al., 2004a), it also plays a role in proliferation of SMCs. *Prx-1* expression was induced during the development of pulmonary vascular disease in adult rats, and Prx-1 enhanced the proliferation rate of cultured rat A10 SMCs via the induction of tenascin-C expression (Jones et al., 2001). Taken together, results suggest that Prx-1 plays multiple roles in the regulation of differentiation status and the regulation of proliferation status in SMCs. This is consistent with the idea that differentiation and proliferation are not necessarily mutually exclusive processes (Owens & Thompson, 1986; Owens et al., 2004). However, it remains unknown whether Prx-1 exhibits these two roles simultaneously or Prx-1 exhibits distinct roles in a developmental stagespecific manner. Of interest, *Prx-1* knockout mice have been made and shown to exhibit major defects in skeletogenesis and die soon after birth (Martin et al., 1995). Mice null for both *Prx-1* and its homologue, *Prx-2*, showed a vascular abnormality with an abnormal positioning and awkward curvature of the aortic arch and a misdirected and elongated ductus arteriosus (Bergwerff et al., 2000). Moreover, expression of endothelial markers such as Flk-1 and VCAM-1 and von Willebrand factor-positive cells were decreased in the lung of *Prx-1* null newborn mice (Ihida-Stansbury et al., 2004), suggesting that Prx-1 is required for lung vascularization *in vivo*. It will be of interest to directly test the role of Prx-1 in CArG-

Another homeodomain protein related to SMC differentiation is Hex. Hex was originally isolated from hematopoietic tissues by PCR using degenerate oligonucleotide primers corresponding to the conserved homeodomain sequences and has been shown to play an

*-actin* gene by EMSA (Hautmann et al., 1997). Similarly, Prx-1 enhanced the binding of SRF to *c-fos* CArG element by EMSA (Grueneberg et al., 1992). However, the formation of a stable higher order complex comprised of Prx-1, SRF, and CArG element was not detected by EMSA. Rather, Prx-1 enhanced both the rate of association and the rate of dissociation between SRF and CArG element, thereby increasing the rate of exchange of SRF on the CArG element. Although further studies are required to clarify these mechanisms in detail, results thus far suggest that Prx-1 plays a key role in the transcription of CArG-dependent genes through

α

α

*-actin*, as well as *Prx-1* expression in

*-actin* gene (Yoshida et al.,

EMSA and chromatin immunoprecipitation assays (Wang et al., 2004), this sequence is not the consensus binding site for Elk-1 (Treisman et al., 1992). By using "the site selection method" to purify DNA capable of forming ternary complexes from a pool of randomized oligonucleotides, the consensus binding motif for Elk-1 and Sap-1 was determined as 5'- (C/A)(C/A)GGA(A/T)-3' previously (Treisman et al., 1992). The putative TCF-binding site within the *SM22*α gene (sense: 5'-TTCCCG-3' and antisense: 5'-CGGGAA-3') does not match this sequence completely. In addition, although over-expression of Elk-1 downregulated the *SM22*α promoter-luciferase activity through the competition with myocardin, this competition was still observed when the mutational *SM22*α-luciferase construct, in which the putative TCF-binding site was abolished, was used. Furthermore, there is no putative Elk-1 binding site near the CArG elements within the *SM* α*-actin* promoter (Mack & Owens, 1999). Because chromatin immunoprecipitation assays can detect not only the direct binding of protein to DNA sequence, but also the binding of protein to protein, it is highly possible that the attachment of Elk-1 to the TCF-binding site may not be absolutely required for the competition with myocardin for SRF binding. Nevertheless, the *SM22*α promoter with a mutation in the TCF-binding site has been reported to direct ectopic transcription in the heart in a later embryonic stage, as compared with the wild-type *SM22*α promoter *in vivo* (Wang et al., 2004). Further studies are needed to determine if these findings are applicable to multiple SMC differentiation marker genes.

It is also of interest to determine whether the activation of Elk-1 can recruit histone deacetylases to the promoter regions of SMC differentiation marker genes. Elk-1 contains two transcriptional repression domains, an N-terminal transcriptional repression domain and an R motif located in the C-terminal transcriptional activation domain (Buchwalter et al., 2004). It has been shown that HDAC1 and HDAC2 were recruited to the N-terminal transcriptional repression domain of Elk-1 on the *c-fos* promoter followed by the activation of the MEK1/2-Erk-1/2 pathway, and this recruitment kinetically correlated with the shutoff of the *c-fos* gene expression after growth factor stimulation (Yang et al., 2001; Yang & Sharrocks, 2004). We previously showed that repression of SMC differentiation marker genes after stimulation with PDGF-BB was accompanied by the recruitment of multiple HDACs, HDAC2, HDAC4, and HDAC5 in cultured SMCs (Yoshida et al., 2007). It is possible that the association between Elk-1 and these HDACs on the SMC promoters is one of the mechanisms for repression of SMC differentiation marker gene expression. Moreover, it was reported that SUMO modification of the R motif in Elk-1 could antagonize the MEK1/2-Erk1/2 pathway and repress the transcription of the *c-fos* gene (Yang et al., 2003). Thus, it is also possible that PDGF-BB can induce sumoylation of Elk-1 and exhibit the repressive effects on SMC differentiation marker genes.

In summary, the preceding results indicate that Elk-1 plays dual roles in the transcription of CArG-dependent genes as both an activator and a repressor. However, there are still some questions as discussed above. Clearly, one of the most fascinating questions is to determine if knockdown of Elk-1 abolishes PDGF-BB-induced repression of SMC differentiation marker genes both *in vivo* and *in vitro*.

#### **4. Multiple homeodomain proteins regulate SMC differentiation**

Homeodomain proteins are a family of transcription factors with a highly conserved DNAbinding domain that regulate cell proliferation, differentiation, and migration in many cell

EMSA and chromatin immunoprecipitation assays (Wang et al., 2004), this sequence is not the consensus binding site for Elk-1 (Treisman et al., 1992). By using "the site selection method" to purify DNA capable of forming ternary complexes from a pool of randomized oligonucleotides, the consensus binding motif for Elk-1 and Sap-1 was determined as 5'- (C/A)(C/A)GGA(A/T)-3' previously (Treisman et al., 1992). The putative TCF-binding site

match this sequence completely. In addition, although over-expression of Elk-1

construct, in which the putative TCF-binding site was abolished, was used. Furthermore,

promoter (Mack & Owens, 1999). Because chromatin immunoprecipitation assays can detect not only the direct binding of protein to DNA sequence, but also the binding of protein to protein, it is highly possible that the attachment of Elk-1 to the TCF-binding site may not be absolutely required for the competition with myocardin for SRF binding. Nevertheless, the

transcription in the heart in a later embryonic stage, as compared with the wild-type *SM22*

promoter *in vivo* (Wang et al., 2004). Further studies are needed to determine if these

It is also of interest to determine whether the activation of Elk-1 can recruit histone deacetylases to the promoter regions of SMC differentiation marker genes. Elk-1 contains two transcriptional repression domains, an N-terminal transcriptional repression domain and an R motif located in the C-terminal transcriptional activation domain (Buchwalter et al., 2004). It has been shown that HDAC1 and HDAC2 were recruited to the N-terminal transcriptional repression domain of Elk-1 on the *c-fos* promoter followed by the activation of the MEK1/2-Erk-1/2 pathway, and this recruitment kinetically correlated with the shutoff of the *c-fos* gene expression after growth factor stimulation (Yang et al., 2001; Yang & Sharrocks, 2004). We previously showed that repression of SMC differentiation marker genes after stimulation with PDGF-BB was accompanied by the recruitment of multiple HDACs, HDAC2, HDAC4, and HDAC5 in cultured SMCs (Yoshida et al., 2007). It is possible that the association between Elk-1 and these HDACs on the SMC promoters is one of the mechanisms for repression of SMC differentiation marker gene expression. Moreover, it was reported that SUMO modification of the R motif in Elk-1 could antagonize the MEK1/2-Erk1/2 pathway and repress the transcription of the *c-fos* gene (Yang et al., 2003). Thus, it is also possible that PDGF-BB can induce sumoylation of Elk-1 and exhibit the

In summary, the preceding results indicate that Elk-1 plays dual roles in the transcription of CArG-dependent genes as both an activator and a repressor. However, there are still some questions as discussed above. Clearly, one of the most fascinating questions is to determine if knockdown of Elk-1 abolishes PDGF-BB-induced repression of SMC differentiation

Homeodomain proteins are a family of transcription factors with a highly conserved DNAbinding domain that regulate cell proliferation, differentiation, and migration in many cell

**4. Multiple homeodomain proteins regulate SMC differentiation** 

promoter with a mutation in the TCF-binding site has been reported to direct ectopic

myocardin, this competition was still observed when the mutational *SM22*

findings are applicable to multiple SMC differentiation marker genes.

there is no putative Elk-1 binding site near the CArG elements within the *SM* 

gene (sense: 5'-TTCCCG-3' and antisense: 5'-CGGGAA-3') does not

promoter-luciferase activity through the competition with

α


α*-actin*

α

within the *SM22*

*SM22*α

downregulated the *SM22*

α

α

repressive effects on SMC differentiation marker genes.

marker genes both *in vivo* and *in vitro*.

types during embryogenesis (Gorski & Walsh, 2003). This family is comprised of over 160 genes, and it has been reported that several homeodomain proteins are able to regulate differentiation of SMCs by interacting with the CArG-SRF complex.

One of these factors is Prx-1 (Paired-related homeobox gene-1), which is also known as MHox and Phox (Cserjesi et al., 1992; Grueneberg et al., 1992). Expression of Prx-1 is completely restricted to mesodermally derived cell types during embryogenesis and to cell lines of mesodermal origin including cultured aortic SMCs (Blank et al., 1995; Cserjesi et al., 1992). Previous studies from our laboratory and others showed that Prx-1 was capable of inducing the transcription of the CArG-SRF dependent genes (Grueneberg et al., 1992; Hautmann et al., 1997; Yoshida et al., 2004a). Indeed, we found that angiotensin II increased expression of multiple SMC differentiation marker genes including *SM* α*-actin*, as well as *Prx-1* expression in cultured SMCs (Hautmann et al., 1997; Turla et al., 1991; Yoshida et al., 2004a). Of major interest, we provided evidence that siRNA-induced suppression of Prx-1 dramatically reduced both basal and angiotensin II-induced transcription of the *SM* α*-actin* gene (Yoshida et al., 2004a). In addition, Prx-1 increased the SRF binding to degenerate CArG B element within the *SM* α*-actin* gene by EMSA (Hautmann et al., 1997). Similarly, Prx-1 enhanced the binding of SRF to *c-fos* CArG element by EMSA (Grueneberg et al., 1992). However, the formation of a stable higher order complex comprised of Prx-1, SRF, and CArG element was not detected by EMSA. Rather, Prx-1 enhanced both the rate of association and the rate of dissociation between SRF and CArG element, thereby increasing the rate of exchange of SRF on the CArG element. Although further studies are required to clarify these mechanisms in detail, results thus far suggest that Prx-1 plays a key role in the transcription of CArG-dependent genes through regulating the binding of SRF to CArG elements.

Although the preceding results suggest that Prx-1 is involved in the regulation of SMC differentiation marker gene expression (Hautmann et al., 1997; Yoshida et al., 2004a), it also plays a role in proliferation of SMCs. *Prx-1* expression was induced during the development of pulmonary vascular disease in adult rats, and Prx-1 enhanced the proliferation rate of cultured rat A10 SMCs via the induction of tenascin-C expression (Jones et al., 2001). Taken together, results suggest that Prx-1 plays multiple roles in the regulation of differentiation status and the regulation of proliferation status in SMCs. This is consistent with the idea that differentiation and proliferation are not necessarily mutually exclusive processes (Owens & Thompson, 1986; Owens et al., 2004). However, it remains unknown whether Prx-1 exhibits these two roles simultaneously or Prx-1 exhibits distinct roles in a developmental stagespecific manner. Of interest, *Prx-1* knockout mice have been made and shown to exhibit major defects in skeletogenesis and die soon after birth (Martin et al., 1995). Mice null for both *Prx-1* and its homologue, *Prx-2*, showed a vascular abnormality with an abnormal positioning and awkward curvature of the aortic arch and a misdirected and elongated ductus arteriosus (Bergwerff et al., 2000). Moreover, expression of endothelial markers such as Flk-1 and VCAM-1 and von Willebrand factor-positive cells were decreased in the lung of *Prx-1* null newborn mice (Ihida-Stansbury et al., 2004), suggesting that Prx-1 is required for lung vascularization *in vivo*. It will be of interest to directly test the role of Prx-1 in CArGdependent SMC differentiation marker gene expression in these mice.

Another homeodomain protein related to SMC differentiation is Hex. Hex was originally isolated from hematopoietic tissues by PCR using degenerate oligonucleotide primers corresponding to the conserved homeodomain sequences and has been shown to play an

Molecular Control of Smooth Muscle Cell Differentiation

**5. A number of factors associate with SRF** 

differentiation and maturation (Morrisey et al., 1998).

downregulation of SMC differentiation marker genes including *SM* 

development.

briefly.

**5.1 GATA-6** 

Marker Genes by Serum Response Factor and Its Interacting Proteins 33

Fig. 3. Pitx2 transactivates SMC differentiation marker genes through three mechanisms. Pitx2 induces expression of SMC differentiation marker genes by: (1) binding to a consensus TAATC(C/T) *cis*-element; (2) interacting with SRF; and (3) mediating exchange of HDACs with p300 at the promoter region of SMC differentiation marker genes. These mechanisms are important for the initial induction of SMC differentiation during the early embryonic

In addition to the factors described above, there are a number of transcription factors known to interact with SRF. These factors also play key roles in the control of SMC differentiation marker gene expression. In this section, some of these transcription factors will be discussed

GATA proteins are a family of zinc finger transcription factors, and play essential roles in development through their interaction with a DNA consensus element, "WGATAR" (Molkentin, 2000). Six GATA transcription factors have been identified in vertebrates, and GATA-4, GATA-5, and GATA-6 are thought to be involved in the formation of the heart, gut, and vessels. During the early murine embryonic development, expression patterns of GATA-6 and GATA-4 were similar, with expression being detected in the precardiac mesoderm, the embryonic heart tube, and the primitive gut (Morrisey et al., 1996). However, during the late development, GATA-6 became the only GATA factor to be expressed in vascular SMCs. Knockout of the *GATA-6* gene in mice resulted in embryonic lethality between embryonic day 6.5 and 7.5, precluding the evaluation of the role of GATA-6 in SMC

As described in a previous section, GATA-6 has shown to interact with SRF and Nkx-3.2 and to induce SMC differentiation marker gene expression (Morrisey et al., 1998; Nishida et al., 2002). *GATA-6* expression in SMCs was rapidly downregulated after vascular injury in rat carotid arteries, and adenovirus-mediated transfer of GATA-6 to the vessel wall after the balloon injury partially inhibited the formation of intimal thickening and reversed the

(Mano et al., 1999). These results suggest the important role of GATA-6 in regulating SMC

α

*-actin* and *SM-MHC*

important role in inducing differentiation of vascular endothelial cells (Thomas et al., 1998). In SMCs, Hex protein expression was induced in the neointima after balloon injury of rat aorta, while it was undetectable in normal aorta (Sekiguchi et al., 2001). The expression pattern of Hex was similar to that of SMemb/NMHC-B, a marker of phenotypically modulated SMCs. Hex induced the transcription of the SMemb promoter, and cAMPresponsive element (CRE) located at -481 bp within the promoter was critical for Hex responsiveness. However, Hex failed to bind to CRE directly, thus the precise mechanisms whereby Hex activated the *SMemb* promoter are still unclear. Of interest, subsequent studies showed that Hex also induced expression of a subset of SMC differentiation marker genes including *SM* α*-actin* and *SM22*α, but not *SM-MHC* and *h1-calponin* (Oyama et al., 2004). Hex induced the transcription of the *SM22*α gene in a CArG-dependent manner, and it enhanced the binding of SRF to CArG-near element within the *SM22*α promoter, as determined by EMSA. In addition, immunoprecipitation assays revealed the physical association between SRF and Hex. As such, the mechanisms whereby Hex induces SMC differentiation marker genes seem to be similar to those of Prx-1. However, results showing that Hex simultaneously activated expression of both SMC differentiation marker genes and those characteristic of phenotypically modulated SMCs are paradoxical, and further studies are clearly needed to precisely define the pathophysiological role of Hex in SMCs.

Nkx-3.2 is also a homeodomain protein that regulates expression of SMC differentiation marker genes (Nishida et al., 2002). It has been demonstrated that a triad of SRF, GATA-6, and Nkx-3.2 formed a complex with their corresponding *cis*-elements and cooperatively transactivated SMC differentiation marker genes including α*1-integrin*, *SM22*α, and *caldesmon*. Because co-localization of GATA-6, Nkx-3.2, and SRF was exclusively observed in SMCs, SMC-specific gene expression does not appear to be the result of any single transcription factor that is unique to SMCs, but rather is due to unique combinatorial interactions of factors that may be expressed in multiple cell types but only found together in SMCs.

Furthermore, we recently identified Pitx2 as a homeodomain protein which is required for the initial induction of SMC differentiation by using a subtraction hybridization screen (Shang et al., 2008). Over-expression of Pitx2 induced expression of CArG-dependent SMC differentiation marker genes, whereas knockdown of Pitx2 attenuated retinoic acid-induced differentiation of SMCs from undifferentiated SMC precursor cells. Furthermore, *Pitx2* knockout mouse embryos exhibited impaired induction of SMC differentiation markers in the dorsal aorta and branchial arch arteries. We identified three mechanisms for Pitx2 induced transcription of SMC differentiation marker genes (Figure 3). First, Pitx2 bound to its consensus TAATC(C/T) element in the promoter region of SMC differentiation marker genes. Second, Pitx2 physically associated with SRF. Third, Pitx2 mediated exchange of HDACs with p300 to increase acetylation levels of histone H4 at the SMC promoters. These results provide compelling evidence that Pitx2 plays a critical role in the induction of SMC differentiation during the early embryogenesis. Further studies are needed to determine if Pitx2 also contributes to the pathogenesis of vascular diseases including atherosclerosis.

As such, several homeodomain proteins are involved in the regulation of CArG-SRF dependent SMC differentiation marker gene expression, and some of the mechanisms appear to be mediated by common pathways. Further studies are needed to clarify the temporal and spatial roles of each of these homeodomain proteins in SMC differentiation.

Fig. 3. Pitx2 transactivates SMC differentiation marker genes through three mechanisms. Pitx2 induces expression of SMC differentiation marker genes by: (1) binding to a consensus TAATC(C/T) *cis*-element; (2) interacting with SRF; and (3) mediating exchange of HDACs with p300 at the promoter region of SMC differentiation marker genes. These mechanisms are important for the initial induction of SMC differentiation during the early embryonic development.

## **5. A number of factors associate with SRF**

In addition to the factors described above, there are a number of transcription factors known to interact with SRF. These factors also play key roles in the control of SMC differentiation marker gene expression. In this section, some of these transcription factors will be discussed briefly.

## **5.1 GATA-6**

32 The Cardiovascular System – Physiology, Diagnostics and Clinical Implications

important role in inducing differentiation of vascular endothelial cells (Thomas et al., 1998). In SMCs, Hex protein expression was induced in the neointima after balloon injury of rat aorta, while it was undetectable in normal aorta (Sekiguchi et al., 2001). The expression pattern of Hex was similar to that of SMemb/NMHC-B, a marker of phenotypically modulated SMCs. Hex induced the transcription of the SMemb promoter, and cAMPresponsive element (CRE) located at -481 bp within the promoter was critical for Hex responsiveness. However, Hex failed to bind to CRE directly, thus the precise mechanisms whereby Hex activated the *SMemb* promoter are still unclear. Of interest, subsequent studies showed that Hex also induced expression of a subset of SMC differentiation marker genes

α

determined by EMSA. In addition, immunoprecipitation assays revealed the physical association between SRF and Hex. As such, the mechanisms whereby Hex induces SMC differentiation marker genes seem to be similar to those of Prx-1. However, results showing that Hex simultaneously activated expression of both SMC differentiation marker genes and those characteristic of phenotypically modulated SMCs are paradoxical, and further studies

Nkx-3.2 is also a homeodomain protein that regulates expression of SMC differentiation marker genes (Nishida et al., 2002). It has been demonstrated that a triad of SRF, GATA-6, and Nkx-3.2 formed a complex with their corresponding *cis*-elements and cooperatively

*caldesmon*. Because co-localization of GATA-6, Nkx-3.2, and SRF was exclusively observed in SMCs, SMC-specific gene expression does not appear to be the result of any single transcription factor that is unique to SMCs, but rather is due to unique combinatorial interactions of factors that may be expressed in multiple cell types but only found together

Furthermore, we recently identified Pitx2 as a homeodomain protein which is required for the initial induction of SMC differentiation by using a subtraction hybridization screen (Shang et al., 2008). Over-expression of Pitx2 induced expression of CArG-dependent SMC differentiation marker genes, whereas knockdown of Pitx2 attenuated retinoic acid-induced differentiation of SMCs from undifferentiated SMC precursor cells. Furthermore, *Pitx2* knockout mouse embryos exhibited impaired induction of SMC differentiation markers in the dorsal aorta and branchial arch arteries. We identified three mechanisms for Pitx2 induced transcription of SMC differentiation marker genes (Figure 3). First, Pitx2 bound to its consensus TAATC(C/T) element in the promoter region of SMC differentiation marker genes. Second, Pitx2 physically associated with SRF. Third, Pitx2 mediated exchange of HDACs with p300 to increase acetylation levels of histone H4 at the SMC promoters. These results provide compelling evidence that Pitx2 plays a critical role in the induction of SMC differentiation during the early embryogenesis. Further studies are needed to determine if Pitx2 also contributes to the pathogenesis of vascular diseases including atherosclerosis.

As such, several homeodomain proteins are involved in the regulation of CArG-SRF dependent SMC differentiation marker gene expression, and some of the mechanisms appear to be mediated by common pathways. Further studies are needed to clarify the temporal and spatial roles of each of these homeodomain proteins in SMC differentiation.

, but not *SM-MHC* and *h1-calponin* (Oyama et al., 2004).

gene in a CArG-dependent manner, and it

α

α

*1-integrin*, *SM22*

promoter, as

α, and

including *SM* 

in SMCs.

α

*-actin* and *SM22*

Hex induced the transcription of the *SM22*

α

transactivated SMC differentiation marker genes including

enhanced the binding of SRF to CArG-near element within the *SM22*

are clearly needed to precisely define the pathophysiological role of Hex in SMCs.

GATA proteins are a family of zinc finger transcription factors, and play essential roles in development through their interaction with a DNA consensus element, "WGATAR" (Molkentin, 2000). Six GATA transcription factors have been identified in vertebrates, and GATA-4, GATA-5, and GATA-6 are thought to be involved in the formation of the heart, gut, and vessels. During the early murine embryonic development, expression patterns of GATA-6 and GATA-4 were similar, with expression being detected in the precardiac mesoderm, the embryonic heart tube, and the primitive gut (Morrisey et al., 1996). However, during the late development, GATA-6 became the only GATA factor to be expressed in vascular SMCs. Knockout of the *GATA-6* gene in mice resulted in embryonic lethality between embryonic day 6.5 and 7.5, precluding the evaluation of the role of GATA-6 in SMC differentiation and maturation (Morrisey et al., 1998).

As described in a previous section, GATA-6 has shown to interact with SRF and Nkx-3.2 and to induce SMC differentiation marker gene expression (Morrisey et al., 1998; Nishida et al., 2002). *GATA-6* expression in SMCs was rapidly downregulated after vascular injury in rat carotid arteries, and adenovirus-mediated transfer of GATA-6 to the vessel wall after the balloon injury partially inhibited the formation of intimal thickening and reversed the downregulation of SMC differentiation marker genes including *SM* α*-actin* and *SM-MHC* (Mano et al., 1999). These results suggest the important role of GATA-6 in regulating SMC

Molecular Control of Smooth Muscle Cell Differentiation

(Yoshida et al., 2008).

*actin*, *SM-MHC*, *SM22*

differentiation.

**5.4 PIAS-1** 

α

Marker Genes by Serum Response Factor and Its Interacting Proteins 35

Fig. 4. Conditional knockout of the *Klf4* gene in mice accelerates neointimal formation following vascular injury. Klf4 is a potent repressor of SMC differentiation marker genes. Interestingly, conditional knockout of the *Klf4* gene in mice delays downregulation of SMC differentiation markers, but also accelerates neointimal formation after vascular injury

it has been reported that CRP1 and CRP2 are also able to function as transcriptional cofactors (Chang et al., 2003). Over-expression of three factors, SRF, GATA-6, and CRP1/CRP2 strongly activated the transcription of SMC differentiation marker genes including *SM* 

CRP1/2 interacted with SRF, and that the C-terminal LIM domain of CRP1/2 interacted with GATA-6, and that SRF and GATA-6 also interacted each other. These results suggest a critical role of CRP1/2 in organizing multiprotein complexes onto the SMC promoters for SMC differentiation. However, it is still unclear how CRP1 and CRP2 are translocated from the cytoplasm to the nucleus and what signaling pathways control their nuclear localization. Moreover, there is a lack of evidence that these factors play a role in control of SMC differentiation marker gene expression *in vivo* in SMCs. Indeed, results of recent studies showed that SMC differentiation in *CRP1* knockout mice or *CRP2* knockout mice appeared to be normal, although neointimal formation was altered after vascular injury (Lilly et al., 2010; Wei et al., 2005). Results raised a question as to the role of CRP1/2 in SMC

Results of previous studies showed that over-expression of class I basic Helix-Loop-Helix

promoter-enhancer in BALBc/3T3 cells (Kumar et al., 2003). However, direct interaction between E2-2 and SRF was undetectable by EMSA using the recombinant proteins. We isolated PIAS-1 (protein inhibitor of activated STAT-1) as an interacting protein for E2-2 by a yeast two-hybrid screen (Kawai-Kowase et al., 2005). We also found that PIAS-1 interacted with SRF, suggesting that PIAS-1 works as a bridging molecule between E2-2 and SRF. Interestingly, PIAS-1 belongs to a family of E3 ligases which promote SUMO modifications of target proteins (Schmidt & Müller, 2002). Indeed, recent studies showed that transcription factors involved in SMC differentiation, such as myocardin and Klf4, were sumoylation

proteins, E2-2, and SRF exhibited a synergistic effect on the transcription of the *SM* 

, *h1-calponin*, and *h-caldesmon*. The N-terminal LIM domain of

α*-*

α*-actin*

differentiation. Of interest, results of studies (Yin & Herring, 2005) showed that GATA-6 increased the transcriptional activity of the *SM* α*-actin* and *SM-MHC* genes, whereas it reduced the transcriptional activity of the *telokin* gene. They found that the GATA-6 binding site was located adjacent to CArG element in the *telokin* promoter and that over-expression of GATA-6 interfered the interaction between myocardin and SRF by mammalian twohybrid assays. However, it is unclear why GATA-6 has positive and negative effects on CArG-dependent SMC differentiation marker genes. It is possible that these opposite effects are due to the number of CArG elements or the distance between the GATA-6 binding site and the CArG element. Further studies are needed to test these possibilities.

#### **5.2 Klf4**

Klf4 is a member of Krüppel-like transcriptional factors that have recently received increased attention. Previously, Klf4 was identified as a binding factor for the transforming growth factor-β1 control element (TCE) found in the promoter region of the *SM* α*-actin* and *SM22*α genes, based on a yeast one-hybrid screen (Adam et al., 2000). Klf4 exhibited a profound inhibitory effect on expression of SMC differentiation marker genes via a TCEdependent and a CArG-SRF-dependent manner (Liu et al., 2003, 2005). For example, adenovirus-mediated over-expression of Klf4 repressed endogenous expression of *SM* α*actin* and *SM-MHC* genes, as well as expression of *myocardin*, in cultured SMCs as measured by real-time reverse transcription-PCR (Liu et al., 2005). In addition, over-expression of Klf4 completely abolished myocardin-induced activation of SMC differentiation marker genes. Co-immunoprecipitation assays revealed that Klf4 physically interacted with SRF, and chromatin immunoprecipitation assays showed that over-expression of Klf4 markedly reduced the binding of SRF to CArG elements on the *SM* α*-actin* promoter in intact chromatin of cultured SMCs (Liu et al., 2005). Moreover, PDGF-BB treatment induced *Klf4* mRNA expression in cultured SMCs, and siRNA-induced suppression of Klf4 partially blocked PDGF-BB-induced suppression of SMC differentiation marker genes (Liu et al., 2005). Of significant interest, we demonstrated that conditional knockout of the *Klf4* gene in mice exhibited a delay in suppression of SMC differentiation markers, and an enhanced neointimal formation following vascular injury (Figure 4) (Yoshida et al., 2008b). Additionally, we showed that Klf4, Elk-1, and HDACs cooperatively suppress oxidized phospholipid-induced suppression of SMC differentiation marker genes in cultured SMCs (Yoshida et al., 2008a). Taken together, these results suggest that Klf4 plays a key role in mediating phenotypic switching of SMCs.

#### **5.3 Cysteine-rich LIM-only proteins, CRP1 and CRP2**

The members of the cysteine-rich LIM-only protein (CRP) family, CRP1 and CRP2, are expressed predominantly in SMCs and contain two LIM domains in the structure (Henderson et al., 1999; Jain et al., 1996). It is known that the functions of LIM domains are to mediate protein-protein interactions, to target proteins to distinct subcellular locations, and to mediate assembly of multimeric protein complexes. One of the functions of CRP1 and CRP2 is to interact with both the actin crosslinking protein, α-actinin, and the adhesion plaque protein, zyxin, and to regulate the stability and structure of adhesion complexes (Arber & Caroni, 1996; Schmeichel & Beckerle, 1994). In addition to such a cytoplasmic role,

Fig. 4. Conditional knockout of the *Klf4* gene in mice accelerates neointimal formation following vascular injury. Klf4 is a potent repressor of SMC differentiation marker genes. Interestingly, conditional knockout of the *Klf4* gene in mice delays downregulation of SMC differentiation markers, but also accelerates neointimal formation after vascular injury (Yoshida et al., 2008).

it has been reported that CRP1 and CRP2 are also able to function as transcriptional cofactors (Chang et al., 2003). Over-expression of three factors, SRF, GATA-6, and CRP1/CRP2 strongly activated the transcription of SMC differentiation marker genes including *SM* α*actin*, *SM-MHC*, *SM22*α, *h1-calponin*, and *h-caldesmon*. The N-terminal LIM domain of CRP1/2 interacted with SRF, and that the C-terminal LIM domain of CRP1/2 interacted with GATA-6, and that SRF and GATA-6 also interacted each other. These results suggest a critical role of CRP1/2 in organizing multiprotein complexes onto the SMC promoters for SMC differentiation. However, it is still unclear how CRP1 and CRP2 are translocated from the cytoplasm to the nucleus and what signaling pathways control their nuclear localization. Moreover, there is a lack of evidence that these factors play a role in control of SMC differentiation marker gene expression *in vivo* in SMCs. Indeed, results of recent studies showed that SMC differentiation in *CRP1* knockout mice or *CRP2* knockout mice appeared to be normal, although neointimal formation was altered after vascular injury (Lilly et al., 2010; Wei et al., 2005). Results raised a question as to the role of CRP1/2 in SMC differentiation.

## **5.4 PIAS-1**

34 The Cardiovascular System – Physiology, Diagnostics and Clinical Implications

differentiation. Of interest, results of studies (Yin & Herring, 2005) showed that GATA-6

reduced the transcriptional activity of the *telokin* gene. They found that the GATA-6 binding site was located adjacent to CArG element in the *telokin* promoter and that over-expression of GATA-6 interfered the interaction between myocardin and SRF by mammalian twohybrid assays. However, it is unclear why GATA-6 has positive and negative effects on CArG-dependent SMC differentiation marker genes. It is possible that these opposite effects are due to the number of CArG elements or the distance between the GATA-6 binding site

Klf4 is a member of Krüppel-like transcriptional factors that have recently received increased attention. Previously, Klf4 was identified as a binding factor for the transforming

profound inhibitory effect on expression of SMC differentiation marker genes via a TCEdependent and a CArG-SRF-dependent manner (Liu et al., 2003, 2005). For example, adenovirus-mediated over-expression of Klf4 repressed endogenous expression of *SM* 

*actin* and *SM-MHC* genes, as well as expression of *myocardin*, in cultured SMCs as measured by real-time reverse transcription-PCR (Liu et al., 2005). In addition, over-expression of Klf4 completely abolished myocardin-induced activation of SMC differentiation marker genes. Co-immunoprecipitation assays revealed that Klf4 physically interacted with SRF, and chromatin immunoprecipitation assays showed that over-expression of Klf4 markedly

chromatin of cultured SMCs (Liu et al., 2005). Moreover, PDGF-BB treatment induced *Klf4* mRNA expression in cultured SMCs, and siRNA-induced suppression of Klf4 partially blocked PDGF-BB-induced suppression of SMC differentiation marker genes (Liu et al., 2005). Of significant interest, we demonstrated that conditional knockout of the *Klf4* gene in mice exhibited a delay in suppression of SMC differentiation markers, and an enhanced neointimal formation following vascular injury (Figure 4) (Yoshida et al., 2008b). Additionally, we showed that Klf4, Elk-1, and HDACs cooperatively suppress oxidized phospholipid-induced suppression of SMC differentiation marker genes in cultured SMCs (Yoshida et al., 2008a). Taken together, these results suggest that Klf4 plays a key role in

The members of the cysteine-rich LIM-only protein (CRP) family, CRP1 and CRP2, are expressed predominantly in SMCs and contain two LIM domains in the structure (Henderson et al., 1999; Jain et al., 1996). It is known that the functions of LIM domains are to mediate protein-protein interactions, to target proteins to distinct subcellular locations, and to mediate assembly of multimeric protein complexes. One of the functions of CRP1 and CRP2 is to interact with both the actin crosslinking protein, α-actinin, and the adhesion plaque protein, zyxin, and to regulate the stability and structure of adhesion complexes (Arber & Caroni, 1996; Schmeichel & Beckerle, 1994). In addition to such a cytoplasmic role,

genes, based on a yeast one-hybrid screen (Adam et al., 2000). Klf4 exhibited a

and the CArG element. Further studies are needed to test these possibilities.

growth factor-β1 control element (TCE) found in the promoter region of the *SM* 

reduced the binding of SRF to CArG elements on the *SM* 

mediating phenotypic switching of SMCs.

**5.3 Cysteine-rich LIM-only proteins, CRP1 and CRP2** 

α

*-actin* and *SM-MHC* genes, whereas it

α

α

*-actin* promoter in intact

*-actin* and

α*-*

increased the transcriptional activity of the *SM* 

**5.2 Klf4** 

*SM22*α

> Results of previous studies showed that over-expression of class I basic Helix-Loop-Helix proteins, E2-2, and SRF exhibited a synergistic effect on the transcription of the *SM* α*-actin* promoter-enhancer in BALBc/3T3 cells (Kumar et al., 2003). However, direct interaction between E2-2 and SRF was undetectable by EMSA using the recombinant proteins. We isolated PIAS-1 (protein inhibitor of activated STAT-1) as an interacting protein for E2-2 by a yeast two-hybrid screen (Kawai-Kowase et al., 2005). We also found that PIAS-1 interacted with SRF, suggesting that PIAS-1 works as a bridging molecule between E2-2 and SRF. Interestingly, PIAS-1 belongs to a family of E3 ligases which promote SUMO modifications of target proteins (Schmidt & Müller, 2002). Indeed, recent studies showed that transcription factors involved in SMC differentiation, such as myocardin and Klf4, were sumoylation

Molecular Control of Smooth Muscle Cell Differentiation

**7. Acknowledgments** 

Science Foundation.

**8. References** 

Marker Genes by Serum Response Factor and Its Interacting Proteins 37

This work was supported in part by Keio Gijuku Academic Development Funds and Takeda

Adam, P.J.; Regan, C.P.; Hautmann, M.B. & Owens, G.K. (2000) Positive- and negative-

Arber, S. & Caroni, P. (1996) Specificity of single LIM motifs in targeting and LIM/LIM

Arsenian, S.; Weinhold, B.; Oelgeschläger, M.; Rüther, U. & Nordheim, A. (1998) Serum

Bergwerff, M.; Gittenberger-de Groot, A.C.; Wisse, L.J.; DeRuiter, M.C.; Wessels, A.; Martin,

Blank, R.S.; Swartz, E.A.; Thompson, M.M.; Olson, E.N. & Owens, G.K. (1995) A retinoic

Buchwalter, G.; Gross, C. & Wasylyk, B. (2004) Ets ternary complex transcription factors.

Cao, D.; Wang, Z.; Zhang, C.L.; Oh, J.; Xing, W.; Li, S. et al. (2005) Modulation of smooth

Cen, B.; Selvaraj, A.; Burgess, R.C.; Hitzler, J.K.; Ma, Z.; Morris, S.W. et al. (2003)

Chang, D.F.; Belaguli, N.S.; Iyer, D.; Roberts, W.B.; Wu, S.P.; Dong, X.R. et al. (2003)

Chang, P.S.; Li, L.; McAnally, J. & Olson, E.N. (2001) Muscle specificity encoded by specific

Chen, J.; Kitchen, C.M.; Streb, J.W. & Miano, J.M. (2002) Myocardin: a component of a

Creemers, E.E.; Sutherland, L.B.; Oh, J.; Barbosa, A.C. & Olson, E.N. (2006) Coactivation of MEF2 by the SAP domain proteins myocardin and MASTR. Mol Cell. 23:83-96 Cserjesi, P.; Lilly, B.; Bryson, L.; Wang, Y.; Sassoon, D.A. & Olson, E.N. (1992) MHox: a

Doi, H.; Iso, T.; Yamazaki, M.; Akiyama, H.; Kanai, H.; Sato, H. et al. (2005) HERP1 inhibits

SRF binding to CArG box. *Arterioscler Thromb Vasc Biol*. 25:2328-2334

serum response factor-binding sites. *J Biol Chem*. 276:17206-17212

muscle creatine kinase enhancer. *Development*. 115:1087-1101

cells expresses smooth muscle characteristics. *Circ Res*. 76:742-749

deacetylases with myocardin. *Mol Cell Biol*. 25:364-376

differentiation cofactors. *Dev Cell*. 4:107-118

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acting Krüppel-like transcription factors bind a transforming growth factor β control element required for expression of the smooth muscle cell differentiation

response factor is essential for mesoderm formation during mouse embryogenesis.

J.F. et al. (2000) Loss of function of the *Prx1* and *Prx2* homeobox genes alters architecture of the great elastic arteries and ductus arteriosus. *Virchows Arch*.

acid-induced clonal cell line derived from multipotential P19 embryonal carcinoma

muscle gene expression by association of histone acetyltransferases and

Megakaryoblastic leukemia 1, a potent transcriptional coactivator for serum response factor (SRF), is required for serum induction of SRF target genes. *Mol Cell* 

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mesodermally restricted homeodomain protein that binds an essential site in the

myocardin-induced vascular smooth muscle cell differentiation by interering with

targets of PIAS-1. Myocardin sumoylation by PIAS-1 transactivated cardiogenic genes in 10T1/2 fibroblasts (Wang et al., 2007), whereas sumoylation of Klf4 by PIAS-1 promoted transforming growth factor-β induced activation of SM α-actin expression in SMCs (Kawai-Kowase et al., 2009). Further studies are needed to determine effects of *PIAS-1* knockout on SMC differentiation as well as phenotypic switching of SMCs.

## **6. Conclusion and perspectives**

As discussed above, it is clear that the CArG-SRF complex plays a central role in the regulation of SMC differentiation marker gene expression. However, it is also clear that expression of SMC differentiation marker genes is not controlled by the CArG-SRF complex alone, nor by any single transcription factor that is expressed exclusively in SMCs. Rather, SMC-selective gene expression appears to be mediated by complex combinatorial interactions of multiple transcription factors and co-factors, including some that are ubiquitously expressed like SRF and PIAS-1, as well as others that are selective for SMCs like myocardin, Prx-1, CRP-1/2, and GATA-6. In addition to the transcription factors described above, several novel factors, including Fhl2 (Philippar et al., 2004), HERP1 (Doi et al., 2005) and lupaxin (Sundberg-Smith et al., 2008), have also been identified as factors interacting with SRF.

However, our knowledge is immature regarding the overall connection among multiple transcription factors and co-factors that can modify the activity of SRF. Most of studies analyzing the protein-protein interaction thus far have been focused on the relationship among two or three proteins. However, a number of factors should be coordinately regulated and interacted by a single environmental cue. It is of interest to determine whether all of SRF-interacting factors are simultaneously required for SMC differentiation marker gene expression or these factors independently contribute to SMC differentiation marker gene expression in time- and position-specific manner. Thus, in the long term, future studies in the SMC field are needed not only to screen out other key transcription factors, but also to map out the connection networks of these factors.

During the past decade, there is a tremendous progress in our understanding of the roles of chromatin modifying enzymes and chromatin structure in gene transcription in all cell types. Accumulating evidence indicates that the N-terminal tails of histones are the target of numerous modifications, including acetylation, methylation, phosphorylation, ubiqutination, and ADP ribosylation, and that these modifications control gene transcription (Fischle et al., 2003). However, this issue in the SMC field is obviously in its infancy. Thus far, only several transcription factors have been reported to be involved in chromatin remodeling. Clearly, more detailed studies are required to determine the mechanisms whereby SRF and its interacting factors coordinately contribute to chromatin remodeling.

Finally, although much progress has been made in our understanding of the role of transcription factors in the control of SMC differentiation marker gene expression, some of these studies are performed only in cultured SMCs or SM-like systems. Studies of these factors *in vivo* will provide more compelling information to enhance our knowledge about SMC differentiation and development.

## **7. Acknowledgments**

This work was supported in part by Keio Gijuku Academic Development Funds and Takeda Science Foundation.

## **8. References**

36 The Cardiovascular System – Physiology, Diagnostics and Clinical Implications

targets of PIAS-1. Myocardin sumoylation by PIAS-1 transactivated cardiogenic genes in 10T1/2 fibroblasts (Wang et al., 2007), whereas sumoylation of Klf4 by PIAS-1 promoted transforming growth factor-β induced activation of SM α-actin expression in SMCs (Kawai-Kowase et al., 2009). Further studies are needed to determine effects of *PIAS-1* knockout on

As discussed above, it is clear that the CArG-SRF complex plays a central role in the regulation of SMC differentiation marker gene expression. However, it is also clear that expression of SMC differentiation marker genes is not controlled by the CArG-SRF complex alone, nor by any single transcription factor that is expressed exclusively in SMCs. Rather, SMC-selective gene expression appears to be mediated by complex combinatorial interactions of multiple transcription factors and co-factors, including some that are ubiquitously expressed like SRF and PIAS-1, as well as others that are selective for SMCs like myocardin, Prx-1, CRP-1/2, and GATA-6. In addition to the transcription factors described above, several novel factors, including Fhl2 (Philippar et al., 2004), HERP1 (Doi et al., 2005) and lupaxin (Sundberg-Smith et al., 2008), have also been identified as factors

However, our knowledge is immature regarding the overall connection among multiple transcription factors and co-factors that can modify the activity of SRF. Most of studies analyzing the protein-protein interaction thus far have been focused on the relationship among two or three proteins. However, a number of factors should be coordinately regulated and interacted by a single environmental cue. It is of interest to determine whether all of SRF-interacting factors are simultaneously required for SMC differentiation marker gene expression or these factors independently contribute to SMC differentiation marker gene expression in time- and position-specific manner. Thus, in the long term, future studies in the SMC field are needed not only to screen out other key transcription factors,

During the past decade, there is a tremendous progress in our understanding of the roles of chromatin modifying enzymes and chromatin structure in gene transcription in all cell types. Accumulating evidence indicates that the N-terminal tails of histones are the target of numerous modifications, including acetylation, methylation, phosphorylation, ubiqutination, and ADP ribosylation, and that these modifications control gene transcription (Fischle et al., 2003). However, this issue in the SMC field is obviously in its infancy. Thus far, only several transcription factors have been reported to be involved in chromatin remodeling. Clearly, more detailed studies are required to determine the mechanisms whereby SRF and its interacting factors coordinately contribute to chromatin

Finally, although much progress has been made in our understanding of the role of transcription factors in the control of SMC differentiation marker gene expression, some of these studies are performed only in cultured SMCs or SM-like systems. Studies of these factors *in vivo* will provide more compelling information to enhance our knowledge about

SMC differentiation as well as phenotypic switching of SMCs.

but also to map out the connection networks of these factors.

**6. Conclusion and perspectives** 

interacting with SRF.

remodeling.

SMC differentiation and development.


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**3** 

*Trans* **Fatty Acids and Human Health** 

According to various studies, fats of animal and vegetable origins satisfy 22% to 42% of the daily energy demands of human beings (Srinivasan et al., 2006; Wagner et al., 2008; Willet, 2006). Some fats, and especially those that are hydrogenated, contain *trans* fatty acids (TFAs), i.e. unsaturated fatty acids with at least one double bond in a *trans* configuration (Craig-Schmidt, 2006). This *trans*-double-bond configuration results in a greater bond angle than for the *cis* configuration, thus producing a more extended fatty-acid carbon chain that is more similar to that of the saturated fatty acids (SFAs), rather than to that of the *cis*unsaturated double-bond-containing fatty acids (Fig. 1) (Moss, 2006; Oomen et al., 2001).

Fig. 1. Structure of different isomers of C16 (Willett, 2006)

**1. Introduction** 

Sebastjan Filip and Rajko Vidrih

 *Department of Food Science and Technology,* 

*Biotechnical Faculty,* 

 *Slovenia* 

 *University of Ljubljana,* 


## *Trans* **Fatty Acids and Human Health**

Sebastjan Filip and Rajko Vidrih

*Biotechnical Faculty, Department of Food Science and Technology, University of Ljubljana, Slovenia* 

#### **1. Introduction**

42 The Cardiovascular System – Physiology, Diagnostics and Clinical Implications

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C895

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formation in cysteine-rich protein 2-deficient mice in response to vascular injury.

recruitment of the mSin3A-histone deacetylase corepressor complex to the ETS

According to various studies, fats of animal and vegetable origins satisfy 22% to 42% of the daily energy demands of human beings (Srinivasan et al., 2006; Wagner et al., 2008; Willet, 2006). Some fats, and especially those that are hydrogenated, contain *trans* fatty acids (TFAs), i.e. unsaturated fatty acids with at least one double bond in a *trans* configuration (Craig-Schmidt, 2006). This *trans*-double-bond configuration results in a greater bond angle than for the *cis* configuration, thus producing a more extended fatty-acid carbon chain that is more similar to that of the saturated fatty acids (SFAs), rather than to that of the *cis*unsaturated double-bond-containing fatty acids (Fig. 1) (Moss, 2006; Oomen et al., 2001).

Fig. 1. Structure of different isomers of C16 (Willett, 2006)

*Trans* Fatty Acids and Human Health 45

et al., 2006), and lowering the intake of TFAs can also reduce the incidence of CHD (Willett, 2006). Estimates based on changes in plasma concentrations of LDL and high-density lipoprotein (HDL) indicate around a 4% reduction in CHD incidence, while based on epidemiological associations, when TFA intake is lowered by 2% (5 g/day), the estimates indicate a >20% reduction in CHD incidence (Katan, 2006; Moss, 2006). In The Netherlands, a major reduction in the TFA content of retail foods was achieved in the 1990s through the efforts of the industry and with minimal government intervention. Society pressure is also now helping to reduce the TFA content of 'fast foods'. This illustrates the feasibility of reducing TFAs in fast foods without increasing the saturated fats, with the daily intake kept

Comparison of the different recommendations for macronutrients in some European countries, for the World Health Organisation/ Food and Agriculture Organisation of the United Nations (WHO/FAO), and in the USA and Canada, are given in Table 1. Most of the recommendations are the same, or are in similar ranges. The recommendations for protein, however, are expressed differently, either as grams per day or grams per kilogram per day, and usually without any indication of a representative weight at each age to allow conversion of one to the other. The Joint FAO/WHO/United Nations University (UNU) Expert Consultation of 1985 (WHO, 1985) defined the protein requirement of an individual as "the lowest level of dietary protein intake that will balance the losses of nitrogen from the body in persons maintaining an energy balance at modest levels of physical activity". The human body can synthesise both SFAs and mono-unsaturated fatty acids (MUFAs) from acetate, whereas PUFAs (in both the n–6 linoleic acid and n–3 linolenic acid series) are required in the diet, and they are therefore known as essential fatty acids. These essential fatty acids are important for various cell-membrane functions, such as fluidity, permeability, activity of membrane-bound enzymes and receptors, and signal transduction. Linoleic and linolenic acids can be elongated and desaturated in the body, and transformed into biologically active substances, like prostaglandins, prostacyclins and leukotrienes. These substances participate in the regulation of blood pressure, renal function, blood coagulation, inflammatory and immunological reactions, and many other functions (Nordic Nutrition Recommendations, 2004). The DACH Reference Values for Nutrient Supply (DACH, 2000) for total fat intake in adults (not more than 30% of the energy intake) are related to light work, heavy muscle work (not more than 35% of energy intake) and extremely heavy work (not more than 40% of energy intake). SFAs should not exceed 10% of energy intake. PUFAs should provide about 7%, and up to 10% if SFAs provide more than 10% of energy intake. MUFAs should constitute the rest. TFAs should contribute not more than 1% of the daily energy. The ratio of n–6 linoleic acid to n–3 linolenic acid should be about 5:1 (WHO/FAO, 2002). These fatty acids compete for the metabolic enzymes, and it is therefore important to maintain a balance between them (Nordic Nutrition Recommendations, 2004). The Nordic Nutrition Recommendations indicate the limiting of the intake of SFAs plus TFAs to about 10% of the daily energy and the total fat intake to 30% of the daily energy (25%-30%) (Filip et al., 2010). The recommendations for carbohydrate intake are from 50%of the daily energy in the DACH (2000) reference values, to 55% (50%-60%) in the Nordic Nutrition Recommendations (2004), 55%-75% by WHO/FAO, and 45%-65% in the USA/ Canada

as low as possible, to minimise the health risks (Stender et al., 2006).

recommendations, as detailed in Table 1.

Fat is a thus major source of energy for the body, and it also aids in the absorption of vitamins A, D, E and K, and of the carotenoids. Both animal-derived and plant-derived food products contain fat, and when eaten in moderation, fat is important for correct growth and development, and for the maintenance of good health. As a food ingredient, fat provides taste, consistency and stability, and helps us to feel 'full'. In addition, parents should be aware that fats are an especially important source of calories and nutrients for infants and toddlers (up to 2 years of age), who have the highest energy needs per unit body weight of any age group.

However, SFAs and TFAs raise low-density lipoprotein (LDL; or 'bad') cholesterol levels in the blood, thereby increasing the risk of heart disease. Indeed, prospective epidemiological studies and case-control studies support a major role for TFAs in the risk of cardiovascular disease, and therefore dietary cholesterol can also contribute to heart disease (see below). Unsaturated fats, which can be mono-unsaturated or polyunsaturated, do not raise LDL cholesterol and are beneficial to health when consumed in moderation.

Hydrogenated oils tend to have a higher TFA content than oils that do not contain hydrogenated fats. In the partially hydrogenated soybean oil, which is the major source of TFAs worldwide, the main isomer is *trans*-10 C18:1. In the European countries with the highest TFA intake (The Netherlands and Norway), consumption of partially hydrogenated fish oils was common until the mid-1990s, after which they largely disappeared from the dietary fat intake. These partially hydrogenated fish oils included a variety of very-long-chain TFAs. Recent findings from Asian countries (India and Iran) have indicated a very high intake of TFAs from partially hydrogenated soybean oil (4% of energy). Thus, TFAs appear to be a particular problem in developing countries where soybean oil is used.

Formation of these *trans* double bonds thus impacts on the physical properties of a fatty acid. Fatty acids that contain a *trans* double bond have the potential for closer packing and alignment of their acyl chains, which will result in decreased molecular mobility (Willett, 2006). Therefore, the oil fluidity will be reduced when compared to that of fatty acids that contain a *cis* double bond. Partial hydrogenation of unsaturated oils results in the isomerisation of some of the remaining double bonds and the migration of others, producing an increase in the TFA content and a hardening of the fat. It has been shown that foods that contain hydrogenated oils tend to have a higher TFA content than those that do not contain hydrogenated oils (Moss, 2006; Oomen et al., 2001). Nevertheless, the hydrogenation of oils, such as corn oil, can result in both *cis* and *trans* double bonds, which are generally located anywhere between carbon 4 and carbon 16 of the fatty acids. One of the major TFAs is elaidic acid (*trans*-9 C18:1), although during hydrogenation of polyunsaturated fatty acids (PUFAs), small amounts of several other TFAs are produced, including: *trans*-9,*cis*-12 C18:2; *cis*-9,*trans*-12 C18:2; *cis*-9,*cis*-12,*trans*-15 C18:3; and *cis*-5,*cis*-8,*cis*-11,*cis*-14,*trans*-17 C20:5 (Craig-Schmidt, 2006; Wagner et al., 2008). Conversely, one way to produce 'zero' levels of TFAs is through the *trans*-esterification reaction between vegetable oils and solid fatty acids, like C8:0, C12:0, C14:0 and C16:0.

Correlations between high intake of industrially produced TFAs (IP-TFAs) and increased risk of coronary heart disease (CHD) have been reported (Stender et al., 2006; Tarrago-Trani

Fat is a thus major source of energy for the body, and it also aids in the absorption of vitamins A, D, E and K, and of the carotenoids. Both animal-derived and plant-derived food products contain fat, and when eaten in moderation, fat is important for correct growth and development, and for the maintenance of good health. As a food ingredient, fat provides taste, consistency and stability, and helps us to feel 'full'. In addition, parents should be aware that fats are an especially important source of calories and nutrients for infants and toddlers (up to 2 years of age), who have the highest energy needs per unit body weight of

However, SFAs and TFAs raise low-density lipoprotein (LDL; or 'bad') cholesterol levels in the blood, thereby increasing the risk of heart disease. Indeed, prospective epidemiological studies and case-control studies support a major role for TFAs in the risk of cardiovascular disease, and therefore dietary cholesterol can also contribute to heart disease (see below). Unsaturated fats, which can be mono-unsaturated or polyunsaturated, do not raise LDL

Hydrogenated oils tend to have a higher TFA content than oils that do not contain hydrogenated fats. In the partially hydrogenated soybean oil, which is the major source of TFAs worldwide, the main isomer is *trans*-10 C18:1. In the European countries with the highest TFA intake (The Netherlands and Norway), consumption of partially hydrogenated fish oils was common until the mid-1990s, after which they largely disappeared from the dietary fat intake. These partially hydrogenated fish oils included a variety of very-long-chain TFAs. Recent findings from Asian countries (India and Iran) have indicated a very high intake of TFAs from partially hydrogenated soybean oil (4% of energy). Thus, TFAs appear to be a particular problem in developing countries where

Formation of these *trans* double bonds thus impacts on the physical properties of a fatty acid. Fatty acids that contain a *trans* double bond have the potential for closer packing and alignment of their acyl chains, which will result in decreased molecular mobility (Willett, 2006). Therefore, the oil fluidity will be reduced when compared to that of fatty acids that contain a *cis* double bond. Partial hydrogenation of unsaturated oils results in the isomerisation of some of the remaining double bonds and the migration of others, producing an increase in the TFA content and a hardening of the fat. It has been shown that foods that contain hydrogenated oils tend to have a higher TFA content than those that do not contain hydrogenated oils (Moss, 2006; Oomen et al., 2001). Nevertheless, the hydrogenation of oils, such as corn oil, can result in both *cis* and *trans* double bonds, which are generally located anywhere between carbon 4 and carbon 16 of the fatty acids. One of the major TFAs is elaidic acid (*trans*-9 C18:1), although during hydrogenation of polyunsaturated fatty acids (PUFAs), small amounts of several other TFAs are produced, including: *trans*-9,*cis*-12 C18:2; *cis*-9,*trans*-12 C18:2; *cis*-9,*cis*-12,*trans*-15 C18:3; and *cis*-5,*cis*-8,*cis*-11,*cis*-14,*trans*-17 C20:5 (Craig-Schmidt, 2006; Wagner et al., 2008). Conversely, one way to produce 'zero' levels of TFAs is through the *trans*-esterification reaction between

Correlations between high intake of industrially produced TFAs (IP-TFAs) and increased risk of coronary heart disease (CHD) have been reported (Stender et al., 2006; Tarrago-Trani

cholesterol and are beneficial to health when consumed in moderation.

vegetable oils and solid fatty acids, like C8:0, C12:0, C14:0 and C16:0.

any age group.

soybean oil is used.

et al., 2006), and lowering the intake of TFAs can also reduce the incidence of CHD (Willett, 2006). Estimates based on changes in plasma concentrations of LDL and high-density lipoprotein (HDL) indicate around a 4% reduction in CHD incidence, while based on epidemiological associations, when TFA intake is lowered by 2% (5 g/day), the estimates indicate a >20% reduction in CHD incidence (Katan, 2006; Moss, 2006). In The Netherlands, a major reduction in the TFA content of retail foods was achieved in the 1990s through the efforts of the industry and with minimal government intervention. Society pressure is also now helping to reduce the TFA content of 'fast foods'. This illustrates the feasibility of reducing TFAs in fast foods without increasing the saturated fats, with the daily intake kept as low as possible, to minimise the health risks (Stender et al., 2006).

Comparison of the different recommendations for macronutrients in some European countries, for the World Health Organisation/ Food and Agriculture Organisation of the United Nations (WHO/FAO), and in the USA and Canada, are given in Table 1. Most of the recommendations are the same, or are in similar ranges. The recommendations for protein, however, are expressed differently, either as grams per day or grams per kilogram per day, and usually without any indication of a representative weight at each age to allow conversion of one to the other. The Joint FAO/WHO/United Nations University (UNU) Expert Consultation of 1985 (WHO, 1985) defined the protein requirement of an individual as "the lowest level of dietary protein intake that will balance the losses of nitrogen from the body in persons maintaining an energy balance at modest levels of physical activity". The human body can synthesise both SFAs and mono-unsaturated fatty acids (MUFAs) from acetate, whereas PUFAs (in both the n–6 linoleic acid and n–3 linolenic acid series) are required in the diet, and they are therefore known as essential fatty acids. These essential fatty acids are important for various cell-membrane functions, such as fluidity, permeability, activity of membrane-bound enzymes and receptors, and signal transduction. Linoleic and linolenic acids can be elongated and desaturated in the body, and transformed into biologically active substances, like prostaglandins, prostacyclins and leukotrienes. These substances participate in the regulation of blood pressure, renal function, blood coagulation, inflammatory and immunological reactions, and many other functions (Nordic Nutrition Recommendations, 2004). The DACH Reference Values for Nutrient Supply (DACH, 2000) for total fat intake in adults (not more than 30% of the energy intake) are related to light work, heavy muscle work (not more than 35% of energy intake) and extremely heavy work (not more than 40% of energy intake). SFAs should not exceed 10% of energy intake. PUFAs should provide about 7%, and up to 10% if SFAs provide more than 10% of energy intake. MUFAs should constitute the rest. TFAs should contribute not more than 1% of the daily energy. The ratio of n–6 linoleic acid to n–3 linolenic acid should be about 5:1 (WHO/FAO, 2002). These fatty acids compete for the metabolic enzymes, and it is therefore important to maintain a balance between them (Nordic Nutrition Recommendations, 2004). The Nordic Nutrition Recommendations indicate the limiting of the intake of SFAs plus TFAs to about 10% of the daily energy and the total fat intake to 30% of the daily energy (25%-30%) (Filip et al., 2010). The recommendations for carbohydrate intake are from 50%of the daily energy in the DACH (2000) reference values, to 55% (50%-60%) in the Nordic Nutrition Recommendations (2004), 55%-75% by WHO/FAO, and 45%-65% in the USA/ Canada recommendations, as detailed in Table 1.

*Trans* Fatty Acids and Human Health 47

contain, TFAs (Borra et al., 2007). Then in late 2006, New York City became the first major city in the United States to pass a regulation limiting IP-TFAs in restaurants. This has served as a model for others to follow, with these regulations including: a maximum level per serving size of 0.5 g TFAs; a distinction between frying and baking, with a phased-in implementation; a help centre to assist restaurants to make the switch to more healthy options; and plans to evaluate the regulation and its impact on CHD (Borra et al., 2007).

Accurate quantification of C18:1 TFAs in food products is thus an important issue, with policies recently implemented in different countries to limit their consumption and their occurrence in food products because of their relationship with CHD (Carriquiry et al., 2008;

Margarine was invented in 1869 by Hippolyte Mège Mouriès, a French food research chemist, in response to a request by Napoleon III for a wholesome alternative to butter. It is not entirely clear whether the primary aim was the betterment of the working classes or the economics of the food supply to the French army. In the laboratory, Mège Mouriès solidified purified fat, after which the resulting substance was pressed in a thin cloth, which formed stearine and discharged oil. This oil formed the basis of the butter substitute. For the new product, Mège Mouriès used margaric acid, a fatty-acid component isolated in 1813 by the Frenchman Michel Eugène Chevreuil. While analysing the fatty acids that are the building blocks of fats, he singled out this one and named it margaric acid, because of the lustrous pearly drops that reminded him of the Greek word for pearls, i.e. margarites (Chen et al.,

In 1871, Mège Mouriès sold this know-how to the Dutch firm Jurgens, which is now part of Unilever. In the early days, margarine contained two types of fat: a large proportion of animal fat and a small proportion of vegetable fat. As time passed, the small vegetable-fat element increased, through two specific stages in the process. First, by improving the process of refining vegetable oils, use could be made of a greater variety of liquid oils and a higher proportion of solid vegetable fats. Secondly, through the development of processes for turning liquid oils into solid fats on a commercial scale, use could be made of larger

During the early years of this period, in the late 1800s, TFA intake from partially hydrogenated vegetable oils was minimal. Indeed, it was not until the late 1800s that the process of partial hydrogenation of oils was invented in Europe. These partially hydrogenated oils apparently entered the United States food supply by 1920. Although the rate of increase before 1950 is not completely clear, by 1950 the amount of IP-TFAs in the food supply was quite substantial. Partly because of economic effects during World War II, margarine production rose rapidly as a replacement for butter (Chen et al., 2007). Then during the 1960s, margarine became viewed as a healthy alternative to butter because of its absence of cholesterol and its low content of SFAs. Thus, consumption increased further, and so margarine, which was heavily hydrogenated at that time, became widespread in the food supply and was the major source of IP-TFAs. This phenomenon is illustrated in Figure 2. The total TFAs consumption was approximately 2% to 3% of the food energy. Since then, the sources of TFAs have changed, from mainly margarine to mainly deep-fried fast foods and commercially baked products,

although per capita, the intake has remained roughly the same (Willett, 2006).

Chen et al., 2007).

2008; Craig-Schmidt, 2006).

quantities of liquid vegetable oils (Filip, 2010).

**2. History** 


NNR, Nordic Nutrition Recommendations; DACH, Austria–Germany–Switzerland Reference Values for Nutrient Supply; WHO, World Health Organisation; FAO, Food and Agriculture Organisation of the United Nations; AMDR; acceptable macronutrient distribution; FAs, fatty acids; SFAs, saturated fatty acids; PUFAs, polyunsaturated fatty acids; TFA, *trans* fatty acids; MUFAs, mono-unsaturated fatty acids.

Table 1. Comparison of reference daily intakes for adults according to different recommendations around the World (Pavlovic et al., 2007)

As indicated above, prospective epidemiological studies and case-control studies using adipose-tissue analyses have confirmed a major role for TFAs in the risk of CHD. The magnitude of the association with CHD is considerably stronger than for SFAs, and it is stronger than that predicted for the effects of TFAs on LDL and HDL cholesterol (Katan, 2006; Tarrago-Trani et al., 2006). In this context, it needs to be considered that data for the Russian Federation show that every year 1,005 people per 100,000 of the population between 25 and 64 years of age die because of circulatory system diseases (WHO, 2008). As a consequence influence of TFAs on CHD, in 2003, the United States FDA issued a ruling that required food manufacturers to list the TFAs in the nutritional facts labels of all packaged food products (FDA, 2003), with the food industry being given until 1 January, 2006 to comply. Along with these growing health concerns about TFAs, this mandate led to marked changes in the fat and oil industries, with newer technologies developed to reduce the TFA contents of fats and oils used in the manufacture of food products. Conversely, given the labelling mandate and these technological advances, it is possible that food products traditionally considered to be sources of TFAs are now much lower in, or indeed do not

contain, TFAs (Borra et al., 2007). Then in late 2006, New York City became the first major city in the United States to pass a regulation limiting IP-TFAs in restaurants. This has served as a model for others to follow, with these regulations including: a maximum level per serving size of 0.5 g TFAs; a distinction between frying and baking, with a phased-in implementation; a help centre to assist restaurants to make the switch to more healthy options; and plans to evaluate the regulation and its impact on CHD (Borra et al., 2007).

Accurate quantification of C18:1 TFAs in food products is thus an important issue, with policies recently implemented in different countries to limit their consumption and their occurrence in food products because of their relationship with CHD (Carriquiry et al., 2008; Chen et al., 2007).

## **2. History**

46 The Cardiovascular System – Physiology, Diagnostics and Clinical Implications

**WHO/FAO (2002)** 

in SFAs 1 <1 <2 Minimise

**Euro Diet (2000)** 

**USA/Canada AMDR (2002)** 

25-38 (14 g/1000 kcal)

**DACH (2000)** 

(%) 30 (25-35) 30 15-30 <30 20-35 SFAs (%) ≤10 10 <10 <10 Minimise PUFAs (%) 5 (10) 7-10 6-10 - n-6 FAs (%) 4 (9) 2.5 5-8 4-8 5-10 (linoleic) n-3 FAs (%) 1 0.5 1-2 2 (linolenic) 0.6-1.2

MUFAs (%) 10-15 The rest of the total -

carbohydrates (%) 55 (50-60) 50 55-75 >55 45-65

(%) <10 30 <10 <25

(%) 15 (10-20) 8-10 10-15 - 10-35 Cholesterol (mg/day) 300 <300 Minimise

NNR, Nordic Nutrition Recommendations; DACH, Austria–Germany–Switzerland Reference Values for Nutrient Supply; WHO, World Health Organisation; FAO, Food and Agriculture Organisation of the United Nations; AMDR; acceptable macronutrient distribution; FAs, fatty acids; SFAs, saturated fatty acids; PUFAs, polyunsaturated fatty acids; TFA, *trans* fatty acids; MUFAs, mono-unsaturated fatty

As indicated above, prospective epidemiological studies and case-control studies using adipose-tissue analyses have confirmed a major role for TFAs in the risk of CHD. The magnitude of the association with CHD is considerably stronger than for SFAs, and it is stronger than that predicted for the effects of TFAs on LDL and HDL cholesterol (Katan, 2006; Tarrago-Trani et al., 2006). In this context, it needs to be considered that data for the Russian Federation show that every year 1,005 people per 100,000 of the population between 25 and 64 years of age die because of circulatory system diseases (WHO, 2008). As a consequence influence of TFAs on CHD, in 2003, the United States FDA issued a ruling that required food manufacturers to list the TFAs in the nutritional facts labels of all packaged food products (FDA, 2003), with the food industry being given until 1 January, 2006 to comply. Along with these growing health concerns about TFAs, this mandate led to marked changes in the fat and oil industries, with newer technologies developed to reduce the TFA contents of fats and oils used in the manufacture of food products. Conversely, given the labelling mandate and these technological advances, it is possible that food products traditionally considered to be sources of TFAs are now much lower in, or indeed do not

Fibre (g/day) 25-35 (3 g/MJ) (12.5 g/1000 kcal)

Salt (sodium) (g/day) 5–6 (2.3-2.7) <5 (2)

Table 1. Comparison of reference daily intakes for adults according to different

recommendations around the World (Pavlovic et al., 2007)

**Component NNR** 

TFAs (%) Included

Total energy from fat

Total energy from

Energy from sugars

Energy from proteins

acids.

**(2004)** 

Margarine was invented in 1869 by Hippolyte Mège Mouriès, a French food research chemist, in response to a request by Napoleon III for a wholesome alternative to butter. It is not entirely clear whether the primary aim was the betterment of the working classes or the economics of the food supply to the French army. In the laboratory, Mège Mouriès solidified purified fat, after which the resulting substance was pressed in a thin cloth, which formed stearine and discharged oil. This oil formed the basis of the butter substitute. For the new product, Mège Mouriès used margaric acid, a fatty-acid component isolated in 1813 by the Frenchman Michel Eugène Chevreuil. While analysing the fatty acids that are the building blocks of fats, he singled out this one and named it margaric acid, because of the lustrous pearly drops that reminded him of the Greek word for pearls, i.e. margarites (Chen et al., 2008; Craig-Schmidt, 2006).

In 1871, Mège Mouriès sold this know-how to the Dutch firm Jurgens, which is now part of Unilever. In the early days, margarine contained two types of fat: a large proportion of animal fat and a small proportion of vegetable fat. As time passed, the small vegetable-fat element increased, through two specific stages in the process. First, by improving the process of refining vegetable oils, use could be made of a greater variety of liquid oils and a higher proportion of solid vegetable fats. Secondly, through the development of processes for turning liquid oils into solid fats on a commercial scale, use could be made of larger quantities of liquid vegetable oils (Filip, 2010).

During the early years of this period, in the late 1800s, TFA intake from partially hydrogenated vegetable oils was minimal. Indeed, it was not until the late 1800s that the process of partial hydrogenation of oils was invented in Europe. These partially hydrogenated oils apparently entered the United States food supply by 1920. Although the rate of increase before 1950 is not completely clear, by 1950 the amount of IP-TFAs in the food supply was quite substantial. Partly because of economic effects during World War II, margarine production rose rapidly as a replacement for butter (Chen et al., 2007). Then during the 1960s, margarine became viewed as a healthy alternative to butter because of its absence of cholesterol and its low content of SFAs. Thus, consumption increased further, and so margarine, which was heavily hydrogenated at that time, became widespread in the food supply and was the major source of IP-TFAs. This phenomenon is illustrated in Figure 2. The total TFAs consumption was approximately 2% to 3% of the food energy. Since then, the sources of TFAs have changed, from mainly margarine to mainly deep-fried fast foods and commercially baked products, although per capita, the intake has remained roughly the same (Willett, 2006).

*Trans* Fatty Acids and Human Health 49

subcutaneous fat, because visceral deposits release free fatty acids directly into the portal vein (Bray, 2003). The fatty acid pattern carried to the portal circulation is of great importance, because different fatty acids show distinct atherogenicities, depending on the chain length and degree of unsaturation. Here, SFAs have been associated with increased cardio-metabolic risk, while n-3 and n-9 unsaturated fatty acids have been proposed as

In milk fat, TFAs are produced by anaerobic fermentation of PUFAs in the rumen of lactating cows (Destaillats et al., 2007; Fournier et al., 2006). This fermentation process is called biohydrogenation, and it results in TFAs that can be further metabolised in the mammary gland. Accurate estimations of fatty-acid compositions are vital not only for the definition of the nutrient composition of foods, but also to accurately determine treatment effects that can alter the fatty-acid composition of the foods (Ascherio, 2002; Burdge et al.,

There is a considerable overlap of TFA isomers in fats of ruminant origin and in partially hydrogenated vegetable oils, as they have many isomers in common. However, there are considerable differences in the amounts of individual TFAs in these sources. While there is evidence of unfavourable effects of TFAs from hydrogenated vegetable oils on LDL and other risk factors for atherosclerosis, at present it is not certain which of the component(s) of the TFAs created by chemical hydrogenation are responsible for these a negative metabolic effects (Ascherio, 2002). Prospective studies addressing the effects of TFA intake on CHD risk, where estimates of TFA intake were based on dietary protocols, have mostly been carried out in populations with a relatively low intake of dairy or ruminant TFAs (Pfeuffer & Schrezenmeir, 2006). Nevertheless, the biggest effects of fatty-acid composition and the nutritive quality of foods of animal origin, like meat and milk products, depend on the feed

These TFA-containing fats can be incorporated into both foetal and adult tissues, although the transfer rate through the placenta continues to be a contradictory subject. In preterm infants and healthy term babies, the *trans* isomers have been inversely correlated with infant birth mass (Koletzko & Müller, 1990). Maternal milk reflects precisely the mother's daily dietary intake of TFAs, with presence of 2% to 5% total TFAs in human milk. The levels of linoleic acid in human milk are increased by a high *trans* diet, although long-chain polyunsaturated TFAs remain mostly unaffected (Koletzko, 1992; Koletzko & Desci, 1994). Alterations in the maternal dietary intake of PUFAs cause similar changes in the PUFA content of their milk. Several investigations have shown that supplementation of the consumed fat with fish oils increases the amounts of C20:5n-3 and C22:6n-3 in the milk and in the maternal and infant erythrocyte lipids. Likewise, infant tissues incorporate the TFAs from the maternal milk, increasing the levels of linoleic acid and decreasing arachidonic acid and docosahexaenoic acid. This suggests an inhibitory effect of TFAs on the liver n-6 fatty-

protective agents against these alterations (Garaulet et al., 2011).

2005; Kummerow et al., 2004; Murrieta et al., 2003; Triantafillou et al., 2003).

**3.1 Studies in animals** 

quality and the health of the animals.

acid-desaturase activity (Jensen et al., 1992).

**3.2 Studies in humans** 

Fig. 2. Relative food energy supplied by the different fatty acids, and the predicted changes for the food industry and fat hydrogenation (Simopoulos, 2004)

After World War II, the process of making hydrogenated and hardened fats from cheaper sources of vegetable oils was widely adopted. Margarines were developed and marketed as alternatives to butter, and vegetable shortening increasingly replaced animal fats in cooking (Albers et al., 2008). However, as early as 1975, at what is now the University of Glamorgan in South Wales, a group of scientists led by Leo Thomas suspected that deaths from CHD were connected with this eating of partially hydrogenated fats. It is now generally accepted that TFAs are actually worse for health than the SFAs that they were designed to replace (Blake, 2009).

#### **3. Studies of** *trans* **fatty acids**

Increases in 'civilization diseases' in the developed world led scientists to investigate why this was happening. While there is enough food that is also cheaper and more accessible than ever before in the developed world, we are witnessing more and more overweight and obese populations. Modern populations have worse nutrition habits than ever before, except for some specific small social groups e.g. through religion, ecology and ethnic aspects (WHO, 2008). Obesity is a severe health issue that is characterised by fat accumulation and defined by means of the body mass index (BMI), as body weight [kg]/ (height [m])2. According to this index, different obesity levels have been described, ranging from overweight (BMI, 25.0-29.9), through obese (BMI, 30.0-40.0) to the most detrimental stage, morbid obesity (BMI, ≥40) (Garaulet et al., 2011). The relevance of this classification is that as the BMI increases, the morbidity and mortality risks also increase (Bray, 2003). Furthermore, regional fat accumulation is an important factor in the development of obesity-related alterations. It has been suggested that excess visceral fat is more detrimental than excess

Fig. 2. Relative food energy supplied by the different fatty acids, and the predicted changes

After World War II, the process of making hydrogenated and hardened fats from cheaper sources of vegetable oils was widely adopted. Margarines were developed and marketed as alternatives to butter, and vegetable shortening increasingly replaced animal fats in cooking (Albers et al., 2008). However, as early as 1975, at what is now the University of Glamorgan in South Wales, a group of scientists led by Leo Thomas suspected that deaths from CHD were connected with this eating of partially hydrogenated fats. It is now generally accepted that TFAs are actually worse for health than the SFAs that they were designed to replace

Increases in 'civilization diseases' in the developed world led scientists to investigate why this was happening. While there is enough food that is also cheaper and more accessible than ever before in the developed world, we are witnessing more and more overweight and obese populations. Modern populations have worse nutrition habits than ever before, except for some specific small social groups e.g. through religion, ecology and ethnic aspects (WHO, 2008). Obesity is a severe health issue that is characterised by fat accumulation and defined by means of the body mass index (BMI), as body weight [kg]/ (height [m])2. According to this index, different obesity levels have been described, ranging from overweight (BMI, 25.0-29.9), through obese (BMI, 30.0-40.0) to the most detrimental stage, morbid obesity (BMI, ≥40) (Garaulet et al., 2011). The relevance of this classification is that as the BMI increases, the morbidity and mortality risks also increase (Bray, 2003). Furthermore, regional fat accumulation is an important factor in the development of obesity-related alterations. It has been suggested that excess visceral fat is more detrimental than excess

for the food industry and fat hydrogenation (Simopoulos, 2004)

(Blake, 2009).

**3. Studies of** *trans* **fatty acids** 

subcutaneous fat, because visceral deposits release free fatty acids directly into the portal vein (Bray, 2003). The fatty acid pattern carried to the portal circulation is of great importance, because different fatty acids show distinct atherogenicities, depending on the chain length and degree of unsaturation. Here, SFAs have been associated with increased cardio-metabolic risk, while n-3 and n-9 unsaturated fatty acids have been proposed as protective agents against these alterations (Garaulet et al., 2011).

#### **3.1 Studies in animals**

In milk fat, TFAs are produced by anaerobic fermentation of PUFAs in the rumen of lactating cows (Destaillats et al., 2007; Fournier et al., 2006). This fermentation process is called biohydrogenation, and it results in TFAs that can be further metabolised in the mammary gland. Accurate estimations of fatty-acid compositions are vital not only for the definition of the nutrient composition of foods, but also to accurately determine treatment effects that can alter the fatty-acid composition of the foods (Ascherio, 2002; Burdge et al., 2005; Kummerow et al., 2004; Murrieta et al., 2003; Triantafillou et al., 2003).

There is a considerable overlap of TFA isomers in fats of ruminant origin and in partially hydrogenated vegetable oils, as they have many isomers in common. However, there are considerable differences in the amounts of individual TFAs in these sources. While there is evidence of unfavourable effects of TFAs from hydrogenated vegetable oils on LDL and other risk factors for atherosclerosis, at present it is not certain which of the component(s) of the TFAs created by chemical hydrogenation are responsible for these a negative metabolic effects (Ascherio, 2002). Prospective studies addressing the effects of TFA intake on CHD risk, where estimates of TFA intake were based on dietary protocols, have mostly been carried out in populations with a relatively low intake of dairy or ruminant TFAs (Pfeuffer & Schrezenmeir, 2006). Nevertheless, the biggest effects of fatty-acid composition and the nutritive quality of foods of animal origin, like meat and milk products, depend on the feed quality and the health of the animals.

#### **3.2 Studies in humans**

These TFA-containing fats can be incorporated into both foetal and adult tissues, although the transfer rate through the placenta continues to be a contradictory subject. In preterm infants and healthy term babies, the *trans* isomers have been inversely correlated with infant birth mass (Koletzko & Müller, 1990). Maternal milk reflects precisely the mother's daily dietary intake of TFAs, with presence of 2% to 5% total TFAs in human milk. The levels of linoleic acid in human milk are increased by a high *trans* diet, although long-chain polyunsaturated TFAs remain mostly unaffected (Koletzko, 1992; Koletzko & Desci, 1994). Alterations in the maternal dietary intake of PUFAs cause similar changes in the PUFA content of their milk. Several investigations have shown that supplementation of the consumed fat with fish oils increases the amounts of C20:5n-3 and C22:6n-3 in the milk and in the maternal and infant erythrocyte lipids. Likewise, infant tissues incorporate the TFAs from the maternal milk, increasing the levels of linoleic acid and decreasing arachidonic acid and docosahexaenoic acid. This suggests an inhibitory effect of TFAs on the liver n-6 fattyacid-desaturase activity (Jensen et al., 1992).

*Trans* Fatty Acids and Human Health 51

thousands of premature deaths in the United States alone (Mensink & Nestel, 2009). Although dramatic, this effect is substantially smaller than the increase in cardiovascular mortality associated with TFA intake in epidemiological studies, suggesting that other mechanisms are likely to contribute to the toxicity of TFAs (Ascherio, 2006). Thus, although there is accumulating evidence linking inflammatory proteins and other biomarkers to CHD, lipid concentrations in the blood remain one of the strongest and most consistent predictors of risk. Therefore, the LDL/HDL cholesterol ratio is probably the best marker to date for estimating the effects of TFAs on plasma lipids, which are most likely relevant to

Further rigorous randomised trials to establish the effects of hydrogenated fats and TFA intake on individual lipoprotein classes started in 1990, when a report from The Netherlands suggested that a diet enriched in elaidic acid (*trans*-9 C18:1) increases the total and LDL cholesterol concentrations and decreased HDL cholesterol concentrations, compared to a diet enriched in oleic acid. In contrast, enrichment of the diet with SFAs increases LDL cholesterol, but has no effect on HDL cholesterol, thus resulting in a smaller adverse change

In one study (Filip et al., 2011), the effects of natural antioxidants on formation of TFAs during heat treatment of sunflower oil was investigated. The data from the fatty acid analyses are summarized in Table 2. Here, the non-treated control sunflower oil had a 7.5% palmitic acid content, with 4.5% stearic acid, 25.0% oleic acid, and 60.5% linoleic acid, as is usual for the common (not high in oleic acid) sunflower oils; these data compare well with those of other studies (Sánchez-Gimeno et al., 2008; Bansal, Zhou, Tan, Neo, & Lo, 2009). This sunflower oil was purchased directly from a supplier of oils that are used mainly by small food enterprises (Zvijezda d.d., Zagreb, Croatia). The natural antioxidant extract of rosemary (*Rosmarinus officinalis* L.) that was added to this sunflower oil (SOR) was purchased directly from Vitiva d.d., Markovci, Slovenia (INOLENS4®; Product N° 301770; Batch N°. LAB. 09-779004), and had a carnosic acid content of 4.30%. Similarly, the lutein added to this sunflower oil (SOL) was from pelargonium (2.2% mixture), as obtained from Etol, d.o.o., Celje, Slovenia (NovaSoL® Lutein; Aquanova AG, Birkenweg 8-10, Germany).

The initial levels of the total TFAs in the samples was 0.91% (±0.01%). This compares with the range from 0.15% to 6.03% reported by Bansal et al. (2009) for TFAs in refined oils (soybean, corn, sunflower, high oleic sunflower, low erucic rapeseed and high erucic rapeseed oils). The aim in this study with the sunflower oil was to evaluate the effects of heat on this TFA composition of the oil when subjected to treatment representative of deepfat frying (185 ±5°C). Since sunflower oil is in common use for deep-fat frying, it is particularly important to know what species and levels of TFA isomers appear during such

In this study, we focussed mainly on these effects of heat on the TFAs with 18 carbon atoms, which were the most represented. Prior to the treatment, the content of *trans* C 18:1, t-9 was 0.67% (±0.08%). At the end of the heat treatment (120 h at 185 ±5°C), in the control sunflower oil the *trans* C 18:1, t-9 increased to 1.12% (±0.14%), in SOR, to 0.99% (±0.04%), and in SOL, to 0.91% (±0.01%). Within each treatment, these increases were significantly different from the

than in the case of elaidic acid (Mensink & Katan, 1993; Mensink & Nestel, 2009).

CHD incidence and mortality (Larqué & Zamora, 2001).

heat treatment (Filip et al., 2011; Martin et al., 2007).

**3.3 Studies of antioxidant effects** 

As opposed to blood and liver, the brain appears to be protected from TFA accumulation in experimental animals, although no data have yet been reported for newborn humans (Larqué, 2001). A significant interaction between diet and pregnancy was shown for the activities of Δ6-desaturase and glucose 6-phosphatase in liver microsomes: dietary TFAs decreased the activities of both of these enzymes, although only in pregnant rats (Larqué et al., 2000; Larqué & Zamora, 2000; Larqué et al., 2003). In Spain, TFAs in human milk were investigated by Boatella et al*.* (Boatella et al., 1993), and they showed that the average content of TFAs in 38 samples was 0.98% of the milk fatty acids. This value is lower than that for human milk from other developed countries, where consumption of hydrogenated fats is higher. In a study by Chen et al*.* (Chen et al., 1995) on TFAs in human milk in Canada, the mean total TFA content was 7.19% (±3.03%) of the total milk fatty acids, with a range from 0.10% to 17.15%.

The compelling data linking dietary TFAs to increased risk of CHD have originated from large, prospective, population-based studies, which included from 667 to 80,082 men and women across different age groups who were monitored for six to 20 years. This link has also been seen in controlled feeding trials (Oomen et al., 2001). Among these studies, there are: the United States Health Professional's follow-up study; the Finnish alpha-tocopherol, β-carotene Cancer Prevention Study; the United States nurse's health study (with 14-year and 20-year follow-up) (Willett, 2006); and the Dutch Zutphen elderly study (Oomen et al., 2001). These studies are consistent in their finding of a strong positive association between TFA intake and the risk of CHD. Interestingly, a weaker correlation between SFA intake and the risk of CHD also has been reported (Willettt, 2006).

The Zutphen elderly study included 667 men from 64 to 84 years of age who were free of CHD at baseline (Oomen et al., 2001). Dietary surveys were used to establish the food consumption patterns of the participants. Information on risk factors and diet were obtained in 1985, 1990 and 1995. After a 10-year follow-up, from 1985-1995, there were 98 cases of fatal or non-fatal CHD. The findings showed that over this period, the mean TFA intake decreased from 4.3% to 1.9% of the food energy. After adjustments for age, BMI, smoking and dietary covariates, TFA intake at baseline was positively associated with 10-year risk of CHD. Thus, a high intake of TFAs, which included all types of isomers, contributed to the risk of CHD. A substantial decrease in TFA intake, which was mainly due to the lowering of the TFA content in edible fats in the Dutch industry, therefore had a large impact on public health (Craig-Schmidt, 2006; Larqué et al., 2001).

In multiple and rigorous randomised trials, the intake of TFAs has been consistently shown to have adverse effects on blood lipids, and most notably on the LDL/HDL cholesterol ratio, which is a strong marker of cardiovascular risk. When a mixture of TFA isomers obtained by partial hydrogenation of vegetable oils is used to replace oleic acid, there is a dosedependent increase in the LDL/HDL ratio. The relationship between the levels of TFAs as the percentage of energy and the increase in the LDL/HDL ratio appears to be approximately linear, with no evidence of a threshold at low levels of TFA intake, and with a slope that is twice as steep as that observed by replacing oleic acid with a SFA (Borra et al., 2007; Mensink & Nestel, 2009). Studies comparing animal and vegetable TFAs have shown similar effects on the total/HDL cholesterol ratio. The effects of TFAs on lipoproteins from both sources appeared at doses exceeding 2% of energy (Mensink & Nestel, 2009). The average impact of TFA-induced changes in the LDL/HDL ratio corresponds to tens of

As opposed to blood and liver, the brain appears to be protected from TFA accumulation in experimental animals, although no data have yet been reported for newborn humans (Larqué, 2001). A significant interaction between diet and pregnancy was shown for the activities of Δ6-desaturase and glucose 6-phosphatase in liver microsomes: dietary TFAs decreased the activities of both of these enzymes, although only in pregnant rats (Larqué et al., 2000; Larqué & Zamora, 2000; Larqué et al., 2003). In Spain, TFAs in human milk were investigated by Boatella et al*.* (Boatella et al., 1993), and they showed that the average content of TFAs in 38 samples was 0.98% of the milk fatty acids. This value is lower than that for human milk from other developed countries, where consumption of hydrogenated fats is higher. In a study by Chen et al*.* (Chen et al., 1995) on TFAs in human milk in Canada, the mean total TFA content was 7.19% (±3.03%) of the total milk fatty acids, with a range

The compelling data linking dietary TFAs to increased risk of CHD have originated from large, prospective, population-based studies, which included from 667 to 80,082 men and women across different age groups who were monitored for six to 20 years. This link has also been seen in controlled feeding trials (Oomen et al., 2001). Among these studies, there are: the United States Health Professional's follow-up study; the Finnish alpha-tocopherol, β-carotene Cancer Prevention Study; the United States nurse's health study (with 14-year and 20-year follow-up) (Willett, 2006); and the Dutch Zutphen elderly study (Oomen et al., 2001). These studies are consistent in their finding of a strong positive association between TFA intake and the risk of CHD. Interestingly, a weaker correlation between SFA intake and

The Zutphen elderly study included 667 men from 64 to 84 years of age who were free of CHD at baseline (Oomen et al., 2001). Dietary surveys were used to establish the food consumption patterns of the participants. Information on risk factors and diet were obtained in 1985, 1990 and 1995. After a 10-year follow-up, from 1985-1995, there were 98 cases of fatal or non-fatal CHD. The findings showed that over this period, the mean TFA intake decreased from 4.3% to 1.9% of the food energy. After adjustments for age, BMI, smoking and dietary covariates, TFA intake at baseline was positively associated with 10-year risk of CHD. Thus, a high intake of TFAs, which included all types of isomers, contributed to the risk of CHD. A substantial decrease in TFA intake, which was mainly due to the lowering of the TFA content in edible fats in the Dutch industry, therefore had a large impact on public

In multiple and rigorous randomised trials, the intake of TFAs has been consistently shown to have adverse effects on blood lipids, and most notably on the LDL/HDL cholesterol ratio, which is a strong marker of cardiovascular risk. When a mixture of TFA isomers obtained by partial hydrogenation of vegetable oils is used to replace oleic acid, there is a dosedependent increase in the LDL/HDL ratio. The relationship between the levels of TFAs as the percentage of energy and the increase in the LDL/HDL ratio appears to be approximately linear, with no evidence of a threshold at low levels of TFA intake, and with a slope that is twice as steep as that observed by replacing oleic acid with a SFA (Borra et al., 2007; Mensink & Nestel, 2009). Studies comparing animal and vegetable TFAs have shown similar effects on the total/HDL cholesterol ratio. The effects of TFAs on lipoproteins from both sources appeared at doses exceeding 2% of energy (Mensink & Nestel, 2009). The average impact of TFA-induced changes in the LDL/HDL ratio corresponds to tens of

from 0.10% to 17.15%.

the risk of CHD also has been reported (Willettt, 2006).

health (Craig-Schmidt, 2006; Larqué et al., 2001).

thousands of premature deaths in the United States alone (Mensink & Nestel, 2009). Although dramatic, this effect is substantially smaller than the increase in cardiovascular mortality associated with TFA intake in epidemiological studies, suggesting that other mechanisms are likely to contribute to the toxicity of TFAs (Ascherio, 2006). Thus, although there is accumulating evidence linking inflammatory proteins and other biomarkers to CHD, lipid concentrations in the blood remain one of the strongest and most consistent predictors of risk. Therefore, the LDL/HDL cholesterol ratio is probably the best marker to date for estimating the effects of TFAs on plasma lipids, which are most likely relevant to CHD incidence and mortality (Larqué & Zamora, 2001).

Further rigorous randomised trials to establish the effects of hydrogenated fats and TFA intake on individual lipoprotein classes started in 1990, when a report from The Netherlands suggested that a diet enriched in elaidic acid (*trans*-9 C18:1) increases the total and LDL cholesterol concentrations and decreased HDL cholesterol concentrations, compared to a diet enriched in oleic acid. In contrast, enrichment of the diet with SFAs increases LDL cholesterol, but has no effect on HDL cholesterol, thus resulting in a smaller adverse change than in the case of elaidic acid (Mensink & Katan, 1993; Mensink & Nestel, 2009).

#### **3.3 Studies of antioxidant effects**

In one study (Filip et al., 2011), the effects of natural antioxidants on formation of TFAs during heat treatment of sunflower oil was investigated. The data from the fatty acid analyses are summarized in Table 2. Here, the non-treated control sunflower oil had a 7.5% palmitic acid content, with 4.5% stearic acid, 25.0% oleic acid, and 60.5% linoleic acid, as is usual for the common (not high in oleic acid) sunflower oils; these data compare well with those of other studies (Sánchez-Gimeno et al., 2008; Bansal, Zhou, Tan, Neo, & Lo, 2009). This sunflower oil was purchased directly from a supplier of oils that are used mainly by small food enterprises (Zvijezda d.d., Zagreb, Croatia). The natural antioxidant extract of rosemary (*Rosmarinus officinalis* L.) that was added to this sunflower oil (SOR) was purchased directly from Vitiva d.d., Markovci, Slovenia (INOLENS4®; Product N° 301770; Batch N°. LAB. 09-779004), and had a carnosic acid content of 4.30%. Similarly, the lutein added to this sunflower oil (SOL) was from pelargonium (2.2% mixture), as obtained from Etol, d.o.o., Celje, Slovenia (NovaSoL® Lutein; Aquanova AG, Birkenweg 8-10, Germany).

The initial levels of the total TFAs in the samples was 0.91% (±0.01%). This compares with the range from 0.15% to 6.03% reported by Bansal et al. (2009) for TFAs in refined oils (soybean, corn, sunflower, high oleic sunflower, low erucic rapeseed and high erucic rapeseed oils). The aim in this study with the sunflower oil was to evaluate the effects of heat on this TFA composition of the oil when subjected to treatment representative of deepfat frying (185 ±5°C). Since sunflower oil is in common use for deep-fat frying, it is particularly important to know what species and levels of TFA isomers appear during such heat treatment (Filip et al., 2011; Martin et al., 2007).

In this study, we focussed mainly on these effects of heat on the TFAs with 18 carbon atoms, which were the most represented. Prior to the treatment, the content of *trans* C 18:1, t-9 was 0.67% (±0.08%). At the end of the heat treatment (120 h at 185 ±5°C), in the control sunflower oil the *trans* C 18:1, t-9 increased to 1.12% (±0.14%), in SOR, to 0.99% (±0.04%), and in SOL, to 0.91% (±0.01%). Within each treatment, these increases were significantly different from the

*Trans* Fatty Acids and Human Health 53

start to the end of the treatment (P <0.001), and also the decreases in *trans* C 18:1, t-9 production with the addition of rosemary oil and lutein were statistically significant in comparison with the control (SOR vs. sunflower oil: 0.32% vs. 0.45%; SOL vs. sunflower oil: 0.24% vs. 0.45%; P <0.001 for both). These data are consistent with an earlier report where there were reductions in *trans*-isomerisation and polar compounds in model oils when α-

When the content of the total TFAs is expressed as the sum of the unsaturated FAs with at least one *trans* double bond, these increased significantly from the initial control sunflower oil of 0.91% (±0.03%), to 1.71% (±0.07%) at 120 h, with significantly lower increases for SOR and SOL, to 1.55% (±0.16%) and 1.43% (±0.04%), respectively (Table 2). Indeed, these differences among treatments were statistically significant (P <0.001) at each step of the heat treatment (24, 48, 72, 96, 120 h). These data relating particularly to the increases in TFAs are comparable to those of Gamel et al. (1999), where they looked at the effects of phenol extracts on TFA formation during frying. A linear relationship between the amounts of elaidic acid and the number of frying cycles has also been reported (Bansal et al., 2009).

According to the nutritional recommendations of the various health authorities, the content of SFAs should not exceed 30% in dietary fats. Sunflower oil thus fits into this recommendation, even though its content in the control sunflower oil increased from 12.43% (±0.13%) to 14.76% (±0.35%), and in the SOR and SOL to 14.68% (±0.61%) and 14.84%

The initial PUFA:SFA ratio here was 4.94 (±0.10), and after the full time of the heat exposure for the control sunflower oil, this was significantly decreased to 3.64 (±0.14) (P ≤0.05). Meanwhile, , for the SOR and SOL at 120 h of heat treatment, the PUFA:SFA ratio decreased to 3.75 (±0.09; P <0.001) and 3.54 (±0.18; P <0.001). As higher PUFA/SFA ratios are more nutritionally appropriate, these data confirm that the heat treatments of this sunflower oil

Governments are increasingly recognising that the risks to consumers from the increased consumption of TFAs cannot be ignored. In 2003, Denmark became the first country to introduce laws to control the sale of foods containing TFAs. This started with the publication of a study in The Lancet by Willett in 1993. Then the Danish Nutrition Council, which was established in 1992, was the driving force behind the campaign that convinced Danish politicians that IP-TFAs can be removed from foods without any effects on their taste, price or availability. The Nutrition Council argued that as no positive health effects of IP-TFAs had ever been reported, then just the suspicion that a high intake has harmful effects on health justified the ban (Astrup, 2006; Mjøs, 2003). The Danish success story might be interesting for other countries, where this unnecessary health hazard could also be

Then in January 2006, it became law in the United States that the contents of TFAs have to be specifically listed on food labels. There is a complication to this, however, because there were two reasons why the consumers might not see a TFA content on the label of a food product. First, although products entering interstate commerce on or after 1 January, 2006, had to be labelled, the FDA realized that it would take some time for food products to move

tocopherol (1%) was added as an antioxidant (Tsuzuki et al., 2008).

(±0.96%), respectively (Table 2).

also worsened this nutritional factor.

eliminated from the foods.

**4.** *Trans* **fatty acids and legislation** 


SFAs, saturated fatty acids; MUFAs, mono-unsaturated fatty acids; PUFAs, polyunsaturated fatty acids; TFAs, trans fatty acids; a, b, c, d Values followed by a different letter are significantly different along each row according to the Duncan test (P <0.05);

Table 2. Effect of cooking heat (185 ±5°C) on the fatty acids composition of sunflower oil, with the addition of the natural antioxidants of a rosemary extract (SOR) and of lutein (SOL) (Filip et al., 2011)

 **0 24 48 72 96 120 Sunflower oil (control)**  SFAs (%) 12.43 ±0.13b 12.54 ±0.12b 12.77 ±0.58b 14.02 ±0.49ab 14.62 ±2.63b 14.76 ±0.35a

PUFAs (%) 61.44 ±0.75a 58.50 ±1.23b 57.83 ±1.33b 56.14 ±1.15bc 56.20 ±3.01bc 53.64 ±2.08c

(%) 61.13 ±0.76a 58.20 ±1.23b 57.82 ±1.34b 55.64 ±1.17bc 55.66 ±3.03bc 53.05 ±2.08c

(%) 0.31 ±0.02b 0.30 ±0.00b 0.31 ±0.02b 0.34 ±0.01a 0.35 ±0.02a 0.36 ±0.01a

163.68 ±7.73b

179.91 ±9.26bc

177.99 ±19.87c

185.41 ±11.56a

TFAs (%) 0.91 ±0.03d 0.99 ±0.06d 1.25 ±0.07c 1.46 ±0.15b 1.56 ±0.09b 1.71 ±0.07a **Sunflower oil with rosemary extract (SOR; 1.0g/kg oil)**  SFAs (%) 12.39 ±0.57c 12.70 ±0.48c 12.74 ±0.41c 13.80 ±0.29b 14.02 ±0.70ab 14.68 ±0.61a

(%) 0.31 ±0.02b 0.31 ±0.01b 0.31 ±0.01b 0.33 ±0.01a 0.33 ±0.01a 0.35 ±0.01a

186.36 ±5.67b

TFAs (%) 0.91 ±0.09d 0.82 ±0.02d 1.02 ±0.05c 1.24 ±0.20c 1.35 ±0.10b 1.55 ±0.16a **Sunflower oil with lutein (SOL; 0.1g/kg oil)**  SFAs (%) 12.51 ±0.72c 12.36 ±0.35c 13.06 ±0.36bc 13.21 ±0.65bc 13.91 ±0.54ab 14.84 ±0.96a MUFAs (%) 26.25 ±2.60b 25.05 ±0.64b 28.22 ±2.49b 27.55 ±2.79b 28.07 ±0.85b 32.79 ±1.63a PUFAs (%) 61.24 ±2.82ab 62.59 ±0.72a 58.72 ±2.47b 59.24 ±3.15b 58.02 ±1.37b 52.37 ±0.74c

(%) 60.93 ±2.83ab 62.29 ±0.72a 58.31 ±2.48bc 58.80 ±3.15bc 57.51 ±1.40c 51.80 ±0.73d

(%) 0.31 ±0.02b 0.31 ±0.01b 0.32 ±0.01ab 0.33 ±0.02ab 0.33 ±0.02ab 0.35 ±0.03a

180.24 ±9.67bc

TFAs (%) 0.91 ±0.06c 0.84 ±0.03c 1.01 ±0.02b 1.23 ±0.16b 1.28 ±0.11b 1.43 ±0.04a SFAs, saturated fatty acids; MUFAs, mono-unsaturated fatty acids; PUFAs, polyunsaturated fatty acids; TFAs, trans fatty acids; a, b, c, d Values followed by a different letter are significantly different along each

Table 2. Effect of cooking heat (185 ±5°C) on the fatty acids composition of sunflower oil, with the addition of the natural antioxidants of a rosemary extract (SOR) and of lutein (SOL)

±0.85abc 31.18 ±0.96ab 31.59 ±0.35a

159.78 ±16.01b

±0.81ab 25.69 ±3.46bc 28.87 ±0.40ab 30.25 ±2.24a

±0.57bc 60.51 ±3.33ab 57.11 ±0.55cd 55.07 ±1.71d

±0.56bc 60.07 ±3.32ab 56.56 ±0.57cd 54.45 ±1.72d

169.54 ±7.89c

174.65 ±12.92c 149.37 ±6.99b

154.65 ±3.96d

149.07 ±10.26d

**Component Time (h)** 

MUFAs (%) 26.13 ±0.68d 28.56 ±1.22c 29.40 ±1.06cb 29.84

192.92 ±4.71a

±1.92bc 25.37 ±1.67c 28.73

198.56 ±6.72a

203.99 ±5.54a

PUFAs (%) 61.88 ±1.67a 61.93 ±1.43a 58.53

(%) 61.58 ±1.66a 61.62 ±1.43a 58.13

n6 PUFAs

n3 PUFAs

n6 PUFAs

n3 PUFAs

n6 PUFAs

n3 PUFAs

(Filip et al., 2011)

n6/n3 199.93

MUFAs (%) 25.72

n6/n3 201.53

n6/n3 199.10

±10.73a

±16.69ab

row according to the Duncan test (P <0.05);

±15.84a

start to the end of the treatment (P <0.001), and also the decreases in *trans* C 18:1, t-9 production with the addition of rosemary oil and lutein were statistically significant in comparison with the control (SOR vs. sunflower oil: 0.32% vs. 0.45%; SOL vs. sunflower oil: 0.24% vs. 0.45%; P <0.001 for both). These data are consistent with an earlier report where there were reductions in *trans*-isomerisation and polar compounds in model oils when αtocopherol (1%) was added as an antioxidant (Tsuzuki et al., 2008).

When the content of the total TFAs is expressed as the sum of the unsaturated FAs with at least one *trans* double bond, these increased significantly from the initial control sunflower oil of 0.91% (±0.03%), to 1.71% (±0.07%) at 120 h, with significantly lower increases for SOR and SOL, to 1.55% (±0.16%) and 1.43% (±0.04%), respectively (Table 2). Indeed, these differences among treatments were statistically significant (P <0.001) at each step of the heat treatment (24, 48, 72, 96, 120 h). These data relating particularly to the increases in TFAs are comparable to those of Gamel et al. (1999), where they looked at the effects of phenol extracts on TFA formation during frying. A linear relationship between the amounts of elaidic acid and the number of frying cycles has also been reported (Bansal et al., 2009).

According to the nutritional recommendations of the various health authorities, the content of SFAs should not exceed 30% in dietary fats. Sunflower oil thus fits into this recommendation, even though its content in the control sunflower oil increased from 12.43% (±0.13%) to 14.76% (±0.35%), and in the SOR and SOL to 14.68% (±0.61%) and 14.84% (±0.96%), respectively (Table 2).

The initial PUFA:SFA ratio here was 4.94 (±0.10), and after the full time of the heat exposure for the control sunflower oil, this was significantly decreased to 3.64 (±0.14) (P ≤0.05). Meanwhile, , for the SOR and SOL at 120 h of heat treatment, the PUFA:SFA ratio decreased to 3.75 (±0.09; P <0.001) and 3.54 (±0.18; P <0.001). As higher PUFA/SFA ratios are more nutritionally appropriate, these data confirm that the heat treatments of this sunflower oil also worsened this nutritional factor.

## **4.** *Trans* **fatty acids and legislation**

Governments are increasingly recognising that the risks to consumers from the increased consumption of TFAs cannot be ignored. In 2003, Denmark became the first country to introduce laws to control the sale of foods containing TFAs. This started with the publication of a study in The Lancet by Willett in 1993. Then the Danish Nutrition Council, which was established in 1992, was the driving force behind the campaign that convinced Danish politicians that IP-TFAs can be removed from foods without any effects on their taste, price or availability. The Nutrition Council argued that as no positive health effects of IP-TFAs had ever been reported, then just the suspicion that a high intake has harmful effects on health justified the ban (Astrup, 2006; Mjøs, 2003). The Danish success story might be interesting for other countries, where this unnecessary health hazard could also be eliminated from the foods.

Then in January 2006, it became law in the United States that the contents of TFAs have to be specifically listed on food labels. There is a complication to this, however, because there were two reasons why the consumers might not see a TFA content on the label of a food product. First, although products entering interstate commerce on or after 1 January, 2006, had to be labelled, the FDA realized that it would take some time for food products to move

*Trans* Fatty Acids and Human Health 55

The fatty acid composition of food is usually determined using gas-liquid chromatography of the corresponding fatty acid methyl esters (FAMEs) (Baggio et al., 2005; Bondia-Pons et al., 2004; Chen et al., 1999; Ratnayake, 1995; Ulberth & Henninger; 1992). Usually, the FAMEs can be conveniently prepared by heating lipids with a large excess of either acidcatalysed or base-catalysed reagents. However, most of the analytical methods are time consuming and impractical for the processing of large numbers of samples, because the lipids have to be extracted prior to preparation of the FAMEs. For this reason, some procedures have been developed that can be used to prepare FAMEs directly from fresh

Vaccenic acid (*trans*-11 C18:1) accounts for over 60% of the natural TFAs, whereas with IP-TFAs, a broad mixture of TFAs is produced, with elaidic acid (*trans-*9 C18:1) as the main product (Oomen et al., 2001). In recent years, new technologies have been developed to reduce the TFA content in fats and oils used in the manufacture of food products. As indicated above, the content of TFAs in Danish food has been monitored for the last 30 years. In margarine and shortening, the TFA content has steadily declined, from about 10 g per 100 g of margarine in the 1970s, to practically no TFAs in margarine in 1999, to

In North America, the daily TFA intake has been estimated using food frequency questionnaires, and it was found to be 3-4 g per person (ADA, 2007), while by extrapolation of human milk data, it was said to be greater than 10 g per person (Chardigny et al., 1995). The data also show that the levels of TFAs can vary considerably among foods within any specific category, reflecting the differences in the fats and oils used in the manufacturing or preparation processes. For example, the range of TFAs in 17 brands of crackers was from 23% to 51% of the total fatty acids, which represents differences of 1 g to 13 g TFAs per 100 g of crackers. These data thus show that the wide variability in the TFA content of different foods can result in large errors in the estimation of the TFA intake of individuals, and

TFA consumption in European countries varies considerably. The diet in northern European countries traditionally contains more TFAs than that in the Mediterranean countries, where olive oil is commonly used. The diet in France has always been relatively low in TFAs, because France has traditionally used predominantly ruminant fats, as compared to hydrogenated vegetable oils. A more recent decrease in dietary TFAs has been seen due to the modification of commercial fats and changes in consumer choice (Larqué et al., 2001). In the TRANSFAIR study (Poppel et al., 1998), which was based on a market basket analysis of diets across 14 European countries, the mean daily intake of TFAs in European countries ranged from the lowest in Greece (1.4 g TFA per day) to the highest in Iceland (5.4 g TFA per day) (Fig. 3).

The lover daily intake of TFAs was recorded in Greece where 1.4 g of TFAs are consumed per day what represent 0.6 % of daily energy intake. The highest daily intake of TFAs was recorded in Iceland where 5.4 g of TFAs are consumed per day what represent 2.0 % of daily energy intake. As shown by researches (Innis et al., 1999; Leth et al., 2006; Poppel et al., 1998) the lowest TFA intake is more often in countries with Mediterranean type of nutrition

**5. Analytical methods for** *trans* **fatty acid determination** 

tissue (Park & Goins, 1994; Garchés & Mancha, 1993).

**6. Consumption of** *trans* **fatty acids** 

efficiently reduce the health risk related to TFAs.

potentially, of groups (Innis, 2006).

habits (Mediterranean diet).

through the distribution chain to a store shelf. Then, foods that contain less than 0.5 g TFAs per serving can be labelled as being free from TFAs. Furthermore, in Europe, the declaring of TFAs on food labels is still not obligatory in many countries. At the same time, these regulations only applied to food that was labelled; food sold in restaurants and canteens was not covered by this law (FDA, 2003; Moss, 2006; Stender et al., 2006). Thus many still feel that foods that contain more than 4 g/100 g SFAs and TFAs together should not be claimed to be healthy food. Indeed, Danish law prohibits the sale of foods that contain more than 2 g TFAs per 100 g of fat, excluding food that naturally contains more TFAs (Filip et al., 2010). Denmark decided to impose this maximum level of IP-TFAs as labelling was deemed insufficient to protect consumers, and especially for risk groups like children and adults with a high intake of fast foods (Garchés & Mancha, 1993; Leth et al., 2006).

Then, in December 2006, the Board of Health of New York City banned many TFAs from restaurants in the city, prompting similar moves in Philadelphia, Montgomery County in Maryland, and the Boston suburb of Brooklyn. The first phase of the regulation applies to oils, shortening and margarine, used in cooking and as spreads, and for recipes that contain more than 0.5 g TFA per serving. Since 1 July, 2007, New York City officials have also called for restaurants to clearly display calorie counts next to their menu items, in a bid to increase consumer awareness of the nutritional content of their food. By 1 July, 2008, the ban had been extended to include TFAs used in baked goods, including bread and cakes, in prepared foods, salad dressings and oils used for deep frying, and in dough and cake batter. Similar bans are being proposed in Chicago and in the state of Illinois; other cities may follow suit, most likely in California (Albers et al., 2008; Blake, 2009).

The American Heart Association recommends a healthy dietary pattern and lifestyle to combat heart disease, limiting TFA consumption to less than 1% (or approximately 2 g on a 2,000-calorie diet), and saturated fat consumption to less than 7% of the total daily calories (Borra et al., 2007). This is consistent with the TFA recommendations made by the American Dietetic Association and the Dietitians of Canada (ADA, 2007).

The benefits of adding TFAs on food Nutrition Facts labels in the United States means that consumers now know the levels of SFAs, TFAs and cholesterol in the foods that they choose to eat. This enables them to make heart-healthy food choices, to help them to reduce their risk of CHD. This labelling is also of particular interest to those concerned about high blood cholesterol. However, to gain the full benefit of this system, all of the consumers need be aware of the risk posed by consuming too high levels of SFAs, TFAs and cholesterol.

At the same time, about half of the convenience products on the Austrian market that have been tested contained less than 1% TFAs, and one third less than 5% (Wagner et al., 2008). However, almost 5% of the products tested contained more than 20% TFAs. A similar level was seen for fast food products, with the highest TFA levels of 8.9%, while the total TFAs of household fats were significantly lower (1.45% ±1.99%) than fats for industrial use (7.83% ±10.0%; P <0.001). Compared to investigations in Austria (and Germany) around 10 years ago, the TFA contents of foods have decreased significantly. About half of the investigated products contained less than 1% of TFAs or total fatty acids, although very high levels of TFAs (>15%) are still detected, and an intake of more than 5 g TFA per portion is possible, which has been shown to significantly increase the risk of CHD (Oomen et al., 2001; Wagner et al., 2008; Wilett, 2006).

through the distribution chain to a store shelf. Then, foods that contain less than 0.5 g TFAs per serving can be labelled as being free from TFAs. Furthermore, in Europe, the declaring of TFAs on food labels is still not obligatory in many countries. At the same time, these regulations only applied to food that was labelled; food sold in restaurants and canteens was not covered by this law (FDA, 2003; Moss, 2006; Stender et al., 2006). Thus many still feel that foods that contain more than 4 g/100 g SFAs and TFAs together should not be claimed to be healthy food. Indeed, Danish law prohibits the sale of foods that contain more than 2 g TFAs per 100 g of fat, excluding food that naturally contains more TFAs (Filip et al., 2010). Denmark decided to impose this maximum level of IP-TFAs as labelling was deemed insufficient to protect consumers, and especially for risk groups like children and adults

Then, in December 2006, the Board of Health of New York City banned many TFAs from restaurants in the city, prompting similar moves in Philadelphia, Montgomery County in Maryland, and the Boston suburb of Brooklyn. The first phase of the regulation applies to oils, shortening and margarine, used in cooking and as spreads, and for recipes that contain more than 0.5 g TFA per serving. Since 1 July, 2007, New York City officials have also called for restaurants to clearly display calorie counts next to their menu items, in a bid to increase consumer awareness of the nutritional content of their food. By 1 July, 2008, the ban had been extended to include TFAs used in baked goods, including bread and cakes, in prepared foods, salad dressings and oils used for deep frying, and in dough and cake batter. Similar bans are being proposed in Chicago and in the state of Illinois; other cities may follow suit,

The American Heart Association recommends a healthy dietary pattern and lifestyle to combat heart disease, limiting TFA consumption to less than 1% (or approximately 2 g on a 2,000-calorie diet), and saturated fat consumption to less than 7% of the total daily calories (Borra et al., 2007). This is consistent with the TFA recommendations made by the American

The benefits of adding TFAs on food Nutrition Facts labels in the United States means that consumers now know the levels of SFAs, TFAs and cholesterol in the foods that they choose to eat. This enables them to make heart-healthy food choices, to help them to reduce their risk of CHD. This labelling is also of particular interest to those concerned about high blood cholesterol. However, to gain the full benefit of this system, all of the consumers need be

At the same time, about half of the convenience products on the Austrian market that have been tested contained less than 1% TFAs, and one third less than 5% (Wagner et al., 2008). However, almost 5% of the products tested contained more than 20% TFAs. A similar level was seen for fast food products, with the highest TFA levels of 8.9%, while the total TFAs of household fats were significantly lower (1.45% ±1.99%) than fats for industrial use (7.83% ±10.0%; P <0.001). Compared to investigations in Austria (and Germany) around 10 years ago, the TFA contents of foods have decreased significantly. About half of the investigated products contained less than 1% of TFAs or total fatty acids, although very high levels of TFAs (>15%) are still detected, and an intake of more than 5 g TFA per portion is possible, which has been shown to significantly increase the risk of CHD (Oomen et al., 2001; Wagner

aware of the risk posed by consuming too high levels of SFAs, TFAs and cholesterol.

with a high intake of fast foods (Garchés & Mancha, 1993; Leth et al., 2006).

most likely in California (Albers et al., 2008; Blake, 2009).

Dietetic Association and the Dietitians of Canada (ADA, 2007).

et al., 2008; Wilett, 2006).

## **5. Analytical methods for** *trans* **fatty acid determination**

The fatty acid composition of food is usually determined using gas-liquid chromatography of the corresponding fatty acid methyl esters (FAMEs) (Baggio et al., 2005; Bondia-Pons et al., 2004; Chen et al., 1999; Ratnayake, 1995; Ulberth & Henninger; 1992). Usually, the FAMEs can be conveniently prepared by heating lipids with a large excess of either acidcatalysed or base-catalysed reagents. However, most of the analytical methods are time consuming and impractical for the processing of large numbers of samples, because the lipids have to be extracted prior to preparation of the FAMEs. For this reason, some procedures have been developed that can be used to prepare FAMEs directly from fresh tissue (Park & Goins, 1994; Garchés & Mancha, 1993).

## **6. Consumption of** *trans* **fatty acids**

Vaccenic acid (*trans*-11 C18:1) accounts for over 60% of the natural TFAs, whereas with IP-TFAs, a broad mixture of TFAs is produced, with elaidic acid (*trans-*9 C18:1) as the main product (Oomen et al., 2001). In recent years, new technologies have been developed to reduce the TFA content in fats and oils used in the manufacture of food products. As indicated above, the content of TFAs in Danish food has been monitored for the last 30 years. In margarine and shortening, the TFA content has steadily declined, from about 10 g per 100 g of margarine in the 1970s, to practically no TFAs in margarine in 1999, to efficiently reduce the health risk related to TFAs.

In North America, the daily TFA intake has been estimated using food frequency questionnaires, and it was found to be 3-4 g per person (ADA, 2007), while by extrapolation of human milk data, it was said to be greater than 10 g per person (Chardigny et al., 1995). The data also show that the levels of TFAs can vary considerably among foods within any specific category, reflecting the differences in the fats and oils used in the manufacturing or preparation processes. For example, the range of TFAs in 17 brands of crackers was from 23% to 51% of the total fatty acids, which represents differences of 1 g to 13 g TFAs per 100 g of crackers. These data thus show that the wide variability in the TFA content of different foods can result in large errors in the estimation of the TFA intake of individuals, and potentially, of groups (Innis, 2006).

TFA consumption in European countries varies considerably. The diet in northern European countries traditionally contains more TFAs than that in the Mediterranean countries, where olive oil is commonly used. The diet in France has always been relatively low in TFAs, because France has traditionally used predominantly ruminant fats, as compared to hydrogenated vegetable oils. A more recent decrease in dietary TFAs has been seen due to the modification of commercial fats and changes in consumer choice (Larqué et al., 2001). In the TRANSFAIR study (Poppel et al., 1998), which was based on a market basket analysis of diets across 14 European countries, the mean daily intake of TFAs in European countries ranged from the lowest in Greece (1.4 g TFA per day) to the highest in Iceland (5.4 g TFA per day) (Fig. 3).

The lover daily intake of TFAs was recorded in Greece where 1.4 g of TFAs are consumed per day what represent 0.6 % of daily energy intake. The highest daily intake of TFAs was recorded in Iceland where 5.4 g of TFAs are consumed per day what represent 2.0 % of daily energy intake. As shown by researches (Innis et al., 1999; Leth et al., 2006; Poppel et al., 1998) the lowest TFA intake is more often in countries with Mediterranean type of nutrition habits (Mediterranean diet).

*Trans* Fatty Acids and Human Health 57

population group in the community. All of the fried food and bakery food samples included in this study contained TFAs, the levels of which varied from less than 0.5% to 6.8%. The highest TFA content in the margarines was 5.2%, with 0.3% as the lowest, and a mean margarine TFA content of 2.3%. The main TFAs were the *trans* isomers of mono-unsaturated

Similarly, the findings of Larqué et al. (Larqué et al., 2003) suggest that Spanish margarines have moved to becoming products with a potentially healthier distribution of fatty acids. Even so, the great variability shown in the fatty-acid compositions of margarines and the poor labelling continue to highlight the importance of greater consumer information to

It can be concluded at present that the reduction of TFAs in the food supply is a complex issue that has involved, and still involves, interdependent and interrelated stakeholders. Any further actions taken to reduce TFAs need to be carefully considered, regarding both the intended and unintended consequences related to nutrition and public health. As shown above, the WHO (WHO/FAO, 2002) has already included TFA levels in their recommended daily food intake (Table 1). Many different options of alternative oils and fats can now be used to replace TFAs, as many of these are already available, while others are still being developed. However, decisions on which alternatives to use are complicated and often time consuming, and they involve considerations of health effects, food availability, quality and taste, research and development investments, supply-chain management, operational

As industry responses are now well underway following the policy actions over the past few years, it is possible to take a present-day 'snapshot' of industry activities that provide preliminary answers to these considerations. The first results of most of the anti-*trans* fat campaigns can be seen as modifications that have been made to the fatty-acid compositions of industrial fats. In these fats, there are significantly higher levels of SFAs and possibly a higher index of atherogenicity. Several major food companies have announced efforts to remove TFAs from their leading brands over the past two decades, starting with Unilever in the 1990s, and then more recently with Nestlé in 2002, Kraft in 2003, Campbell's in 2004 (for Goldfish crackers), Kellogg's in 2005, and Frito-Lay in 2006 (for chips). It is of note that the earliest announcements came from European firms, where the use of partially hydrogenated soy was not as common as it was in the United States, and thus this reformulation process

The announcements over the last three years or so have reflected the attention brought to this issue through lawsuits and debates about nutritional labelling regulations. Many companies even chose to implement the disclosure of these *trans*-fat contents earlier than the January 1, 2006, deadline, particularly when they were able to advertise 'zero' *trans* fats on

One aspect for producing such zero TFAs lies in the transesterification reactions between vegetable oils and the SFAs of C8:0, C12:0, C14:0 and C16:0. These reactions can be catalysed by an immobilised sn-1,3 specific *Rhizomucor miehei* lipase. When considering a TFA-free or

avoid detrimental changes to the traditional Mediterranean diet in Spain.

modifications, consumer acceptance, and cost (Borra et al., 2007; FDA, 2003).

octadecenoic acid (C18:1).

has not been as onerous.

their products (Crisco, 2008).

**7. Conclusions and future trends** 

Fig. 3. Mean daily intake of TFAs across the European countries (Innis et al., 1999; Leth et al., 2006; Poppel et al., 1998)

#### **6.1 Dairy products and** *trans* **fatty acids**

Milk fat is also the most abundant source of conjugated linoleic acids (CLAs), which are a group of geometrical and positional isomers of linoleic acid (LA *cis*-9,*cis*-12 C18:2). The major isomer of the CLAs in milk fat is *cis*-9, *trans*-11, and it represents 80 g to 90 g per 100 g of the total CLAs (Chardigny et al., 1995; Ledoux et al., 2005; Seçkim et al., 2005). Some of these fatty acids have biological, physiological and nutritional properties that are very interesting for consumer health, as especially seen for butyric acid and CLAs (Pandya & Ghodke, 2007). The CLAs are synthesised in ruminants both from dietary linoleic acid (*cis*-9,*cis*-12 C18:2) in the rumen by the microbial flora, and from vaccenic acid (*trans*-11 C18:1) in the mammary glands during *de-novo* synthesis (Bauman & Griinari, 2001).

#### **6.2 Industrially produced fat and** *trans* **fatty acids**

Brát and Pokorný (Brát & Pokorný, 2000) investigated a series of 20 margarines, nine cooking fats, and butter that were available on the Czech market. They used the American Oil Chemistry Society standard analysis methods, with capillary gas chromatography. The margarines contained 15.2% to 54.1% cooking fats, and 16.5% to 59.1% SFAs, which was less than the butter. The content of linoleic acid varied between 3.7% and 52.4% in the margarines; small amounts of linolenic acid were present in most samples, while oleic acid prevailed in the cooking fats. Monoenoic TFAs were present only in trace amounts in 10 samples, and *trans*-polyenoic acids were present only in small amounts. Most cooking fats had a high content of TFAs. They summarised these data by indicating that the number of *trans*-free margarines had rapidly increased over a few years.

More recently, Cenčič-Kodba (Cenčič-Kodba, 2007) examined 13 margarines and fatty food samples in Slovenia, which were selected according to the frequency of use among the

Fig. 3. Mean daily intake of TFAs across the European countries (Innis et al., 1999; Leth et al.,

Milk fat is also the most abundant source of conjugated linoleic acids (CLAs), which are a group of geometrical and positional isomers of linoleic acid (LA *cis*-9,*cis*-12 C18:2). The major isomer of the CLAs in milk fat is *cis*-9, *trans*-11, and it represents 80 g to 90 g per 100 g of the total CLAs (Chardigny et al., 1995; Ledoux et al., 2005; Seçkim et al., 2005). Some of these fatty acids have biological, physiological and nutritional properties that are very interesting for consumer health, as especially seen for butyric acid and CLAs (Pandya & Ghodke, 2007). The CLAs are synthesised in ruminants both from dietary linoleic acid (*cis*-9,*cis*-12 C18:2) in the rumen by the microbial flora, and from vaccenic acid (*trans*-11 C18:1) in

Brát and Pokorný (Brát & Pokorný, 2000) investigated a series of 20 margarines, nine cooking fats, and butter that were available on the Czech market. They used the American Oil Chemistry Society standard analysis methods, with capillary gas chromatography. The margarines contained 15.2% to 54.1% cooking fats, and 16.5% to 59.1% SFAs, which was less than the butter. The content of linoleic acid varied between 3.7% and 52.4% in the margarines; small amounts of linolenic acid were present in most samples, while oleic acid prevailed in the cooking fats. Monoenoic TFAs were present only in trace amounts in 10 samples, and *trans*-polyenoic acids were present only in small amounts. Most cooking fats had a high content of TFAs. They summarised these data by indicating that the number of

More recently, Cenčič-Kodba (Cenčič-Kodba, 2007) examined 13 margarines and fatty food samples in Slovenia, which were selected according to the frequency of use among the

the mammary glands during *de-novo* synthesis (Bauman & Griinari, 2001).

**6.2 Industrially produced fat and** *trans* **fatty acids** 

*trans*-free margarines had rapidly increased over a few years.

2006; Poppel et al., 1998)

**6.1 Dairy products and** *trans* **fatty acids** 

population group in the community. All of the fried food and bakery food samples included in this study contained TFAs, the levels of which varied from less than 0.5% to 6.8%. The highest TFA content in the margarines was 5.2%, with 0.3% as the lowest, and a mean margarine TFA content of 2.3%. The main TFAs were the *trans* isomers of mono-unsaturated octadecenoic acid (C18:1).

Similarly, the findings of Larqué et al. (Larqué et al., 2003) suggest that Spanish margarines have moved to becoming products with a potentially healthier distribution of fatty acids. Even so, the great variability shown in the fatty-acid compositions of margarines and the poor labelling continue to highlight the importance of greater consumer information to avoid detrimental changes to the traditional Mediterranean diet in Spain.

## **7. Conclusions and future trends**

It can be concluded at present that the reduction of TFAs in the food supply is a complex issue that has involved, and still involves, interdependent and interrelated stakeholders. Any further actions taken to reduce TFAs need to be carefully considered, regarding both the intended and unintended consequences related to nutrition and public health. As shown above, the WHO (WHO/FAO, 2002) has already included TFA levels in their recommended daily food intake (Table 1). Many different options of alternative oils and fats can now be used to replace TFAs, as many of these are already available, while others are still being developed. However, decisions on which alternatives to use are complicated and often time consuming, and they involve considerations of health effects, food availability, quality and taste, research and development investments, supply-chain management, operational modifications, consumer acceptance, and cost (Borra et al., 2007; FDA, 2003).

As industry responses are now well underway following the policy actions over the past few years, it is possible to take a present-day 'snapshot' of industry activities that provide preliminary answers to these considerations. The first results of most of the anti-*trans* fat campaigns can be seen as modifications that have been made to the fatty-acid compositions of industrial fats. In these fats, there are significantly higher levels of SFAs and possibly a higher index of atherogenicity. Several major food companies have announced efforts to remove TFAs from their leading brands over the past two decades, starting with Unilever in the 1990s, and then more recently with Nestlé in 2002, Kraft in 2003, Campbell's in 2004 (for Goldfish crackers), Kellogg's in 2005, and Frito-Lay in 2006 (for chips). It is of note that the earliest announcements came from European firms, where the use of partially hydrogenated soy was not as common as it was in the United States, and thus this reformulation process has not been as onerous.

The announcements over the last three years or so have reflected the attention brought to this issue through lawsuits and debates about nutritional labelling regulations. Many companies even chose to implement the disclosure of these *trans*-fat contents earlier than the January 1, 2006, deadline, particularly when they were able to advertise 'zero' *trans* fats on their products (Crisco, 2008).

One aspect for producing such zero TFAs lies in the transesterification reactions between vegetable oils and the SFAs of C8:0, C12:0, C14:0 and C16:0. These reactions can be catalysed by an immobilised sn-1,3 specific *Rhizomucor miehei* lipase. When considering a TFA-free or

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low TFA fat that is suitable for use as a confectionery fat, a non-hydrogenated vegetable fat composed of an inter-esterified fat can be used: this can be obtained by subjecting a blend of at least one fat rich in lauric acid and at least one fat without lauric acid to interesterification (Farmani et al., 2007).

For all of the products introduced in 2005 and 2006 that have claimed to contain no *trans* fats, the most commonly used oil ingredients have been canola, sunflower and soybean oils. Palm oil, which is high in saturated fat, also appears among the commonly used ingredients, but not as an alternative to reducing TFAs. Eleven percent of food producers in the United States still use partially hydrogenated oils as ingredient, because the regulations allow 0.5 g per serving of *trans* fats in products that claim to contain 'no *trans* fat', while the use of small amounts of partially hydrogenated oils has facilitated the reformulation of some products (Unnevehr & Jagmanaite, 2008).

Between 2006 and 2007, consumer awareness of *trans* fats increased and attained levels similar to those for saturated fats. This increased awareness has been associated with improved self-reporting behaviour in consumer shopping for groceries (Eckel et al., 2009). However, food labels and food claims that accompany packed foods are still largely incomprehensible for consumers, and therefore they appear to be of very little use at present. Moreover, in Europe, consumers still cannot identify the content of TFAs in the labelling of food products, particularly as the only legislation that restricts the content of TFAs in Europe is in Denmark.

At the same time, we have to be aware that indicators are showing that the world population is still increasing and is expected to reach nearly 8.9 thousand million (8,900,000,000) by the year 2050 (UN, 2004). Knowing of some of the problems that are associated with this increasing population, we are now combating the need that will arise for more and more potential food products to be used for biofuels (Fink & Medved, 2011). Thus, in the future, it will become increasingly difficult to assure food security and food safety, as well as the nutritional quality of food. Indeed, it is the nutritional quality of food and its distribution all over the World that are the main factors that will have a huge impact on human health. In this way, human health is more than just of personal value, as it is also part of the welfare of the whole of our society.

## **8. References**


low TFA fat that is suitable for use as a confectionery fat, a non-hydrogenated vegetable fat composed of an inter-esterified fat can be used: this can be obtained by subjecting a blend of at least one fat rich in lauric acid and at least one fat without lauric acid to inter-

For all of the products introduced in 2005 and 2006 that have claimed to contain no *trans* fats, the most commonly used oil ingredients have been canola, sunflower and soybean oils. Palm oil, which is high in saturated fat, also appears among the commonly used ingredients, but not as an alternative to reducing TFAs. Eleven percent of food producers in the United States still use partially hydrogenated oils as ingredient, because the regulations allow 0.5 g per serving of *trans* fats in products that claim to contain 'no *trans* fat', while the use of small amounts of partially hydrogenated oils has facilitated the reformulation of some products

Between 2006 and 2007, consumer awareness of *trans* fats increased and attained levels similar to those for saturated fats. This increased awareness has been associated with improved self-reporting behaviour in consumer shopping for groceries (Eckel et al., 2009). However, food labels and food claims that accompany packed foods are still largely incomprehensible for consumers, and therefore they appear to be of very little use at present. Moreover, in Europe, consumers still cannot identify the content of TFAs in the labelling of food products, particularly as the only legislation that restricts the content of

At the same time, we have to be aware that indicators are showing that the world population is still increasing and is expected to reach nearly 8.9 thousand million (8,900,000,000) by the year 2050 (UN, 2004). Knowing of some of the problems that are associated with this increasing population, we are now combating the need that will arise for more and more potential food products to be used for biofuels (Fink & Medved, 2011). Thus, in the future, it will become increasingly difficult to assure food security and food safety, as well as the nutritional quality of food. Indeed, it is the nutritional quality of food and its distribution all over the World that are the main factors that will have a huge impact on human health. In this way, human health is more than just of personal value, as it is also

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**4** 

 *Chile* 

**Control and Coordination** 

Pablo S. Gaete and Xavier F. Figueroa

 *Pontificia Universidad Católica de Chile, Santiago,* 

**of Vasomotor Tone in the Microcirculation** 

The blood vascular system consists in a complex network of vessels that is mainly intended to provide oxygen and nutrients to all individual cells of peripheral tissues and help to dispose metabolic wastes. Several distinct functional compartments can be distinguished in the vascular network: arteries, arterioles, capillaries, venules and veins. Conduit arteries (diameter, 1 to several millimeters) carry blood away from the heart through a divergent arborescence that reaches and penetrates into the tissues via the feed arteries (diameter, 100 to 500 µm) (Davis *et al.*, 1986; Segal, 2000, 2005). These muscular vessels give rise to the arterioles (diameter, < l00 µm), which control and coordinate the blood flow distribution in such a way that each capillary is correctly supplied at the proper pressure (Mulvany, 1990; Segal, 2005). This part of the vascular network composed of arterioles, capillaries and venules is embedded within the organ irrigated and is called microcirculation (Davis *et al.*, 1986; Segal, 2005; Lockhart *et al.*, 2009). Finally, veins carry blood back to the heart through a

In general, the vascular wall of arteries consists of an outer tunica adventitia, a central tunica media, and an inner tunica intima. The adventitia mainly contains connective tissue, fibroblasts, mast cells, macrophages, and nerve axons. Although the amount of the wall taken up by adventitia varies with the vascular territory, it is directly proportional to the size of the vessel (Gingras *et al.*, 2009). The media is comprised of circumferentially arranged smooth muscle cells and is bounded on the luminal side by a well-defined internal elastic lamina. An external elastic lamina may also be present between the media and the adventitia or even within the media in larger vessels such as the aorta, but this structure is fragmented in small arteries and absent in arterioles (Mulvany, 1990; London *et al.*, 1998). The number of smooth muscle cell layers decreases with decreasing vessel diameter and, in arterioles, only an unbroken monolayer of smooth muscle cells is found (Davis *et al.*, 1986; Mulvany, 1990; Segal, 2005). In contrast, the structure of the intima is similar in all blood vessels and is formed by a smooth, continuous single layer of endothelial cells that lines the inner surface of the vessels (Mulvany, 1990). These cells are very thin (2 µm thick) and elongated (10 to 20 µm wide and 100 to 150 µm long, in arterioles), and are oriented parallel

to the longitudinal axis of the vessel (Haas & Duling, 1997).

**1. Introduction** 

convergent arborescence.

Mauricio A. Lillo, Francisco R. Pérez, Mariela Puebla,

*Departamento de Fisiología, Facultad de Ciencias Biológicas,* 


## **Control and Coordination of Vasomotor Tone in the Microcirculation**

Mauricio A. Lillo, Francisco R. Pérez, Mariela Puebla, Pablo S. Gaete and Xavier F. Figueroa *Departamento de Fisiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago, Chile* 

## **1. Introduction**

64 The Cardiovascular System – Physiology, Diagnostics and Clinical Implications

UN (2004). World Population to 2300. United Nations. Department of Economic and Social

Unnevehr, L.J., & Jagmanaite, E. (2008). Getting rid of trans fats in the US diet: Policies,

Wagner, K.H., Plasse, E., Proell, C., & Kanzler, S. (2008). Comprehensive studies on the trans

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*Chemistry,* Vol. 108, No. 3, (June 2008), pp. 1054-1060, ISSN 0308-8146 Willett, W.C. (2006). Trans fatty acids and cardiovascular disease – Epidemiological data. *Atherosclerossis Supplements,* Vol. 7, No. 2, (May 2006), pp. 5-8, ISSN 1567-5688 WHO (2008). Atlas of Health in Europe, WHO , Copenhagen, Denmark, pp.125, ISBN 978-

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Affairs, Population Division, New York, USA (2004), pp. 4-10.

ISSN 0306-9192

92-890-1411

WHO/FAO Expert Consultation.

series 724, (Geneva 1985), ISSN 0512-3054

The blood vascular system consists in a complex network of vessels that is mainly intended to provide oxygen and nutrients to all individual cells of peripheral tissues and help to dispose metabolic wastes. Several distinct functional compartments can be distinguished in the vascular network: arteries, arterioles, capillaries, venules and veins. Conduit arteries (diameter, 1 to several millimeters) carry blood away from the heart through a divergent arborescence that reaches and penetrates into the tissues via the feed arteries (diameter, 100 to 500 µm) (Davis *et al.*, 1986; Segal, 2000, 2005). These muscular vessels give rise to the arterioles (diameter, < l00 µm), which control and coordinate the blood flow distribution in such a way that each capillary is correctly supplied at the proper pressure (Mulvany, 1990; Segal, 2005). This part of the vascular network composed of arterioles, capillaries and venules is embedded within the organ irrigated and is called microcirculation (Davis *et al.*, 1986; Segal, 2005; Lockhart *et al.*, 2009). Finally, veins carry blood back to the heart through a convergent arborescence.

In general, the vascular wall of arteries consists of an outer tunica adventitia, a central tunica media, and an inner tunica intima. The adventitia mainly contains connective tissue, fibroblasts, mast cells, macrophages, and nerve axons. Although the amount of the wall taken up by adventitia varies with the vascular territory, it is directly proportional to the size of the vessel (Gingras *et al.*, 2009). The media is comprised of circumferentially arranged smooth muscle cells and is bounded on the luminal side by a well-defined internal elastic lamina. An external elastic lamina may also be present between the media and the adventitia or even within the media in larger vessels such as the aorta, but this structure is fragmented in small arteries and absent in arterioles (Mulvany, 1990; London *et al.*, 1998). The number of smooth muscle cell layers decreases with decreasing vessel diameter and, in arterioles, only an unbroken monolayer of smooth muscle cells is found (Davis *et al.*, 1986; Mulvany, 1990; Segal, 2005). In contrast, the structure of the intima is similar in all blood vessels and is formed by a smooth, continuous single layer of endothelial cells that lines the inner surface of the vessels (Mulvany, 1990). These cells are very thin (2 µm thick) and elongated (10 to 20 µm wide and 100 to 150 µm long, in arterioles), and are oriented parallel to the longitudinal axis of the vessel (Haas & Duling, 1997).

Control and Coordination of Vasomotor Tone in the Microcirculation 67

in endothelial cells (Papassotiriou *et al.*, 2000; Wang *et al.*, 2005). In contrast, SKCa and IKCa are expressed exclusively in endothelial cells (Jackson, 2000; Kohler *et al.*, 2000; Nilius &

All these K+ channels play critical roles in the regulation of vascular function. KATP channels are opened at rest, and then, are very relevant in the control of smooth muscle membrane potential and vasomotor tone in basal unstimulated conditions (Jackson, 1993, 2000). Interestingly, Kir are typically closed at resting conditions, but are activated by hyperpolarization of membrane potential and by increments in extracellular K+ concentration ([K+]o) smaller than 20 mM (Jackson, 2005; Jantzi *et al.*, 2006; Smith *et al.*, 2008). Although BKCa channels are involved in the response to several vasomotor stimuli, the most relevant function of these K+ channels is the tonic control of vasomotor tone by buffering the smooth muscle cell depolarization. The increase in intracellular Ca2+ concentration associated to smooth muscle depolarization activates local Ca2+ transients (i.e. Ca2+ sparks) that result from the opening of tightly clustered ryanodine receptor channels located at extensions of sarcoplasmic reticulum. Ca2+ sparks activate a BKCa-dependent hyperpolarizing current that opposes the smooth muscle depolarization, and thereby, regulates the magnitude of the vasoconstriction (Jaggar *et al.*, 1998; Gollasch *et al.*, 2000; Gordienko *et al.*, 2001; Lohn *et al.*, 2001). SKCa and IKCa channels play a central role in the endothelial cell control of vasomotor tone and peripheral vascular resistance (Busse *et al.*, 2002; Eichler *et al.*, 2003; Taylor *et al.*, 2003; Si *et al.*, 2006; Brahler *et al.*, 2009). However, probably the most recognized function of these K+ channels is their participation in the

One of the most well-characterized mode of communication in the vessel wall is the production of paracrine signals by endothelial cells such as PGs, NO and EDHF (Vanhoutte, 2004; Vanhoutte *et al.*, 2009). The role of these signaling pathways in vascular physiology has been extensively studied and there are several recent reviews that address their involvement in vascular function in normal conditions and disease (Feletou & Vanhoutte, 2009; Vanhoutte *et al.*, 2009; Rafikov *et al.*, 2011). In this section, we will address the most relevant aspects of these signals in relation to the control of vasomotor tone in physiological

PGs are a family of bioactive lipids derived from arachidonic acid (AA or 5,8,11,14 eicosatetraenoic acid), which, in turn, is generated by the enzyme phospholipase A2 (PLA2) from phospholipids of the cell membrane in a Ca2+-dependent manner (Simmons *et al.*, 2004; Fortier *et al.*, 2008). The metabolism of PGs is complex and depends on the hydrolysis of AA by the enzymes cyclooxygenase-1 (COX-1) or cyclooxygenase-2 (COX-2) to form the unstable endoperoxide derivative, prostaglandin G2 (PGG2), and subsequently, prostaglandin H2 (PGH2) (Simmons *et al.*, 2004). PGH2 is the parent compound of all PGs, which are synthesized by specific enzymes: prostaglandin I2 synthase (PGIS), prostaglandin E2 synthase (PGES-1), prostaglandin D2 synthase (PGDS), prostaglandin F2α synthase (PGES-2), and thromboxane A2 synthase (TBXAS-1) that catalyze the production of

Droogmans, 2001; Eichler *et al.*, 2003; Taylor *et al.*, 2003; Brahler *et al.*, 2009).

EDHF signaling (see below) (Busse *et al.*, 2002; Vanhoutte, 2004).

**3. Paracrine signaling in the vessel wall** 

conditions.

**3.1 Prostaglandins** 

Correct supply of blood to the tissues relies on the ability of the vascular system to adjust the resistance of each vessel by controlling its lumen diameter, which is, in turn, a function of the level of tone of the vascular smooth muscle (i.e. vasomotor tone). As blood vessels are complex structures that must work as an unit, control of vasomotor tone depends on the fine synchronization of function of the different cellular components of the vessel wall, mainly smooth muscle cells and endothelial cells (Segal, 2000; Figueroa *et al.*, 2004; Segal, 2005; Figueroa & Duling, 2009). Such synchronization and coordination is accomplished by an intricate system of radial and longitudinal cell-to-cell communication (Beach *et al.*, 1998; Figueroa *et al.*, 2004; Rummery & Hill, 2004; Segal, 2005; Figueroa & Duling, 2009; Bagher & Segal, 2011). In addition, arterioles in the microcirculation form a complex network, and then, the changes in the luminal diameter of different arteriolar segments must also be coordinated to regulate blood flow distribution and peripheral vascular resistance (Figueroa *et al.*, 2004; Rummery & Hill, 2004; Segal, 2005; Figueroa & Duling, 2008). It has typically been assumed that most of the total resistance to blood flow resides on the arterioles. However, it has become apparent that as much as 50% of the precapillary resistance lies proximal to the arterioles (Davis *et al.*, 1986; Mulvany, 1990; Segal, 2000), which situates the feed arteries at a key point for controlling vascular function and highlights the importance of the functional communication between arterioles and feed arteries in the regulation of blood flow distribution.

It is widely recognized that the endothelium plays a critical role controlling function of the vessel wall by the release of paracrine molecules such as nitric oxide (NO), prostaglandins (PGs) and also by the activation of the signaling mechanism known as endothelium-derived hyperpolarizing factor (EDHF) (Moncada *et al.*, 1991; Busse *et al.*, 2002; Feletou & Vanhoutte, 2007; Vanhoutte *et al.*, 2009). However, another mechanism of communication that has emerged as a key pathway to command and coordinate the vascular wall function is the direct cell-to-cell communication via gap junctions (Sandow *et al.*, 2003; Figueroa *et al.*, 2004, 2006). In addition, it is important to note that K+ channels expressed in the endothelium and smooth muscle cells play a central role in the control of vasomotor tone by paracrine or gap junction-mediated signaling mechanisms (Jackson, 2005).

#### **2. Membrane potential and vascular K+ channels**

In contrast to endothelial cells, Ca2+ is a signal for contraction in smooth muscle cells. In smooth muscle cells of blood vessels the L-type voltage-dependent Ca2+ channels play a central role controlling the vasomotor tone (Jackson, 2000). Changes in membrane potential modulate the opening of these Ca2+ channels. Thereby, depolarization produces a Ca2+ influx that leads to vasoconstriction and, on the contrary, hyperpolarization leads to a reduction in intracellular Ca2+ concentration and, subsequently, vasodilation (Jackson, 2000, 2005). In this context, K+ channels play a pivotal role in vascular function by controlling the membrane potential of both endothelial and smooth muscle cells. The main K+ channels expressed in resistance vessels, from a functional point of view, are: the ATP-sensitive K+ channels (KATP), inward rectifying K+ channels (Kir) and Ca2+-activated K+ channels (KCa) of small (SKCa), intermediate (IKCa) and large (BKCa) conductance (Jackson, 2000, 2005). KATP and Kir are expressed in both endothelial and smooth muscle cells (Quayle *et al.*, 1996; Jackson, 2000, 2005; Ko *et al.*, 2008), whereas BKCa are mostly found in smooth muscle cells (Jackson, 2005; Ko *et al.*, 2008), but, on occasion, these K+ channels have also been described

Correct supply of blood to the tissues relies on the ability of the vascular system to adjust the resistance of each vessel by controlling its lumen diameter, which is, in turn, a function of the level of tone of the vascular smooth muscle (i.e. vasomotor tone). As blood vessels are complex structures that must work as an unit, control of vasomotor tone depends on the fine synchronization of function of the different cellular components of the vessel wall, mainly smooth muscle cells and endothelial cells (Segal, 2000; Figueroa *et al.*, 2004; Segal, 2005; Figueroa & Duling, 2009). Such synchronization and coordination is accomplished by an intricate system of radial and longitudinal cell-to-cell communication (Beach *et al.*, 1998; Figueroa *et al.*, 2004; Rummery & Hill, 2004; Segal, 2005; Figueroa & Duling, 2009; Bagher & Segal, 2011). In addition, arterioles in the microcirculation form a complex network, and then, the changes in the luminal diameter of different arteriolar segments must also be coordinated to regulate blood flow distribution and peripheral vascular resistance (Figueroa *et al.*, 2004; Rummery & Hill, 2004; Segal, 2005; Figueroa & Duling, 2008). It has typically been assumed that most of the total resistance to blood flow resides on the arterioles. However, it has become apparent that as much as 50% of the precapillary resistance lies proximal to the arterioles (Davis *et al.*, 1986; Mulvany, 1990; Segal, 2000), which situates the feed arteries at a key point for controlling vascular function and highlights the importance of the functional communication between arterioles and feed arteries in the regulation of

It is widely recognized that the endothelium plays a critical role controlling function of the vessel wall by the release of paracrine molecules such as nitric oxide (NO), prostaglandins (PGs) and also by the activation of the signaling mechanism known as endothelium-derived hyperpolarizing factor (EDHF) (Moncada *et al.*, 1991; Busse *et al.*, 2002; Feletou & Vanhoutte, 2007; Vanhoutte *et al.*, 2009). However, another mechanism of communication that has emerged as a key pathway to command and coordinate the vascular wall function is the direct cell-to-cell communication via gap junctions (Sandow *et al.*, 2003; Figueroa *et al.*, 2004, 2006). In addition, it is important to note that K+ channels expressed in the endothelium and smooth muscle cells play a central role in the control of vasomotor tone by paracrine or gap

 **channels** 

In contrast to endothelial cells, Ca2+ is a signal for contraction in smooth muscle cells. In smooth muscle cells of blood vessels the L-type voltage-dependent Ca2+ channels play a central role controlling the vasomotor tone (Jackson, 2000). Changes in membrane potential modulate the opening of these Ca2+ channels. Thereby, depolarization produces a Ca2+ influx that leads to vasoconstriction and, on the contrary, hyperpolarization leads to a reduction in intracellular Ca2+ concentration and, subsequently, vasodilation (Jackson, 2000, 2005). In this context, K+ channels play a pivotal role in vascular function by controlling the membrane potential of both endothelial and smooth muscle cells. The main K+ channels expressed in resistance vessels, from a functional point of view, are: the ATP-sensitive K+ channels (KATP), inward rectifying K+ channels (Kir) and Ca2+-activated K+ channels (KCa) of small (SKCa), intermediate (IKCa) and large (BKCa) conductance (Jackson, 2000, 2005). KATP and Kir are expressed in both endothelial and smooth muscle cells (Quayle *et al.*, 1996; Jackson, 2000, 2005; Ko *et al.*, 2008), whereas BKCa are mostly found in smooth muscle cells (Jackson, 2005; Ko *et al.*, 2008), but, on occasion, these K+ channels have also been described

blood flow distribution.

junction-mediated signaling mechanisms (Jackson, 2005).

**2. Membrane potential and vascular K+**

in endothelial cells (Papassotiriou *et al.*, 2000; Wang *et al.*, 2005). In contrast, SKCa and IKCa are expressed exclusively in endothelial cells (Jackson, 2000; Kohler *et al.*, 2000; Nilius & Droogmans, 2001; Eichler *et al.*, 2003; Taylor *et al.*, 2003; Brahler *et al.*, 2009).

All these K+ channels play critical roles in the regulation of vascular function. KATP channels are opened at rest, and then, are very relevant in the control of smooth muscle membrane potential and vasomotor tone in basal unstimulated conditions (Jackson, 1993, 2000). Interestingly, Kir are typically closed at resting conditions, but are activated by hyperpolarization of membrane potential and by increments in extracellular K+ concentration ([K+]o) smaller than 20 mM (Jackson, 2005; Jantzi *et al.*, 2006; Smith *et al.*, 2008). Although BKCa channels are involved in the response to several vasomotor stimuli, the most relevant function of these K+ channels is the tonic control of vasomotor tone by buffering the smooth muscle cell depolarization. The increase in intracellular Ca2+ concentration associated to smooth muscle depolarization activates local Ca2+ transients (i.e. Ca2+ sparks) that result from the opening of tightly clustered ryanodine receptor channels located at extensions of sarcoplasmic reticulum. Ca2+ sparks activate a BKCa-dependent hyperpolarizing current that opposes the smooth muscle depolarization, and thereby, regulates the magnitude of the vasoconstriction (Jaggar *et al.*, 1998; Gollasch *et al.*, 2000; Gordienko *et al.*, 2001; Lohn *et al.*, 2001). SKCa and IKCa channels play a central role in the endothelial cell control of vasomotor tone and peripheral vascular resistance (Busse *et al.*, 2002; Eichler *et al.*, 2003; Taylor *et al.*, 2003; Si *et al.*, 2006; Brahler *et al.*, 2009). However, probably the most recognized function of these K+ channels is their participation in the EDHF signaling (see below) (Busse *et al.*, 2002; Vanhoutte, 2004).

## **3. Paracrine signaling in the vessel wall**

One of the most well-characterized mode of communication in the vessel wall is the production of paracrine signals by endothelial cells such as PGs, NO and EDHF (Vanhoutte, 2004; Vanhoutte *et al.*, 2009). The role of these signaling pathways in vascular physiology has been extensively studied and there are several recent reviews that address their involvement in vascular function in normal conditions and disease (Feletou & Vanhoutte, 2009; Vanhoutte *et al.*, 2009; Rafikov *et al.*, 2011). In this section, we will address the most relevant aspects of these signals in relation to the control of vasomotor tone in physiological conditions.

## **3.1 Prostaglandins**

PGs are a family of bioactive lipids derived from arachidonic acid (AA or 5,8,11,14 eicosatetraenoic acid), which, in turn, is generated by the enzyme phospholipase A2 (PLA2) from phospholipids of the cell membrane in a Ca2+-dependent manner (Simmons *et al.*, 2004; Fortier *et al.*, 2008). The metabolism of PGs is complex and depends on the hydrolysis of AA by the enzymes cyclooxygenase-1 (COX-1) or cyclooxygenase-2 (COX-2) to form the unstable endoperoxide derivative, prostaglandin G2 (PGG2), and subsequently, prostaglandin H2 (PGH2) (Simmons *et al.*, 2004). PGH2 is the parent compound of all PGs, which are synthesized by specific enzymes: prostaglandin I2 synthase (PGIS), prostaglandin E2 synthase (PGES-1), prostaglandin D2 synthase (PGDS), prostaglandin F2α synthase (PGES-2), and thromboxane A2 synthase (TBXAS-1) that catalyze the production of

Control and Coordination of Vasomotor Tone in the Microcirculation 69

induces the relaxation of smooth muscle cells by the stimulation of IP receptors (Figure 1) (Gryglewski, 2008; Vanhoutte, 2009). In contrast to COX-1, expression of COX-2 in normal blood vessels is very low (Crofford *et al.*, 1994; Schonbeck *et al.*, 1999). However, Topper et al. (Topper *et al.*, 1996) found that laminar shear stress, but not turbulent flow, up-regulates the levels of COX-2 expression in cultures of vascular endothelial cells. Laminar shear stress is a highly relevant stimulus that is involved in the tonic control of vasomotor tone, which

Probably, the most relevant intercellular communication signal in vascular physiology is the endothelium-dependent NO production. NO is a potent vasodilator synthesized by the enzyme NO synthase (NOS) (Moncada *et al.*, 1991). The substrates for NOS-mediated NO production are the amino acid L-arginine, molecular oxygen and nicotinamide adenine dinucleotide phosphate (NADPH). Three isoforms of NOS have been described: endothelial NOS (eNOS), neuronal NOS (nNOS) and inducible NOS (iNOS) (Moncada *et al.*, 1991; Alderton *et al.*, 2001). The enzyme expressed in endothelial cells (eNOS) is the main NOS isoform found in the vascular system in normal conditions. The NO released by endothelial cells elicits the relaxation of the underlying vascular smooth muscle cells mainly through the initiation of the signaling cascade cGMP/PKG by activation of soluble guanylate cyclase, which has been ascribed as the primary receptor of NO (Moncada *et al.*, 1991). Although certainly the cGMP-dependent signaling pathway has several targets in the vessel wall, the relaxation induced by NO is mainly associated with a reduction in the Ca2+ sensitivity of

Consistent with the importance of NO in vascular function, the activity of eNOS is finely regulated at transcriptional and posttranscriptional level (Fleming & Busse, 2003). Although eNOS was initially characterized as a Ca2+-dependent enzyme and binding of the complex Ca2+-calmodulin plays a central role in the activation of eNOS, NO production is also modulated by phosphorylation and protein-protein interactions (Mount *et al.*, 2007; Rafikov *et al.*, 2011). In this context, the sub-cellular targeting of eNOS is a key process in the regulation of NO production. Two functional pools of eNOS have been identified in vascular endothelial cells: one associated to Golgi complex and other located at caveolae, a subset of invaginated plasmalemmal rafts where the function of key signaling proteins is coordinated (Govers & Rabelink, 2001; Goligorsky *et al.*, 2002; Michel & Vanhoutte, 2010), which provides eNOS with a special proximity to signaling molecules, such as calmodulin, Ca2+ channels, BKCa channels and plasma membrane Ca2+ pumps (Darby *et al.*, 2000; Wang *et al.*, 2005). Although both pools of eNOS have been demonstrated to be functional, it is widely recognized that the integrity of caveolae is critical for the control of Ca2+-mediated activation of NO production. In caveolae, eNOS is found in an inhibitory association with caveolin-1, an integral membrane protein of this signaling microdomain, and the interaction of eNOS with calcium-calmodulin releases the enzyme from its inhibitory association with caveolin-1 (Govers & Rabelink, 2001; Goligorsky *et al.*, 2002; Michel & Vanhoutte, 2010).

The eNOS localization at caveolae seems to be essential for the regulation of eNOS function by controlling L-arginine substrate supply. Typically, regulation of L-arginine availability has been under-appreciated, since intracellular L-arginine concentration is saturating from

highlights the participation of COXs and PGs in the regulation of vascular function.

smooth muscle contractile machinery (Bolz *et al.*, 1999; Bolz *et al.*, 2003).

**3.2 Nitric oxide** 

prostacyclins (PGI2), PGE2, PGD2, PGF2α and thromboxane A2 (TXA2), respectively (Simmons *et al.*, 2004; Gryglewski, 2008). The presence of the different PG synthases varies from tissue to tissue. Finally, PGs are released to the extracellular space and exert their physiological effects by acting on specific membrane receptors (Norel, 2007), as depicted in Figure 1. Then, the production of prostanoids is triggered by an increase in intracellular Ca2+ concentration and the key reaction of this complex enzymatic cascade is catalyzed by the enzymes COXs (Figure 1).

Fig. 1. Biosynthetic pathway of prostaglandins (PGs). An increase in intracellular Ca2+ concentration activates the production of arachidonic acid (AA) by phospholipase A2 (PLA2) from cell membrane phospholipids. The enzymes cyclooxygenase-1 (COX-1) or cyclooxygenase-2 (COX-2) convert AA into the endoperoxide PGH2, which is then metabolized by several synthases to PGs PGD2, PGE2, PGF2α, TXA2 and PGI2 (prostacyclin). Each PG acts on specific membrane receptors located in endothelial and/or smooth muscle cells. The transduction pathways activated by PGs are also depicted in the figure.

COX-1 and COX-2 are very similar and show a 60% homology. However, COX-1 is expressed constitutively, whereas the expression of COX-2 is inducible, since the levels of this COX isoform are very low in normal conditions and its expression increases in response to pro-inflammatory stimuli (Simmons *et al.*, 2004). Consistent with this, in normal physiological conditions, vascular endothelial and smooth muscle cells express COX-1 (Vanhoutte, 2009). In these cells, COX-1 mainly leads to the production of PGI2, which induces the relaxation of smooth muscle cells by the stimulation of IP receptors (Figure 1) (Gryglewski, 2008; Vanhoutte, 2009). In contrast to COX-1, expression of COX-2 in normal blood vessels is very low (Crofford *et al.*, 1994; Schonbeck *et al.*, 1999). However, Topper et al. (Topper *et al.*, 1996) found that laminar shear stress, but not turbulent flow, up-regulates the levels of COX-2 expression in cultures of vascular endothelial cells. Laminar shear stress is a highly relevant stimulus that is involved in the tonic control of vasomotor tone, which highlights the participation of COXs and PGs in the regulation of vascular function.

#### **3.2 Nitric oxide**

68 The Cardiovascular System – Physiology, Diagnostics and Clinical Implications

prostacyclins (PGI2), PGE2, PGD2, PGF2α and thromboxane A2 (TXA2), respectively (Simmons *et al.*, 2004; Gryglewski, 2008). The presence of the different PG synthases varies from tissue to tissue. Finally, PGs are released to the extracellular space and exert their physiological effects by acting on specific membrane receptors (Norel, 2007), as depicted in Figure 1. Then, the production of prostanoids is triggered by an increase in intracellular Ca2+ concentration and the key reaction of this complex enzymatic cascade is catalyzed by the

Fig. 1. Biosynthetic pathway of prostaglandins (PGs). An increase in intracellular Ca2+ concentration activates the production of arachidonic acid (AA) by phospholipase A2 (PLA2) from cell membrane phospholipids. The enzymes cyclooxygenase-1 (COX-1) or cyclooxygenase-2 (COX-2) convert AA into the endoperoxide PGH2, which is then

cells. The transduction pathways activated by PGs are also depicted in the figure.

metabolized by several synthases to PGs PGD2, PGE2, PGF2α, TXA2 and PGI2 (prostacyclin). Each PG acts on specific membrane receptors located in endothelial and/or smooth muscle

COX-1 and COX-2 are very similar and show a 60% homology. However, COX-1 is expressed constitutively, whereas the expression of COX-2 is inducible, since the levels of this COX isoform are very low in normal conditions and its expression increases in response to pro-inflammatory stimuli (Simmons *et al.*, 2004). Consistent with this, in normal physiological conditions, vascular endothelial and smooth muscle cells express COX-1 (Vanhoutte, 2009). In these cells, COX-1 mainly leads to the production of PGI2, which

enzymes COXs (Figure 1).

Probably, the most relevant intercellular communication signal in vascular physiology is the endothelium-dependent NO production. NO is a potent vasodilator synthesized by the enzyme NO synthase (NOS) (Moncada *et al.*, 1991). The substrates for NOS-mediated NO production are the amino acid L-arginine, molecular oxygen and nicotinamide adenine dinucleotide phosphate (NADPH). Three isoforms of NOS have been described: endothelial NOS (eNOS), neuronal NOS (nNOS) and inducible NOS (iNOS) (Moncada *et al.*, 1991; Alderton *et al.*, 2001). The enzyme expressed in endothelial cells (eNOS) is the main NOS isoform found in the vascular system in normal conditions. The NO released by endothelial cells elicits the relaxation of the underlying vascular smooth muscle cells mainly through the initiation of the signaling cascade cGMP/PKG by activation of soluble guanylate cyclase, which has been ascribed as the primary receptor of NO (Moncada *et al.*, 1991). Although certainly the cGMP-dependent signaling pathway has several targets in the vessel wall, the relaxation induced by NO is mainly associated with a reduction in the Ca2+ sensitivity of smooth muscle contractile machinery (Bolz *et al.*, 1999; Bolz *et al.*, 2003).

Consistent with the importance of NO in vascular function, the activity of eNOS is finely regulated at transcriptional and posttranscriptional level (Fleming & Busse, 2003). Although eNOS was initially characterized as a Ca2+-dependent enzyme and binding of the complex Ca2+-calmodulin plays a central role in the activation of eNOS, NO production is also modulated by phosphorylation and protein-protein interactions (Mount *et al.*, 2007; Rafikov *et al.*, 2011). In this context, the sub-cellular targeting of eNOS is a key process in the regulation of NO production. Two functional pools of eNOS have been identified in vascular endothelial cells: one associated to Golgi complex and other located at caveolae, a subset of invaginated plasmalemmal rafts where the function of key signaling proteins is coordinated (Govers & Rabelink, 2001; Goligorsky *et al.*, 2002; Michel & Vanhoutte, 2010), which provides eNOS with a special proximity to signaling molecules, such as calmodulin, Ca2+ channels, BKCa channels and plasma membrane Ca2+ pumps (Darby *et al.*, 2000; Wang *et al.*, 2005). Although both pools of eNOS have been demonstrated to be functional, it is widely recognized that the integrity of caveolae is critical for the control of Ca2+-mediated activation of NO production. In caveolae, eNOS is found in an inhibitory association with caveolin-1, an integral membrane protein of this signaling microdomain, and the interaction of eNOS with calcium-calmodulin releases the enzyme from its inhibitory association with caveolin-1 (Govers & Rabelink, 2001; Goligorsky *et al.*, 2002; Michel & Vanhoutte, 2010).

The eNOS localization at caveolae seems to be essential for the regulation of eNOS function by controlling L-arginine substrate supply. Typically, regulation of L-arginine availability has been under-appreciated, since intracellular L-arginine concentration is saturating from

Control and Coordination of Vasomotor Tone in the Microcirculation 71

Another mechanism that has emerged as an important source of L-arginine supply for NO production is the regeneration of L-arginine from the other product of the eNOS-catalyzed reaction, L-citrulline. This regeneration is catalyzed by the enzymes argininosuccinate synthase (ASS) and argininosuccinate lyase (ASL), which are mostly expressed in caveolae in endothelial cells (Flam *et al.*, 2001; Solomonson *et al.*, 2003) (Figure 2). Interestingly, addition of exogenous L-citrulline results in a larger increase in endothelial NO production than that observed with exogenous L-arginine, without a proportional increase in intracellular L-arginine (Solomonson *et al.*, 2003), suggesting that recycling of L-citrulline to L-arginine is channeled directly to synthesize NO (Figure 2). In addition, it was estimated that under maximum stimulation of NO production with bradykinin, but not in unstimulated conditions, approximately 80% of the eNOS-catalyzed L-arginine was supplied by the recycling of L-citrulline (Solomonson *et al.*, 2003). These findings indicate that eNOS activation is functionally coupled with the L-citrulline recycling system (ASS and ASL) in caveolae (Figure 2). Therefore, NO production seems to be regulated by a complex interaction between different pools of L-arginine, where direct channeling to eNOS of the Larginine regenerated from L-citrulline by the coordinated action of the enzymes ASS and

Although the development of knockout animals has demonstrated the importance of the multiple functions of NO along the whole vascular system, it has become apparent that the relevance of NO in the control of vasomotor tone depends on vessel size. Accordingly, NO is the primary endothelium-dependent vasodilator signal in large, conduit vessels (Shimokawa *et al.*, 1996). However, an additional vasodilator component has also been identified in small resistance arteries and arterioles (Suzuki *et al.*, 1992; Murphy & Brayden, 1995). In these vessels, blockade of NO and PG production only attenuates the response to endothelium-dependent vasodilators such as acetylcholine (ACh) or bradykinin (Vanhoutte, 2004). The relaxant pathway resistant to NOS and COX blockers is associated with smooth muscle hyperpolarization, and thereby, it was attributed to the release of an endotheliumderived hyperpolarizing factor (EDHF). The chemical nature of EDHF remains controversial and seems to depend on vessel size, vascular territory, and species (Vanhoutte, 2004). In this context, several EDHF candidates have been proposed, such as K+ ions (Edwards *et al.*, 1998), epoxyeicosatrienoic acids (EETs) (Archer *et al.*, 2003; Fleming, 2004), hydrogen peroxide (Shimokawa & Morikawa, 2005), and C-type natriuretic peptide (CNP) (Chauhan *et al.*, 2003; Ahluwalia & Hobbs, 2005). However, in most cases, the EDHF-mediated smooth muscle hyperpolarization and vasodilation has been shown to be sensitive to simultaneous blockade of SKCa and IKCa (Doughty *et al.*, 1999; Ghisdal & Morel, 2001; Crane *et al.*, 2003; Eichler *et al.*, 2003; Hilgers *et al.*, 2006). Interestingly, these K+ channels have been reported to be located in two different subcellular domains. While SKCa channels are found in caveolae (Absi *et al.*, 2007; Rath *et al.*, 2009), IKCa channels were proposed to be expressed in the abluminal side of endothelial cells (Figure 3), facing Na+ pumps and Kir channels situated in smooth muscle cells (Edwards *et al.*, 1998; Dora *et al.*, 2008). Then, the opening of IKCa channels may increase the K+ ion concentration in the myoendothelial space, which may couple endothelial cell IKCa signaling to Na+ pump- and Kir channel-mediated smooth muscle

ASL is likely to play a central role (Figure 2).

**3.3 Endothelium-derived hyperpolarizing factor** 

hyperpolarization (Edwards *et al.*, 1998; Dora *et al.*, 2008) (Figure 3).

the perspective of eNOS kinetics (Km = ~5 µM) (Harrison, 1997). However, several reports indicate that increments in extracellular L-arginine levels can enhance NO production in endothelial cells (Zani & Bohlen, 2005; Kakoki *et al.*, 2006), despite a saturating intracellular L-arginine concentration, which was termed as the "Arginine Paradox" (McDonald *et al.*, 1997). This control of NO production by substrate suggests that intracellular L-arginine is not fully available for eNOS, whereas extracellular L-arginine is preferentially delivered to the enzyme. Consistent with this notion, NO production seems to be coupled to L-arginine uptake, because the main carrier that transports 60 – 80% of L-arginine across the plasma membrane of endothelial cells, the cationic amino acid transporter-1 (CAT-1), was found to co-localize with eNOS in caveolae (McDonald *et al.*, 1997) (Figure 2). Interestingly, it was reported that eNOS interacts directly with CAT-1 in bovine aortic endothelial cells (BAECs), and apparently, the eNOS–CAT-1 association in addition to facilitate the delivery of extracellular L-arginine for NO generation, also enhances the eNOS enzymatic activity by increasing the activating phosphorylation of the enzyme at serine 1179 and 635, and by decreasing the association of eNOS with caveolin-1 (Li *et al.*, 2005).

Fig. 2. Local control of eNOS activity by L-arginine. eNOS synthesizes nitric oxide (NO) and the byproduct L-citrulline from L-arginine. The eNOS localization at signaling microdomains known as caveolae provides to this enzyme with a direct, local source of Larginine. In caveolae, eNOS is in direct association with the main carrier of L-arginine in endothelial cells, the cationic amino acid transporter-1 (CAT-1), and is also associated with the enzymes argininosuccinate synthase (ASS) and argininosuccinate lyase (ASL) that regenerate L-arginine from L-citrulline.

the perspective of eNOS kinetics (Km = ~5 µM) (Harrison, 1997). However, several reports indicate that increments in extracellular L-arginine levels can enhance NO production in endothelial cells (Zani & Bohlen, 2005; Kakoki *et al.*, 2006), despite a saturating intracellular L-arginine concentration, which was termed as the "Arginine Paradox" (McDonald *et al.*, 1997). This control of NO production by substrate suggests that intracellular L-arginine is not fully available for eNOS, whereas extracellular L-arginine is preferentially delivered to the enzyme. Consistent with this notion, NO production seems to be coupled to L-arginine uptake, because the main carrier that transports 60 – 80% of L-arginine across the plasma membrane of endothelial cells, the cationic amino acid transporter-1 (CAT-1), was found to co-localize with eNOS in caveolae (McDonald *et al.*, 1997) (Figure 2). Interestingly, it was reported that eNOS interacts directly with CAT-1 in bovine aortic endothelial cells (BAECs), and apparently, the eNOS–CAT-1 association in addition to facilitate the delivery of extracellular L-arginine for NO generation, also enhances the eNOS enzymatic activity by increasing the activating phosphorylation of the enzyme at serine 1179 and 635, and by

Fig. 2. Local control of eNOS activity by L-arginine. eNOS synthesizes nitric oxide (NO) and

microdomains known as caveolae provides to this enzyme with a direct, local source of Larginine. In caveolae, eNOS is in direct association with the main carrier of L-arginine in endothelial cells, the cationic amino acid transporter-1 (CAT-1), and is also associated with the enzymes argininosuccinate synthase (ASS) and argininosuccinate lyase (ASL) that

the byproduct L-citrulline from L-arginine. The eNOS localization at signaling

regenerate L-arginine from L-citrulline.

decreasing the association of eNOS with caveolin-1 (Li *et al.*, 2005).

Another mechanism that has emerged as an important source of L-arginine supply for NO production is the regeneration of L-arginine from the other product of the eNOS-catalyzed reaction, L-citrulline. This regeneration is catalyzed by the enzymes argininosuccinate synthase (ASS) and argininosuccinate lyase (ASL), which are mostly expressed in caveolae in endothelial cells (Flam *et al.*, 2001; Solomonson *et al.*, 2003) (Figure 2). Interestingly, addition of exogenous L-citrulline results in a larger increase in endothelial NO production than that observed with exogenous L-arginine, without a proportional increase in intracellular L-arginine (Solomonson *et al.*, 2003), suggesting that recycling of L-citrulline to L-arginine is channeled directly to synthesize NO (Figure 2). In addition, it was estimated that under maximum stimulation of NO production with bradykinin, but not in unstimulated conditions, approximately 80% of the eNOS-catalyzed L-arginine was supplied by the recycling of L-citrulline (Solomonson *et al.*, 2003). These findings indicate that eNOS activation is functionally coupled with the L-citrulline recycling system (ASS and ASL) in caveolae (Figure 2). Therefore, NO production seems to be regulated by a complex interaction between different pools of L-arginine, where direct channeling to eNOS of the Larginine regenerated from L-citrulline by the coordinated action of the enzymes ASS and ASL is likely to play a central role (Figure 2).

#### **3.3 Endothelium-derived hyperpolarizing factor**

Although the development of knockout animals has demonstrated the importance of the multiple functions of NO along the whole vascular system, it has become apparent that the relevance of NO in the control of vasomotor tone depends on vessel size. Accordingly, NO is the primary endothelium-dependent vasodilator signal in large, conduit vessels (Shimokawa *et al.*, 1996). However, an additional vasodilator component has also been identified in small resistance arteries and arterioles (Suzuki *et al.*, 1992; Murphy & Brayden, 1995). In these vessels, blockade of NO and PG production only attenuates the response to endothelium-dependent vasodilators such as acetylcholine (ACh) or bradykinin (Vanhoutte, 2004). The relaxant pathway resistant to NOS and COX blockers is associated with smooth muscle hyperpolarization, and thereby, it was attributed to the release of an endotheliumderived hyperpolarizing factor (EDHF). The chemical nature of EDHF remains controversial and seems to depend on vessel size, vascular territory, and species (Vanhoutte, 2004). In this context, several EDHF candidates have been proposed, such as K+ ions (Edwards *et al.*, 1998), epoxyeicosatrienoic acids (EETs) (Archer *et al.*, 2003; Fleming, 2004), hydrogen peroxide (Shimokawa & Morikawa, 2005), and C-type natriuretic peptide (CNP) (Chauhan *et al.*, 2003; Ahluwalia & Hobbs, 2005). However, in most cases, the EDHF-mediated smooth muscle hyperpolarization and vasodilation has been shown to be sensitive to simultaneous blockade of SKCa and IKCa (Doughty *et al.*, 1999; Ghisdal & Morel, 2001; Crane *et al.*, 2003; Eichler *et al.*, 2003; Hilgers *et al.*, 2006). Interestingly, these K+ channels have been reported to be located in two different subcellular domains. While SKCa channels are found in caveolae (Absi *et al.*, 2007; Rath *et al.*, 2009), IKCa channels were proposed to be expressed in the abluminal side of endothelial cells (Figure 3), facing Na+ pumps and Kir channels situated in smooth muscle cells (Edwards *et al.*, 1998; Dora *et al.*, 2008). Then, the opening of IKCa channels may increase the K+ ion concentration in the myoendothelial space, which may couple endothelial cell IKCa signaling to Na+ pump- and Kir channel-mediated smooth muscle hyperpolarization (Edwards *et al.*, 1998; Dora *et al.*, 2008) (Figure 3).

Control and Coordination of Vasomotor Tone in the Microcirculation 73

vasodilator components may be confounded. In this context, it is interesting to note that**,** as mentioned above, the EDHF-mediated response is typically studied in presence of NOS and COX blockers. However, NOS inhibition with analogues of L-arginine is a slow, timedependent process and, on occasion, blockade of NO production with these drugs has been observed to be incomplete (Vanheel & Van de Voorde, 2000; Figueroa *et al.*, 2001; Chauhan *et al.*, 2003; Stoen *et al.*, 2003; Stankevicius *et al.*, 2006), and then, the residual NO production observed in presence of NOS inhibitors may contribute to the vasodilation associated with the smooth muscle hyperpolarization attributes to EDHF. In addition, the findings reported recently by Gaete et al. (Gaete *et al.*, 2011) are another important point to take into account in the interaction between EDHF and NO. In this work Gaete et al., demonstrated that SKCa and IKCa channels control the Ca2+-dependent NO release, and thereby, the inactivation of these K+ channels is associated with an increase in NAD(P)H oxidase-mediated superoxide production, which leads to the inhibition of eNOS primarily by its phosphorylation at threonine 495 (Gaete *et al.*, 2011). These findings highlight the relevance of these K+ channels in the control of vascular function and indicate that the participation of superoxide in the

EDHF-mediated response associated to SKCa and IKCa channels must be evaluated.

cardiovascular diseases such as hypertension.

**4. Gap junction communication in the vascular function** 

Furthermore, the regulation of NO and EDHF is different depending on gender. In male animals, NO is the major endothelium-dependent vasodilator signal, but in female EDHF prevails over NO or PGI2 (Scotland *et al.*, 2005). In this context, it is interesting to note that estrogen enhances the EDHF-mediated vasodilation in response to flow (Huang *et al.*, 2001), which suggests that the EDHF-dependent signaling pathway may be more important in the control of blood pressure in female than in male animals. This idea was confirmed using an eNOS/COX-1 double knockout. Deletion of eNOS and COX-1 did not alter the mean arterial blood pressure in female mice, whereas the double knockout resulted in hypertension in male mice (Scotland *et al.*, 2005). In these animals, the endothelium-dependent relaxation was intact in resistance vessels of female mice and was mediated by the smooth muscle hyperpolarization (Scotland *et al.*, 2005), strongly supporting that EDHF plays a predominant role in the tonic control of blood pressure in female. These data suggest that EDHF rather than NO may underlie the higher resistance of premenopausal females to

Gap junctions are intercellular channels that directly connect the cytoplasm of neighboring cells, allowing the passage of current or molecules smaller than ~1.4 nm of diameter such as metabolites (e.g., ADP, glucose, glutamate and glutathione) or second messengers (e.g., Ca2+, cAMP and IP3) (Evans & Martin, 2002; Saez *et al.*, 2003). These intercellular channels are made up by a protein family known as connexins (Cx), which are named according to their predicted molecular mass expressed in kDa. Connexin proteins have four transmembrane domains with the N- and C-termini located on the cytoplasmic membrane face. The radial arrangement of six connexins around a central pore makes a connexon or hemichannel, and the association in the plasma membrane of two hemichannels provided by adjacent cells forms an intercellular gap junction channel (Evans & Martin, 2002; Saez *et al.*, 2003). It is noteworthy that independent hemichannels can also remain unpaired and functional, which have been recognized to release paracrine signals such as ATP, PGE2 or NAD+ (Goodenough & Paul, 2003; Cherian *et al.*, 2005; Saez *et al.*, 2005). The importance of

Fig. 3. K+ channel distribution and endothelium-dependent vasodilation. Ca2+-activated K+ channels of small (SKCa) and intermediate (IKCa) conductance may contribute to the vasodilation associated with an endothelium-mediated smooth muscle hyperpolarization (response typically attributed to an endothelium-derive hyperpolarizing factor, EDHF) by two pathways. First, the hyperpolarization induced by activation of SKCa and IKCa is transmitted electrotonically to the underlying smooth muscle cells (SMC) through the gap junctions located at discrete points of contact between endothelial and smooth muscle cells, structure known as myoendothelial junctions (MEJ). In addition, the small increase in extracellular K+ concentration (<20 mM) resulting from the opening of the IKCa found at the abluminal side of endothelial cells (EC) may activate inward rectifying K+ channels (Kir) and Na+ pump in smooth muscle cells. Between SMC and EC is found the internal elastic lamina (IEL).

Notwithstanding the smooth muscle hyperpolarization is considered to be the hallmark of EDHF action (Vanhoutte, 2004), it is important to note that hyperpolarization of the vessel wall is not a unique characteristic of EDHF. In several vessel preparations, in addition to a reduced Ca2+ sensitivity of the contractile machinery, the NO-dependent vasodilation has also been associated with smooth muscle hyperpolarization (Cohen *et al.*, 1997; Lang & Watson, 1998). Furthermore, consistent with a NO-mediated hyperpolarization, NO has been reported to activate BKCa, Kir and KATP channels on the smooth muscle cells and endothelial cells directly or through the activation of cGMP production (Bolotina *et al.*, 1994; Abderrahmane *et al.*, 1998; Lee & Kang, 2001; Si *et al.*, 2002; Schubert *et al.*, 2004). Therefore, NO and EDHF are not only complementary, but also additive and the effect of both

Fig. 3. K+ channel distribution and endothelium-dependent vasodilation. Ca2+-activated K+ channels of small (SKCa) and intermediate (IKCa) conductance may contribute to the vasodilation associated with an endothelium-mediated smooth muscle hyperpolarization (response typically attributed to an endothelium-derive hyperpolarizing factor, EDHF) by two pathways. First, the hyperpolarization induced by activation of SKCa and IKCa is transmitted electrotonically to the underlying smooth muscle cells (SMC) through the gap junctions located at discrete points of contact between endothelial and smooth muscle cells, structure known as myoendothelial junctions (MEJ). In addition, the small increase in extracellular K+ concentration (<20 mM) resulting from the opening of the IKCa found at the abluminal side of endothelial cells (EC) may activate inward rectifying K+ channels (Kir) and Na+ pump in smooth muscle cells. Between SMC and EC is found the internal elastic lamina

Notwithstanding the smooth muscle hyperpolarization is considered to be the hallmark of EDHF action (Vanhoutte, 2004), it is important to note that hyperpolarization of the vessel wall is not a unique characteristic of EDHF. In several vessel preparations, in addition to a reduced Ca2+ sensitivity of the contractile machinery, the NO-dependent vasodilation has also been associated with smooth muscle hyperpolarization (Cohen *et al.*, 1997; Lang & Watson, 1998). Furthermore, consistent with a NO-mediated hyperpolarization, NO has been reported to activate BKCa, Kir and KATP channels on the smooth muscle cells and endothelial cells directly or through the activation of cGMP production (Bolotina *et al.*, 1994; Abderrahmane *et al.*, 1998; Lee & Kang, 2001; Si *et al.*, 2002; Schubert *et al.*, 2004). Therefore, NO and EDHF are not only complementary, but also additive and the effect of both

(IEL).

vasodilator components may be confounded. In this context, it is interesting to note that**,** as mentioned above, the EDHF-mediated response is typically studied in presence of NOS and COX blockers. However, NOS inhibition with analogues of L-arginine is a slow, timedependent process and, on occasion, blockade of NO production with these drugs has been observed to be incomplete (Vanheel & Van de Voorde, 2000; Figueroa *et al.*, 2001; Chauhan *et al.*, 2003; Stoen *et al.*, 2003; Stankevicius *et al.*, 2006), and then, the residual NO production observed in presence of NOS inhibitors may contribute to the vasodilation associated with the smooth muscle hyperpolarization attributes to EDHF. In addition, the findings reported recently by Gaete et al. (Gaete *et al.*, 2011) are another important point to take into account in the interaction between EDHF and NO. In this work Gaete et al., demonstrated that SKCa and IKCa channels control the Ca2+-dependent NO release, and thereby, the inactivation of these K+ channels is associated with an increase in NAD(P)H oxidase-mediated superoxide production, which leads to the inhibition of eNOS primarily by its phosphorylation at threonine 495 (Gaete *et al.*, 2011). These findings highlight the relevance of these K+ channels in the control of vascular function and indicate that the participation of superoxide in the EDHF-mediated response associated to SKCa and IKCa channels must be evaluated.

Furthermore, the regulation of NO and EDHF is different depending on gender. In male animals, NO is the major endothelium-dependent vasodilator signal, but in female EDHF prevails over NO or PGI2 (Scotland *et al.*, 2005). In this context, it is interesting to note that estrogen enhances the EDHF-mediated vasodilation in response to flow (Huang *et al.*, 2001), which suggests that the EDHF-dependent signaling pathway may be more important in the control of blood pressure in female than in male animals. This idea was confirmed using an eNOS/COX-1 double knockout. Deletion of eNOS and COX-1 did not alter the mean arterial blood pressure in female mice, whereas the double knockout resulted in hypertension in male mice (Scotland *et al.*, 2005). In these animals, the endothelium-dependent relaxation was intact in resistance vessels of female mice and was mediated by the smooth muscle hyperpolarization (Scotland *et al.*, 2005), strongly supporting that EDHF plays a predominant role in the tonic control of blood pressure in female. These data suggest that EDHF rather than NO may underlie the higher resistance of premenopausal females to cardiovascular diseases such as hypertension.

#### **4. Gap junction communication in the vascular function**

Gap junctions are intercellular channels that directly connect the cytoplasm of neighboring cells, allowing the passage of current or molecules smaller than ~1.4 nm of diameter such as metabolites (e.g., ADP, glucose, glutamate and glutathione) or second messengers (e.g., Ca2+, cAMP and IP3) (Evans & Martin, 2002; Saez *et al.*, 2003). These intercellular channels are made up by a protein family known as connexins (Cx), which are named according to their predicted molecular mass expressed in kDa. Connexin proteins have four transmembrane domains with the N- and C-termini located on the cytoplasmic membrane face. The radial arrangement of six connexins around a central pore makes a connexon or hemichannel, and the association in the plasma membrane of two hemichannels provided by adjacent cells forms an intercellular gap junction channel (Evans & Martin, 2002; Saez *et al.*, 2003). It is noteworthy that independent hemichannels can also remain unpaired and functional, which have been recognized to release paracrine signals such as ATP, PGE2 or NAD+ (Goodenough & Paul, 2003; Cherian *et al.*, 2005; Saez *et al.*, 2005). The importance of

Control and Coordination of Vasomotor Tone in the Microcirculation 75

the cytoplasmic Ca2+ concentration and Ca2+ sensitivity of the contractile apparatus. Intracellular Ca2+ concentration is controlled by the smooth muscle cell membrane potential. Then, gap junctions play a central role integrating the smooth muscle cell function because these intercellular channels synchronize changes in both membrane potential and intracellular Ca2+ between adjacent smooth muscle cells (Christ *et al.*, 1991; Christ *et al.*, 1992;

In addition, gap junction communication of vascular smooth muscle cells seems to be involved in the development of myogenic vasomotor tone in resistance arteries (Lagaud *et al.*, 2002; Earley *et al.*, 2004). Interestingly, the participation of gap junction in this process is not related to synchronization of Ca2+ signaling, but rather to earlier signaling events such as coordination of the smooth muscle cell-depolarization or directly the mechanosensitivity of the vascular smooth muscle. This notion is supported by the fact that the gap junctions and connexin hemichannels inhibitors Gap27 (a connexin mimetic peptide) or 18αglycyrrhetinic acid, in addition to block Ca2+ influx and vasoconstriction in mesenteric resistance arteries, also prevented the pressure-induced smooth muscle cell depolarization (Earley *et al.*, 2004). It is important to note that Gap27 and 18α-glycyrrhetinic acid are two well-known gap junction blockers, but they also block connexin-formed hemichannels, which indicates that hemichannels may also be involved in the development of the myogenic response. In any case, the involvement of Cx43-based channels in the control of vasomotor tone is consistent with the finding that tensile stretch increased the expression of this connexin as well as gap junction intercellular communication in vascular smooth muscle cells (Cowan *et al.*, 1998). Interestingly, this response was mediated by the formation of reactive oxygen species (Cowan *et al.*, 1998; Cowan *et al.*, 2003), which has been reported to contribute to the initiation of the myogenic constriction in mouse-tail arterioles (Nowicki

Cx43 has also been involved in the regulation of cell proliferation and migration in the vasculature (Polacek *et al.*, 1997; Yeh *et al.*, 1997; Kwak *et al.*, 2001), which can be appreciated in Cx43-deficient smooth muscle cells. Damage of carotid artery by vascular occlusion or wire injury resulted in an increase in neointima and adventitia formation in smooth muscle cell Cx43 specific knockout mice as compared to wild type animals (Liao *et al.*, 2007), suggesting an accelerated growth of smooth muscle cell with the Cx43 deletion, which was further confirmed using cultured cells. Nevertheless, in apparent opposition to these findings, Chadjichristos et al. (Chadjichristos *et al.*, 2006) show that in heterozygous Cx43 knockout mice the neointimal formation was reduced. However, in those animals, Cx43 was reduced from all cell types expressing Cx43 and the experiments included a high-fat diet, which may have influenced the result by either vascular adaptive response to the diet or complex interactions between different cell types. Although the participation of Cx43 in neointimal formation demands further investigation, these data highlight the relevance of

Cx43 in the feedback control pathways necessary for vascular morphogenesis.

The endothelium plays a key role in the tonic control of blood pressure and the development of knockout animals of vascular connexins has disclosed that gap junction communication of endothelial cells is essential in the coordination and integration of

**4.2 Gap junctions in vascular endothelium** 

Christ *et al.*, 1996).

*et al.*, 2001).

this mode of communication in the vasculature is just starting to be evaluated and, consistent with the participation of vascular hemichannels in paracrine signaling, human microvascular endothelial cell (HMEC-1) monolayers were found to release ATP through Cx43-formed hemichannels (Faigle *et al.*, 2008).

At least twenty connexin isoforms have been described in mammals and one cell type may express more than one connexin (Saez *et al.*, 2003). However, the expression of several connexins in one cell does not seem to be redundant, because gap junctions are not just simple channels that offer a low-resistance intercellular pathway, but connexins mediate highly specific cell-to-cell signaling pathways, and the molecular selectivity as well as subcellular localization differs among connexins (Saez *et al.*, 2003; Figueroa *et al.*, 2004; Locke *et al.*, 2005). Thus, although these proteins may have some overlap in function, they work in concert (Simon & Goodenough, 1998; Figueroa *et al.*, 2004, 2006; Haefliger *et al.*, 2006) and, consequently, it has been observed that many times the function of one connexin cannot be replaced by other connexin isoform (White, 2003; Haefliger *et al.*, 2006; Zheng-Fischhofer *et al.*, 2006; Wolfle *et al.*, 2007). In addition, hemichannels can be composed by one or a mixture of connexin proteins, which provides an additional mechanism for fine regulation of gap junction-mediated signaling processes (White & Bruzzone, 1996; He *et al.*, 1999; Beyer *et al.*, 2000; Cottrell *et al.*, 2002; Moreno, 2004).

Five connexin proteins have been found to be expressed in the vasculature: Cx32, Cx37, Cx40, Cx43, and Cx45 (Severs *et al.*, 2001; Figueroa *et al.*, 2004; Haefliger *et al.*, 2004; Okamoto *et al.*, 2009). The expression of connexins in the different cell types of the vessel wall is not uniform and vary with vessel size, vascular territory, and species (van Kempen *et al.*, 1995; van Kempen & Jongsma, 1999; Hill *et al.*, 2002). In most cases, Cx45 is only observed in smooth muscle cells and has mainly been detected in brain vessels (Kruger *et al.*, 2000; Li & Simard, 2001). In contrast, the expression of Cx32 and Cx37 seems to be restricted to the endothelium (Gabriels & Paul, 1998; van Kempen & Jongsma, 1999; Severs *et al.*, 2001; Okamoto *et al.*, 2009), but Cx37 has also been detected in smooth muscle cells (Rummery *et al.*, 2002). Although Cx40 and Cx43 may be expressed in both cell types (Little *et al.*, 1995; Gabriels & Paul, 1998; van Kempen & Jongsma, 1999; Severs *et al.*, 2001), Cx40 is located predominately in endothelial cells (Gabriels & Paul, 1998; van Kempen & Jongsma, 1999) and Cx43 is the most prominent gap junction protein found in smooth muscle cells (van Kempen & Jongsma, 1999). It should be noted, however, that in mouse, Cx40 is expressed exclusively in the endothelium (de Wit *et al.*, 2000; Figueroa *et al.*, 2003; Figueroa & Duling, 2008).

In addition to connexins, another family of three members of membrane proteins named pannexins (Panxs 1-3) has been documented (Bruzzone *et al.*, 2003). Apparently, pannexins only form hemichannels, and then, the main function of pannexin-based channels is paracrine or autocrine communication (Locovei *et al.*, 2006). Although connexins and pannexins share a similar membrane topology, their amino acid sequences present only a 16% homology (Bruzzone *et al.*, 2003). Only the expression of Panx-1 has been identified in blood vessels at the moment and recently this pannexin was found to be involved in the activation of the vasoconstrictor response mediated by α1-adrenoceptor stimulation (Billaud *et al.*, 2011).

#### **4.1 Gap junctions in vascular smooth muscle**

Coordination of vasomotor signals among smooth muscle cells is critical for the function of blood vessels. As mentioned above, the contractile state of smooth muscle cells depends on

this mode of communication in the vasculature is just starting to be evaluated and, consistent with the participation of vascular hemichannels in paracrine signaling, human microvascular endothelial cell (HMEC-1) monolayers were found to release ATP through

At least twenty connexin isoforms have been described in mammals and one cell type may express more than one connexin (Saez *et al.*, 2003). However, the expression of several connexins in one cell does not seem to be redundant, because gap junctions are not just simple channels that offer a low-resistance intercellular pathway, but connexins mediate highly specific cell-to-cell signaling pathways, and the molecular selectivity as well as subcellular localization differs among connexins (Saez *et al.*, 2003; Figueroa *et al.*, 2004; Locke *et al.*, 2005). Thus, although these proteins may have some overlap in function, they work in concert (Simon & Goodenough, 1998; Figueroa *et al.*, 2004, 2006; Haefliger *et al.*, 2006) and, consequently, it has been observed that many times the function of one connexin cannot be replaced by other connexin isoform (White, 2003; Haefliger *et al.*, 2006; Zheng-Fischhofer *et al.*, 2006; Wolfle *et al.*, 2007). In addition, hemichannels can be composed by one or a mixture of connexin proteins, which provides an additional mechanism for fine regulation of gap junction-mediated signaling processes (White & Bruzzone, 1996; He *et al.*, 1999; Beyer *et al.*,

Five connexin proteins have been found to be expressed in the vasculature: Cx32, Cx37, Cx40, Cx43, and Cx45 (Severs *et al.*, 2001; Figueroa *et al.*, 2004; Haefliger *et al.*, 2004; Okamoto *et al.*, 2009). The expression of connexins in the different cell types of the vessel wall is not uniform and vary with vessel size, vascular territory, and species (van Kempen *et al.*, 1995; van Kempen & Jongsma, 1999; Hill *et al.*, 2002). In most cases, Cx45 is only observed in smooth muscle cells and has mainly been detected in brain vessels (Kruger *et al.*, 2000; Li & Simard, 2001). In contrast, the expression of Cx32 and Cx37 seems to be restricted to the endothelium (Gabriels & Paul, 1998; van Kempen & Jongsma, 1999; Severs *et al.*, 2001; Okamoto *et al.*, 2009), but Cx37 has also been detected in smooth muscle cells (Rummery *et al.*, 2002). Although Cx40 and Cx43 may be expressed in both cell types (Little *et al.*, 1995; Gabriels & Paul, 1998; van Kempen & Jongsma, 1999; Severs *et al.*, 2001), Cx40 is located predominately in endothelial cells (Gabriels & Paul, 1998; van Kempen & Jongsma, 1999) and Cx43 is the most prominent gap junction protein found in smooth muscle cells (van Kempen & Jongsma, 1999). It should be noted, however, that in mouse, Cx40 is expressed exclusively in the endothelium (de Wit *et al.*, 2000;

In addition to connexins, another family of three members of membrane proteins named pannexins (Panxs 1-3) has been documented (Bruzzone *et al.*, 2003). Apparently, pannexins only form hemichannels, and then, the main function of pannexin-based channels is paracrine or autocrine communication (Locovei *et al.*, 2006). Although connexins and pannexins share a similar membrane topology, their amino acid sequences present only a 16% homology (Bruzzone *et al.*, 2003). Only the expression of Panx-1 has been identified in blood vessels at the moment and recently this pannexin was found to be involved in the activation of the vasoconstrictor response mediated by α1-adrenoceptor stimulation (Billaud *et al.*, 2011).

Coordination of vasomotor signals among smooth muscle cells is critical for the function of blood vessels. As mentioned above, the contractile state of smooth muscle cells depends on

Cx43-formed hemichannels (Faigle *et al.*, 2008).

2000; Cottrell *et al.*, 2002; Moreno, 2004).

Figueroa *et al.*, 2003; Figueroa & Duling, 2008).

**4.1 Gap junctions in vascular smooth muscle** 

the cytoplasmic Ca2+ concentration and Ca2+ sensitivity of the contractile apparatus. Intracellular Ca2+ concentration is controlled by the smooth muscle cell membrane potential. Then, gap junctions play a central role integrating the smooth muscle cell function because these intercellular channels synchronize changes in both membrane potential and intracellular Ca2+ between adjacent smooth muscle cells (Christ *et al.*, 1991; Christ *et al.*, 1992; Christ *et al.*, 1996).

In addition, gap junction communication of vascular smooth muscle cells seems to be involved in the development of myogenic vasomotor tone in resistance arteries (Lagaud *et al.*, 2002; Earley *et al.*, 2004). Interestingly, the participation of gap junction in this process is not related to synchronization of Ca2+ signaling, but rather to earlier signaling events such as coordination of the smooth muscle cell-depolarization or directly the mechanosensitivity of the vascular smooth muscle. This notion is supported by the fact that the gap junctions and connexin hemichannels inhibitors Gap27 (a connexin mimetic peptide) or 18αglycyrrhetinic acid, in addition to block Ca2+ influx and vasoconstriction in mesenteric resistance arteries, also prevented the pressure-induced smooth muscle cell depolarization (Earley *et al.*, 2004). It is important to note that Gap27 and 18α-glycyrrhetinic acid are two well-known gap junction blockers, but they also block connexin-formed hemichannels, which indicates that hemichannels may also be involved in the development of the myogenic response. In any case, the involvement of Cx43-based channels in the control of vasomotor tone is consistent with the finding that tensile stretch increased the expression of this connexin as well as gap junction intercellular communication in vascular smooth muscle cells (Cowan *et al.*, 1998). Interestingly, this response was mediated by the formation of reactive oxygen species (Cowan *et al.*, 1998; Cowan *et al.*, 2003), which has been reported to contribute to the initiation of the myogenic constriction in mouse-tail arterioles (Nowicki *et al.*, 2001).

Cx43 has also been involved in the regulation of cell proliferation and migration in the vasculature (Polacek *et al.*, 1997; Yeh *et al.*, 1997; Kwak *et al.*, 2001), which can be appreciated in Cx43-deficient smooth muscle cells. Damage of carotid artery by vascular occlusion or wire injury resulted in an increase in neointima and adventitia formation in smooth muscle cell Cx43 specific knockout mice as compared to wild type animals (Liao *et al.*, 2007), suggesting an accelerated growth of smooth muscle cell with the Cx43 deletion, which was further confirmed using cultured cells. Nevertheless, in apparent opposition to these findings, Chadjichristos et al. (Chadjichristos *et al.*, 2006) show that in heterozygous Cx43 knockout mice the neointimal formation was reduced. However, in those animals, Cx43 was reduced from all cell types expressing Cx43 and the experiments included a high-fat diet, which may have influenced the result by either vascular adaptive response to the diet or complex interactions between different cell types. Although the participation of Cx43 in neointimal formation demands further investigation, these data highlight the relevance of Cx43 in the feedback control pathways necessary for vascular morphogenesis.

#### **4.2 Gap junctions in vascular endothelium**

The endothelium plays a key role in the tonic control of blood pressure and the development of knockout animals of vascular connexins has disclosed that gap junction communication of endothelial cells is essential in the coordination and integration of

Control and Coordination of Vasomotor Tone in the Microcirculation 77

Flow (i.e. shear stress) is one of the most important stimuli involved in the tonic regulation of vasomotor tone. Although the response to shear stress is thought to be mediated primarily by NO, shear stress has also been reported to activate an EDHF-dependent vasodilator response (Watanabe *et al.*, 2005), which suggests that a gap junction-mediated EDHF pathway may be involved in the tonic control of peripheral vascular resistance. Consistent with this idea, intrarenal infusion of connexin-mimetic peptides homologous to the second extracellular loop of Cx43 (43Gap 27) or Cx40 (40Gap 27) not only decreased basal renal blood flow, but also increased mean arterial blood pressure of rats, either in presence or absence of NOS and COX blockers (De Vriese *et al.*, 2002), suggesting that connexinmimetic peptides induced vasoconstriction by disrupting or reducing the response to a tonic

Longitudinal conduction of vasomotor responses provides an essential means of coordinating changes in diameter and flow distribution among vessels of the microcirculation. Vasomotor signals spread along the vessel length through gap junctions connecting cells of the vessel wall, and thereby, participate in the minute-to-minute coordination of vascular resistance by integrating function of proximal and distal vascular segments in the microcirculation (de Wit *et al.*, 2000; Figueroa *et al.*, 2004, 2006). Although vasoconstrictor responses are thought to be conducted by smooth muscle cells (Welsh & Segal, 1998; Bartlett & Segal, 2000; Budel *et al.*, 2003), the cellular pathway for conduction of vasodilator signals is more controversial and may be either exclusively by the endothelium (Emerson & Segal, 2000; Segal & Jacobs, 2001) or by both smooth muscle and endothelial cells (Bartlett & Segal, 2000; Budel *et al.*, 2003). The cellular pathway for conduction of vasomotor responses has been studied by selectively damaging a short segment of endothelial cells or smooth muscle cells by injection of an air bubble via a side branch (Bartlett & Segal, 2000; Figueroa *et al.*, 2007) or with a light-dye (fluorescein-conjugated dextran) treatment (Emerson & Segal, 2000). In feed arteries, selective damage of the endothelium completely blocked the ACh-induced conducted vasodilation (Emerson & Segal, 2000; Segal & Jacobs, 2001), but in arterioles, either damage of the endothelium or the smooth muscle did not affect the ACh-induced conducted responses (Bartlett & Segal, 2000; Budel *et al.*, 2003), which led to the proposal that the cellular pathway for conduction of vasodilations depends on the functional location of the vessel in the microvascular network (Segal, 2005). However, the cellular pathway of vasodilator signals may also depend on the stimulus that initiated the response, because, in contrast to ACh, selective damage of the endothelium blocked the vasodilation induced by bradykinin in arterioles (Welsh & Segal,

Direct measurements of membrane potential have shown that conducted vasomotor responses are associated with rapid propagation (milliseconds) of an electrical signal along the vessel length (Xia & Duling, 1995; Welsh & Segal, 1998; Emerson & Segal, 2000). Because many observations have revealed an exponential decay of the conducted electrical signal, it was proposed that longitudinal spread of vasomotor responses reflects the passive, electrotonic conduction of changes in membrane potential via gap junctions connecting cells of the vessel wall (Pacicca *et al.*, 1996; Welsh & Segal, 1998; Gustafsson & Holstein-Rathlou, 1999). Therefore, the decay of the conducted vasomotor responses along the vessel length

vasodilator stimulus such as shear stress.

1998; Budel *et al.*, 2003).

**5. Conduction of vasomotor responses** 

microvascular function. Vascular endothelial cells-specific deletion of Cx43 (VEC Cx43-/-) results in hypotension (Liao *et al.*, 2001) and, in contrast, ablation of Cx40 produces a hypertension associated with an irregular vasomotion (de Wit *et al.*, 2000; de Wit *et al.*, 2003; Figueroa & Duling, 2008) and a dysregulation of renin production (Krattinger *et al.*, 2007; Wagner *et al.*, 2007). Although deletion of Cx37 does not appear to alter vascular function or blood pressure (Figueroa & Duling, 2008), several polymorphisms of this connexin have been associated with myocardial infarction, coronary artery disease and atherosclerosis (Boerma *et al.*, 1999; Yamada *et al.*, 2002; Hirashiki *et al.*, 2003; Yamada *et al.*, 2004). In mice, Cx40 and Cx37 are primarily expressed in the endothelium, which emphasizes the importance of the endothelial cell-gap junction communication in the control of cardiovascular homeostasis.

Although the mechanistic bases of the hypotension observed in VEC Cx43-/- are still unknown, the plasma levels of angiotensin I and II as well as NO were elevated in these animals (Liao *et al.*, 2001), suggesting that a dysregulation of NO production may have been the responsible of the hypotension with the subsequent activation of the renin-angiotensin system. Also, it is interesting to note that shear stress up-regulates the expression of Cx43 in cultured endothelial cells (DePaola *et al.*, 1999; Bao *et al.*, 2000) and in the endothelium of rat cardiac valves (Inai *et al.*, 2004), which suggests that Cx43 may be involved in the response to mechanical stimuli.

#### **4.3 Gap junctions in smooth muscle-endothelium communication**

Smooth muscle cells and endothelial cells have also been found to be electrically and metabolically connected by gap junctions located at discrete points of contact between the two cell types at the myoendothelial junction (MEJ) (Beny & Pacicca, 1994; Little *et al.*, 1995; Emerson & Segal, 2000; Sandow *et al.*, 2003). This heterocellular communication seems to play a pivotal role in the Ca2+-mediated responses induced by endothelium-dependent vasodilators, such as ACh. As mentioned above, these vasodilator responses are typically paralleled by hyperpolarization of the underlying smooth muscle cells (Emerson & Segal, 2000; Goto *et al.*, 2002; Griffith, 2004), which has been attributed to the release of an EDHF (Vanhoutte, 2004; Feletou & Vanhoutte, 2009). However, the direct electrotonic transmission of a hyperpolarizing current from the endothelial cells to the smooth muscle cells via myoendothelial gap junctions may explain the EDHF pathway (Busse *et al.*, 2002; Dora *et al.*, 2003; Griffith, 2004). In this perspective, the increase in endothelial cell intracellular Ca2+ concentration activates SKCa and IKCa channels leading to the endothelium-dependent hyperpolarization of smooth muscle cells via gap junctions located at the MEJ (Busse *et al.*, 2002; Crane *et al.*, 2003; Eichler *et al.*, 2003; Feletou *et al.*, 2003) (Figure 3). Consistent with this hypothesis, the EDHF-dependent vasodilation has been reported to be prevented by connexinmimetic peptides that are thought to specifically block gap junctions (De Vriese *et al.*, 2002; Karagiannis *et al.*, 2004; Chaytor *et al.*, 2005) as well as endothelial cell-selective loading of antibodies directed against the carboxyl-terminal region of Cx40 (Mather *et al.*, 2005). Interestingly, the gap junction-mediated EDHF signal might be controlled by NO through Snitrosylation. Cx43-based channels can be activated by S-nitrosylation (Retamal *et al.*, 2006). Cx43 and eNOS has been found to be express at MEJ and the activation of NO production in this microdomains leads to a S-nitrosylation-associated opening of Cx43-formed myoendothelial gap junction (Straub *et al.*, 2011), which support the idea that EDHF and NO are not parallel, independent vasodilator components, but in contrast, they work in concert.

microvascular function. Vascular endothelial cells-specific deletion of Cx43 (VEC Cx43-/-) results in hypotension (Liao *et al.*, 2001) and, in contrast, ablation of Cx40 produces a hypertension associated with an irregular vasomotion (de Wit *et al.*, 2000; de Wit *et al.*, 2003; Figueroa & Duling, 2008) and a dysregulation of renin production (Krattinger *et al.*, 2007; Wagner *et al.*, 2007). Although deletion of Cx37 does not appear to alter vascular function or blood pressure (Figueroa & Duling, 2008), several polymorphisms of this connexin have been associated with myocardial infarction, coronary artery disease and atherosclerosis (Boerma *et al.*, 1999; Yamada *et al.*, 2002; Hirashiki *et al.*, 2003; Yamada *et al.*, 2004). In mice, Cx40 and Cx37 are primarily expressed in the endothelium, which emphasizes the importance of the endothelial cell-gap junction communication in the control of

Although the mechanistic bases of the hypotension observed in VEC Cx43-/- are still unknown, the plasma levels of angiotensin I and II as well as NO were elevated in these animals (Liao *et al.*, 2001), suggesting that a dysregulation of NO production may have been the responsible of the hypotension with the subsequent activation of the renin-angiotensin system. Also, it is interesting to note that shear stress up-regulates the expression of Cx43 in cultured endothelial cells (DePaola *et al.*, 1999; Bao *et al.*, 2000) and in the endothelium of rat cardiac valves (Inai *et al.*, 2004), which suggests that Cx43 may be involved in the response

Smooth muscle cells and endothelial cells have also been found to be electrically and metabolically connected by gap junctions located at discrete points of contact between the two cell types at the myoendothelial junction (MEJ) (Beny & Pacicca, 1994; Little *et al.*, 1995; Emerson & Segal, 2000; Sandow *et al.*, 2003). This heterocellular communication seems to play a pivotal role in the Ca2+-mediated responses induced by endothelium-dependent vasodilators, such as ACh. As mentioned above, these vasodilator responses are typically paralleled by hyperpolarization of the underlying smooth muscle cells (Emerson & Segal, 2000; Goto *et al.*, 2002; Griffith, 2004), which has been attributed to the release of an EDHF (Vanhoutte, 2004; Feletou & Vanhoutte, 2009). However, the direct electrotonic transmission of a hyperpolarizing current from the endothelial cells to the smooth muscle cells via myoendothelial gap junctions may explain the EDHF pathway (Busse *et al.*, 2002; Dora *et al.*, 2003; Griffith, 2004). In this perspective, the increase in endothelial cell intracellular Ca2+ concentration activates SKCa and IKCa channels leading to the endothelium-dependent hyperpolarization of smooth muscle cells via gap junctions located at the MEJ (Busse *et al.*, 2002; Crane *et al.*, 2003; Eichler *et al.*, 2003; Feletou *et al.*, 2003) (Figure 3). Consistent with this hypothesis, the EDHF-dependent vasodilation has been reported to be prevented by connexinmimetic peptides that are thought to specifically block gap junctions (De Vriese *et al.*, 2002; Karagiannis *et al.*, 2004; Chaytor *et al.*, 2005) as well as endothelial cell-selective loading of antibodies directed against the carboxyl-terminal region of Cx40 (Mather *et al.*, 2005). Interestingly, the gap junction-mediated EDHF signal might be controlled by NO through Snitrosylation. Cx43-based channels can be activated by S-nitrosylation (Retamal *et al.*, 2006). Cx43 and eNOS has been found to be express at MEJ and the activation of NO production in this microdomains leads to a S-nitrosylation-associated opening of Cx43-formed myoendothelial gap junction (Straub *et al.*, 2011), which support the idea that EDHF and NO are not parallel, independent vasodilator components, but in contrast, they work in concert.

**4.3 Gap junctions in smooth muscle-endothelium communication** 

cardiovascular homeostasis.

to mechanical stimuli.

Flow (i.e. shear stress) is one of the most important stimuli involved in the tonic regulation of vasomotor tone. Although the response to shear stress is thought to be mediated primarily by NO, shear stress has also been reported to activate an EDHF-dependent vasodilator response (Watanabe *et al.*, 2005), which suggests that a gap junction-mediated EDHF pathway may be involved in the tonic control of peripheral vascular resistance. Consistent with this idea, intrarenal infusion of connexin-mimetic peptides homologous to the second extracellular loop of Cx43 (43Gap 27) or Cx40 (40Gap 27) not only decreased basal renal blood flow, but also increased mean arterial blood pressure of rats, either in presence or absence of NOS and COX blockers (De Vriese *et al.*, 2002), suggesting that connexinmimetic peptides induced vasoconstriction by disrupting or reducing the response to a tonic vasodilator stimulus such as shear stress.

#### **5. Conduction of vasomotor responses**

Longitudinal conduction of vasomotor responses provides an essential means of coordinating changes in diameter and flow distribution among vessels of the microcirculation. Vasomotor signals spread along the vessel length through gap junctions connecting cells of the vessel wall, and thereby, participate in the minute-to-minute coordination of vascular resistance by integrating function of proximal and distal vascular segments in the microcirculation (de Wit *et al.*, 2000; Figueroa *et al.*, 2004, 2006). Although vasoconstrictor responses are thought to be conducted by smooth muscle cells (Welsh & Segal, 1998; Bartlett & Segal, 2000; Budel *et al.*, 2003), the cellular pathway for conduction of vasodilator signals is more controversial and may be either exclusively by the endothelium (Emerson & Segal, 2000; Segal & Jacobs, 2001) or by both smooth muscle and endothelial cells (Bartlett & Segal, 2000; Budel *et al.*, 2003). The cellular pathway for conduction of vasomotor responses has been studied by selectively damaging a short segment of endothelial cells or smooth muscle cells by injection of an air bubble via a side branch (Bartlett & Segal, 2000; Figueroa *et al.*, 2007) or with a light-dye (fluorescein-conjugated dextran) treatment (Emerson & Segal, 2000). In feed arteries, selective damage of the endothelium completely blocked the ACh-induced conducted vasodilation (Emerson & Segal, 2000; Segal & Jacobs, 2001), but in arterioles, either damage of the endothelium or the smooth muscle did not affect the ACh-induced conducted responses (Bartlett & Segal, 2000; Budel *et al.*, 2003), which led to the proposal that the cellular pathway for conduction of vasodilations depends on the functional location of the vessel in the microvascular network (Segal, 2005). However, the cellular pathway of vasodilator signals may also depend on the stimulus that initiated the response, because, in contrast to ACh, selective damage of the endothelium blocked the vasodilation induced by bradykinin in arterioles (Welsh & Segal, 1998; Budel *et al.*, 2003).

Direct measurements of membrane potential have shown that conducted vasomotor responses are associated with rapid propagation (milliseconds) of an electrical signal along the vessel length (Xia & Duling, 1995; Welsh & Segal, 1998; Emerson & Segal, 2000). Because many observations have revealed an exponential decay of the conducted electrical signal, it was proposed that longitudinal spread of vasomotor responses reflects the passive, electrotonic conduction of changes in membrane potential via gap junctions connecting cells of the vessel wall (Pacicca *et al.*, 1996; Welsh & Segal, 1998; Gustafsson & Holstein-Rathlou, 1999). Therefore, the decay of the conducted vasomotor responses along the vessel length

Control and Coordination of Vasomotor Tone in the Microcirculation 79

cerebral microcirculation must be coupled to neuronal activity, which is known as neurovascular coupling (Hawkins & Davis, 2005; Leybaert, 2005). In this case, however, vasomotor signals seem to be conducted by astrocytes as opposed to smooth muscle or endothelium (Anderson & Nedergaard, 2003; Zonta *et al.*, 2003; Mulligan & MacVicar, 2004; Koehler *et al.*, 2006; Metea & Newman, 2006; Takano *et al.*, 2006). Tight spatial and temporal coupling between neuronal activity and blood flow is essential for brain function (Anderson & Nedergaard, 2003; Hawkins & Davis, 2005; Leybaert, 2005) and astrocytes are found in a strategic location between neurons and the microvasculature, with the astrocytic endfeet ensheathing the vessels. This spatial organization places the astrocytes in a key position to orchestrate the neurovascular coupling and an increasing body of evidence shows that the astrocyte transduces and conducts to the local microvasculature vasomotor signals generated by an increase in synaptic activity (Anderson & Nedergaard, 2003; Zonta *et al.*, 2003; Mulligan & MacVicar, 2004; Metea & Newman, 2006; Takano *et al.*, 2006) (Figure 4). As a result, astrocytes couple neuronal activation to vasodilation of local parenchymal arterioles (Figure 4), which, in turn, leads to an increase in blood-borne energy substrate that rapidly matches the enhanced metabolic demand (Anderson & Nedergaard, 2003; Hawkins

Calcium seems to be the intracellular vasomotor signal of the astrocyte-mediated neurovascular coupling. Astrocytes express receptors for several neurotransmitters such as glutamate, GABA and ATP (Anderson & Nedergaard, 2003; Leybaert, 2005; Koehler *et al.*, 2009), which can initiate Ca2+ signals (Figure 4). Then, the increase in neuronal activity results in an astrocytic calcium signaling that propagates through the astrocytic processes into the endfeet (Anderson & Nedergaard, 2003; Zonta *et al.*, 2003; Filosa *et al.*, 2004; Mulligan & MacVicar, 2004; Straub *et al.*, 2006). The increase in cytosolic calcium concentration in the endfeet ultimately causes the release of vasoactive factors and arteriolar dilation (Anderson & Nedergaard, 2003; Zonta *et al.*, 2003; Mulligan & MacVicar, 2004; Filosa *et al.*, 2006; Straub *et al.*, 2006) (Figure 4). Interestingly, astrocytes express gap junctions (Martinez & Saez, 2000; Saez *et al.*, 2003; Retamal *et al.*, 2006) and a calcium signal may propagate between neighboring astrocytes in a wave-like manner (Cornell-Bell *et al.*, 1990; Nedergaard, 1994; Cai *et al.*, 1998; Nedergaard *et al.*, 2003), coordinating the neurovascular coupling in the local cerebral microcirculation (Anderson & Nedergaard, 2003; Zonta *et al.*, 2003; Filosa *et al.*, 2004; Mulligan & MacVicar, 2004). Some of the Ca2+-dependent vasodilator mechanisms that may be activated at the astrocytic endfeet facing the vessel wall are the production of epoxyeicosatrienoic acid (EETs) by the cytochrome P450 epoxygenase and PGs by the COX enzyme (Anderson & Nedergaard, 2003; Zonta *et al.*, 2003; Zonta *et al.*, 2003; Filosa *et al.*, 2004; Straub *et al.*, 2006; Koehler *et al.*, 2009), and also ATP release (Shi *et al.*, 2008) via connexin or pannexin hemichannels (Figure 4). In addition, astrocytic endfeet express BKCa and Girouard et al. (Girouard *et al.*, 2010) recently showed in mouse cortical brain slices that these K+ channels play a central role in neurovascular coupling through the release of K+ ion into the perivascular space (Figure 4). The small increase in local [K+]o (<20 mM) activates the Kir channels located in the smooth muscle cell membrane facing the endfeet, which leads to hyperpolarization, and subsequently, vasodilation (Girouard *et al.*, 2010) (Figure 4). It is noteworthy that a higher increase in [K+]o would produce smooth muscle cell depolarization

& Davis, 2005; Leybaert, 2005).

and vasoconstriction (Girouard *et al.*, 2010).

should be consistent with the length constant estimated from electrotonic potentials produced by current injection into the smooth muscle or endothelial cells of arterioles, which is between 0.9 and 1.6 mm (Hirst & Neild, 1978; Hirst *et al.*, 1997; Emerson *et al.*, 2002).

Conduction of vasoconstrictor responses typically behaves as predicted by the electrotonic model. However, a simple electrotonic model often fails to predict conduction of vasodilator signals initiated by endothelium-dependent stimuli, such as ACh or bradykinin. These signals have been reported to propagate for many millimeters without showing noticeable decay in magnitude (Emerson & Segal, 2000; Figueroa & Duling, 2008). In addition, the electrical length constant of ACh-induced hyperpolarization has been shown to be longer than that measured for current injection (Emerson *et al.*, 2002) and the hyperpolarizing signal activated by ACh has been also reported to increase during the first 1000 µm of longitudinal conduction (Crane *et al.*, 2004). The lack of decay of these responses suggests that a regenerative, energy-dependent mechanism underlies the conduction process, similar to that described in neurons. Consistent with this idea, electrical stimulation also activates a conducted, non-decremental endothelium-dependent vasodilation that was hypothesized to be mediated by a complex interplay between voltage-gated Na+ channels (Nav) and T type, voltage-gated Ca2+ channels (T-Cav) (Figueroa *et al.*, 2007). In this hypothetic model, Nav channels underlie the conduction of the signal and T-Cav mediates the vasodilation. Interestingly, deletion of Cx40 selectively eliminates the regenerative component of the conducted vasodilation induced by ACh (Figueroa & Duling, 2008), bradykinin (de Wit *et al.*, 2000) or electrical stimulation (Figueroa *et al.*, 2003), leaving a decaying component consistent with the electrotonic model (Figueroa & Duling, 2008), which suggests that Cx40 based gap junctions provide the pathway for the intercellular propagation of the regenerative conducted component of vasodilator signals. Deletion of Cx37 did not affect conduction of vasodilator responses (Figueroa & Duling, 2008) and replacement of Cx40 by Cx45 did not restore the non-decremental component of the conducted vasodilation activated by ACh or bradykinin (Wolfle *et al.*, 2007), supporting the idea that individual connexins have different functions.

The opening of Kir channels induced by the smooth muscle hyperpolarization may be an alternative hypothesis to explain the extended conduction of vasodilator responses. An intrinsic biophysical property of Kir channels is that they increase their activity upon cell hyperpolarization and it has been proposed that the activation of these K+ channels in the smooth muscle cells amplify the hyperpolarizing current initiated by ACh, thereby facilitating the conduction of this signal (Jantzi *et al.*, 2006). However, as mentioned above, current-induced hyperpolarization decays faster than the response induced by ACh (Emerson *et al.*, 2002), which argues against the participation of Kir alone in the nondecremental component of the conducted vasodilation, and suggests that further investigation is needed to elucidate the mechanisms involved in the conduction of vasomotor responses.

#### **6. Neurovascular coupling**

The brain has a very high metabolic demand and its activity depends on the communication between brain cells and local microvessels (i.e. neurovascular unit). Then, the function of

should be consistent with the length constant estimated from electrotonic potentials produced by current injection into the smooth muscle or endothelial cells of arterioles, which is between 0.9 and 1.6 mm (Hirst & Neild, 1978; Hirst *et al.*, 1997; Emerson *et al.*, 2002). Conduction of vasoconstrictor responses typically behaves as predicted by the electrotonic model. However, a simple electrotonic model often fails to predict conduction of vasodilator signals initiated by endothelium-dependent stimuli, such as ACh or bradykinin. These signals have been reported to propagate for many millimeters without showing noticeable decay in magnitude (Emerson & Segal, 2000; Figueroa & Duling, 2008). In addition, the electrical length constant of ACh-induced hyperpolarization has been shown to be longer than that measured for current injection (Emerson *et al.*, 2002) and the hyperpolarizing signal activated by ACh has been also reported to increase during the first 1000 µm of longitudinal conduction (Crane *et al.*, 2004). The lack of decay of these responses suggests that a regenerative, energy-dependent mechanism underlies the conduction process, similar to that described in neurons. Consistent with this idea, electrical stimulation also activates a conducted, non-decremental endothelium-dependent vasodilation that was hypothesized to be mediated by a complex interplay between voltage-gated Na+ channels (Nav) and T type, voltage-gated Ca2+ channels (T-Cav) (Figueroa *et al.*, 2007). In this hypothetic model, Nav channels underlie the conduction of the signal and T-Cav mediates the vasodilation. Interestingly, deletion of Cx40 selectively eliminates the regenerative component of the conducted vasodilation induced by ACh (Figueroa & Duling, 2008), bradykinin (de Wit *et al.*, 2000) or electrical stimulation (Figueroa *et al.*, 2003), leaving a decaying component consistent with the electrotonic model (Figueroa & Duling, 2008), which suggests that Cx40 based gap junctions provide the pathway for the intercellular propagation of the regenerative conducted component of vasodilator signals. Deletion of Cx37 did not affect conduction of vasodilator responses (Figueroa & Duling, 2008) and replacement of Cx40 by Cx45 did not restore the non-decremental component of the conducted vasodilation activated by ACh or bradykinin (Wolfle *et al.*, 2007), supporting the idea that individual

The opening of Kir channels induced by the smooth muscle hyperpolarization may be an alternative hypothesis to explain the extended conduction of vasodilator responses. An intrinsic biophysical property of Kir channels is that they increase their activity upon cell hyperpolarization and it has been proposed that the activation of these K+ channels in the smooth muscle cells amplify the hyperpolarizing current initiated by ACh, thereby facilitating the conduction of this signal (Jantzi *et al.*, 2006). However, as mentioned above, current-induced hyperpolarization decays faster than the response induced by ACh (Emerson *et al.*, 2002), which argues against the participation of Kir alone in the nondecremental component of the conducted vasodilation, and suggests that further investigation is needed to elucidate the mechanisms involved in the conduction of

The brain has a very high metabolic demand and its activity depends on the communication between brain cells and local microvessels (i.e. neurovascular unit). Then, the function of

connexins have different functions.

vasomotor responses.

**6. Neurovascular coupling** 

cerebral microcirculation must be coupled to neuronal activity, which is known as neurovascular coupling (Hawkins & Davis, 2005; Leybaert, 2005). In this case, however, vasomotor signals seem to be conducted by astrocytes as opposed to smooth muscle or endothelium (Anderson & Nedergaard, 2003; Zonta *et al.*, 2003; Mulligan & MacVicar, 2004; Koehler *et al.*, 2006; Metea & Newman, 2006; Takano *et al.*, 2006). Tight spatial and temporal coupling between neuronal activity and blood flow is essential for brain function (Anderson & Nedergaard, 2003; Hawkins & Davis, 2005; Leybaert, 2005) and astrocytes are found in a strategic location between neurons and the microvasculature, with the astrocytic endfeet ensheathing the vessels. This spatial organization places the astrocytes in a key position to orchestrate the neurovascular coupling and an increasing body of evidence shows that the astrocyte transduces and conducts to the local microvasculature vasomotor signals generated by an increase in synaptic activity (Anderson & Nedergaard, 2003; Zonta *et al.*, 2003; Mulligan & MacVicar, 2004; Metea & Newman, 2006; Takano *et al.*, 2006) (Figure 4). As a result, astrocytes couple neuronal activation to vasodilation of local parenchymal arterioles (Figure 4), which, in turn, leads to an increase in blood-borne energy substrate that rapidly matches the enhanced metabolic demand (Anderson & Nedergaard, 2003; Hawkins & Davis, 2005; Leybaert, 2005).

Calcium seems to be the intracellular vasomotor signal of the astrocyte-mediated neurovascular coupling. Astrocytes express receptors for several neurotransmitters such as glutamate, GABA and ATP (Anderson & Nedergaard, 2003; Leybaert, 2005; Koehler *et al.*, 2009), which can initiate Ca2+ signals (Figure 4). Then, the increase in neuronal activity results in an astrocytic calcium signaling that propagates through the astrocytic processes into the endfeet (Anderson & Nedergaard, 2003; Zonta *et al.*, 2003; Filosa *et al.*, 2004; Mulligan & MacVicar, 2004; Straub *et al.*, 2006). The increase in cytosolic calcium concentration in the endfeet ultimately causes the release of vasoactive factors and arteriolar dilation (Anderson & Nedergaard, 2003; Zonta *et al.*, 2003; Mulligan & MacVicar, 2004; Filosa *et al.*, 2006; Straub *et al.*, 2006) (Figure 4). Interestingly, astrocytes express gap junctions (Martinez & Saez, 2000; Saez *et al.*, 2003; Retamal *et al.*, 2006) and a calcium signal may propagate between neighboring astrocytes in a wave-like manner (Cornell-Bell *et al.*, 1990; Nedergaard, 1994; Cai *et al.*, 1998; Nedergaard *et al.*, 2003), coordinating the neurovascular coupling in the local cerebral microcirculation (Anderson & Nedergaard, 2003; Zonta *et al.*, 2003; Filosa *et al.*, 2004; Mulligan & MacVicar, 2004). Some of the Ca2+-dependent vasodilator mechanisms that may be activated at the astrocytic endfeet facing the vessel wall are the production of epoxyeicosatrienoic acid (EETs) by the cytochrome P450 epoxygenase and PGs by the COX enzyme (Anderson & Nedergaard, 2003; Zonta *et al.*, 2003; Zonta *et al.*, 2003; Filosa *et al.*, 2004; Straub *et al.*, 2006; Koehler *et al.*, 2009), and also ATP release (Shi *et al.*, 2008) via connexin or pannexin hemichannels (Figure 4). In addition, astrocytic endfeet express BKCa and Girouard et al. (Girouard *et al.*, 2010) recently showed in mouse cortical brain slices that these K+ channels play a central role in neurovascular coupling through the release of K+ ion into the perivascular space (Figure 4). The small increase in local [K+]o (<20 mM) activates the Kir channels located in the smooth muscle cell membrane facing the endfeet, which leads to hyperpolarization, and subsequently, vasodilation (Girouard *et al.*, 2010) (Figure 4). It is noteworthy that a higher increase in [K+]o would produce smooth muscle cell depolarization and vasoconstriction (Girouard *et al.*, 2010).

Control and Coordination of Vasomotor Tone in the Microcirculation 81

the glia limitans, which isolate these arterioles from the neurons that are located right below. Vasodilation of pial arterioles associated with neuronal activation was blocked by either selective elimination of astrocytes with L-α aminoadipic acid treatment or the inhibition of Cx43-based channels with the specific connexin mimetic peptide gap-27 (Xu *et al.*, 2008). In astrocytes, Cx43 may be found forming unpaired hemichannels or gap junction intercellular channels (Stout *et al.*, 2002; Saez *et al.*, 2003; Retamal *et al.*, 2006). Thus, astrocytic Cx43-based channels could be involved in the coordination of calcium waves between astrocytes, or in the release of vasoactive factors such as ATP that can be metabolized to the potent

Control of vasomotor tone relies on a complex interplay between NO, PGs, K+ channels and gap junction communication. It is typically thought that NO is the most relevant endothelium-dependent vasodilator signal, but, in resistance vessels and arterioles, K+ channels and gap junction communication between the cells of the vessel wall have emerged as major players in the tonic control and coordination of vascular function. While several K+ channels (e.g. BKCa, Kir and KATP channels) may contribute to the vasodilator response induced by NO, the endothelial cell K+ channels, SKCa and IKCa, seem to be involved in the fine regulation of eNOS activation. In addition, it has become apparent that NO production is also modulated by a delicate caveolar control of L-arginine supply. Although myoendothelial gap junction communication probably contributes to the EDHF signaling mediated by SKCa and IKCa channels, the strategic spatial organization of IKCa and Kir may also be involved in the intercellular transmission of an endothelium-initiated smooth muscle hyperpolarization. A similar organization, but between BKCa and Kir channels, is observed in the astrocyte-mediated neurovascular coupling. Connexin- and pannexin-based hemichannels are an attractive signaling mechanism that may be involved in the control of vascular function, but the study of hemichannels in resistance vessels is just beginning.

This work was supported by Grant Anillos ACT-71 from Comisión Nacional de Investigación Científica y Tecnológica – CONICYT and Grant #1100850 and #1111033 from

Abderrahmane A, Salvail D, Dumoulin M, Garon J, Cadieux A & Rousseau E. (1998). Direct

Alderton Wk, Cooper Ce & Knowles RG. (2001). Nitric oxide synthases: structure, function

of a nitrothiosylation mechanism? *Am J Respir Cell Mol Biol* 19, 485-497. Absi M, Burnham Mp, Weston Ah, Harno E, Rogers M & Edwards G. (2007). Effects of

than just a hyperpolarizing factor. *Trends Pharmacol Sci* 26, 162-167.

activation of K(Ca) channel in airway smooth muscle by nitric oxide: involvement

methyl beta-cyclodextrin on EDHF responses in pig and rat arteries; association between SK(Ca) channels and caveolin-rich domains. *Br J Pharmacol* 151, 332-340. Ahluwalia A & Hobbs AJ. (2005). Endothelium-derived C-type natriuretic peptide: more

Fondo Nacional de Desarrollo Científico y Tecnológico – FONDECYT.

and inhibition. *Biochem J* 357, 593-615.

vasodilator, adenosine (Shi *et al.*, 2008)

**7. Conclusion** 

**8. Acknowledgment** 

**9. References**

Fig. 4. Astrocytes-mediated neurovascular coupling. Neurotransmitters may exit the synaptic cleft and activate receptors on astrocytes, which couple neuronal activity with astrocyte signaling. The activation of astrocyte receptors triggers a Ca2+ wave that reaches the astrocytic endfeet, leading to the opening of large conductance Ca2+-activated K+ channels (BKCa). The K+ ion release via BKCa elicits a small increase in extracellular K+ concentration (<20 mM) in the perivascular space that activates the Kir channels located in the smooth muscle cell membrane facing the endfeet, which, in turn, leads to hyperpolarization, and subsequently, vasodilation. The vessel wall hyperpolarizationmediated vasodilation is conducted to upstream arterioles, coupling function of proximal and distal vessels.

As described in the peripheral microcirculation (Segal & Kurjiaka, 1995; Segal, 2000), local vasodilation of cerebral arterioles must be communicated to upstream vascular segments to produce a functional increase of blood flow supply and effectively match the local metabolic demand (Cox *et al.*, 1993; Iadecola *et al.*, 1997). Although vasomotor responses have been observed to be conducted by the wall of cerebral arterioles (Dietrich *et al.*, 1996; Horiuchi *et al.*, 2002), it seems to be that astrocytes also play a central role in integrating function of local arterioles with upstream cerebral vessels involved in the neurovascular coupling. Pial arterioles are important upstream vessels of the parenchymal cerebral arterioles. It is important to note that pial arterioles overlie a thick layer of astrocytic processes, known as the glia limitans, which isolate these arterioles from the neurons that are located right below. Vasodilation of pial arterioles associated with neuronal activation was blocked by either selective elimination of astrocytes with L-α aminoadipic acid treatment or the inhibition of Cx43-based channels with the specific connexin mimetic peptide gap-27 (Xu *et al.*, 2008). In astrocytes, Cx43 may be found forming unpaired hemichannels or gap junction intercellular channels (Stout *et al.*, 2002; Saez *et al.*, 2003; Retamal *et al.*, 2006). Thus, astrocytic Cx43-based channels could be involved in the coordination of calcium waves between astrocytes, or in the release of vasoactive factors such as ATP that can be metabolized to the potent vasodilator, adenosine (Shi *et al.*, 2008)

## **7. Conclusion**

80 The Cardiovascular System – Physiology, Diagnostics and Clinical Implications

Fig. 4. Astrocytes-mediated neurovascular coupling. Neurotransmitters may exit the synaptic cleft and activate receptors on astrocytes, which couple neuronal activity with astrocyte signaling. The activation of astrocyte receptors triggers a Ca2+ wave that reaches the astrocytic endfeet, leading to the opening of large conductance Ca2+-activated K+ channels (BKCa). The K+ ion release via BKCa elicits a small increase in extracellular K+ concentration (<20 mM) in the perivascular space that activates the Kir channels located in

hyperpolarization, and subsequently, vasodilation. The vessel wall hyperpolarizationmediated vasodilation is conducted to upstream arterioles, coupling function of proximal

As described in the peripheral microcirculation (Segal & Kurjiaka, 1995; Segal, 2000), local vasodilation of cerebral arterioles must be communicated to upstream vascular segments to produce a functional increase of blood flow supply and effectively match the local metabolic demand (Cox *et al.*, 1993; Iadecola *et al.*, 1997). Although vasomotor responses have been observed to be conducted by the wall of cerebral arterioles (Dietrich *et al.*, 1996; Horiuchi *et al.*, 2002), it seems to be that astrocytes also play a central role in integrating function of local arterioles with upstream cerebral vessels involved in the neurovascular coupling. Pial arterioles are important upstream vessels of the parenchymal cerebral arterioles. It is important to note that pial arterioles overlie a thick layer of astrocytic processes, known as

the smooth muscle cell membrane facing the endfeet, which, in turn, leads to

and distal vessels.

Control of vasomotor tone relies on a complex interplay between NO, PGs, K+ channels and gap junction communication. It is typically thought that NO is the most relevant endothelium-dependent vasodilator signal, but, in resistance vessels and arterioles, K+ channels and gap junction communication between the cells of the vessel wall have emerged as major players in the tonic control and coordination of vascular function. While several K+ channels (e.g. BKCa, Kir and KATP channels) may contribute to the vasodilator response induced by NO, the endothelial cell K+ channels, SKCa and IKCa, seem to be involved in the fine regulation of eNOS activation. In addition, it has become apparent that NO production is also modulated by a delicate caveolar control of L-arginine supply. Although myoendothelial gap junction communication probably contributes to the EDHF signaling mediated by SKCa and IKCa channels, the strategic spatial organization of IKCa and Kir may also be involved in the intercellular transmission of an endothelium-initiated smooth muscle hyperpolarization. A similar organization, but between BKCa and Kir channels, is observed in the astrocyte-mediated neurovascular coupling. Connexin- and pannexin-based hemichannels are an attractive signaling mechanism that may be involved in the control of vascular function, but the study of hemichannels in resistance vessels is just beginning.

## **8. Acknowledgment**

This work was supported by Grant Anillos ACT-71 from Comisión Nacional de Investigación Científica y Tecnológica – CONICYT and Grant #1100850 and #1111033 from Fondo Nacional de Desarrollo Científico y Tecnológico – FONDECYT.

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**5** 

Ali Nasimi

*Iran* 

**Hemodynamics** 

*Isfahan University of Medical Sciences,* 

F = P/R (1)

Hemodynamics is the study of the relationship among physical factors affecting blood flow through the vessels. In this chapter these factors and their relationship were discussed.

Blood flow (F) through a blood vessel is determined by two main factors: (1) pressure difference (P) between the two ends of the vessel and (2) the resistance (R) to blood flow

Flow (F) is defined as the volume of blood passing each point of the vessel in one unit time. Usually, blood flow is expressed in milliliters per minute or liters per minute, but it is also

Pressure which is the force that pushes the blood through the vessel is defined as the force exerted on a unit surface of the wall of the tube perpendicular to flow. Pressure is expressed as millimeters of mercury (mmHg). Since the pressure is changing over the course of the blood vessel, there is no single pressure to use; therefore the pressure parameter used is pressure difference (P), also called pressure gradient, which is the difference between the pressure at the beginning of the vessel (P1) and the pressure at the end of the vessel (P2), i.e.

**2. Blood flow is a function of pressure difference and resistance (Darcy's** 

**1. Introduction** 

through the vessel (Fig. 1).

Fig. 1. Blood flow through a blood vessel.

The equation relating these parameters is:

expressed in milliliters per second.

This equation is called Darcy's law or Ohm's law.

**law)** 

