Pulmonary Hypertention

*Interventional Pulmonology and Pulmonary Hypertension - Updates on Specific Topics*

[25] Cui Y, Ma D-q, Liu W-h. Value of multiplanar reconstruction in MSCT in demonstrating the relationship between solitary pulmonary nodule and bronchus. Clinical Imaging.

2009;**33**:15-21

[26] Kotlyarov PM. Virtual bronchoscopy in the diagnosis of lung cancer. Radiation Diagnosis and Therapy. 2015. № 1. pp. 56-63. In Russian. (Котляров ПМ. Виртуальная бронхоскопия в диагностике рака легкого. Лучевая диагностика и терапия.

2015. № 1. С. 56-63)

С. 76-81)

micr.2018-suppl 1

[27] Kharchenko VP, Kotlyarov PM, Vinikovetskaya AV et al. Trauma of the right main bronchus (clinical observation). Medical Imaging. 2011. N 4. pp. 76-81. In Russian. (Харченко ВП, Котляров ПМ, Виниковецкая АВ и др. Травматический отрыв правого главного бронха (клиническое наблюдение). Медицинская визуализация. 2011. N 4.

[28] Kotlyarov PM, Chernichenko NV. Virtual bronchoscopy multislice tomography in traumatic injuries of the main bronchi. Journal of Medical Imaging and Case Reports. 2018. Proceedings of the First International Conference on Medical Imaging and Case Reports (MICR-2018);**2**(2):S25-S26. DOI: 10.17756/

**56**

**59**

**Chapter 5**

**Abstract**

heathy individuals.

**1. Introduction**

Pulmonary Vascular Reserve and

Pulmonary circulation has long been known to have specific proprieties of recruitment and distention to keep the hemodynamic pressure low even when facing very high blood flow. Aerobic exercise especially at high intensity has the particularity to increase considerably the cardiac output. The ability of the pulmonary circulation to face high blood flow with maintaining low pressures is considered as the pulmonary vascular reserve. Furthermore, high pulmonary vascular reserve has been shown to be characterized by low pulmonary vascular resistance, high pulmonary vascular distensibility, high pulmonary capillary volume, and high lung diffusing capacity allowing for lower ventilation at a same metabolic cost. The pulmonary vascular reserve thus reflects the capacity of the pulmonary circulation, including the capillary network, to adapt to high exercise levels. Interestingly, a high pulmonary vascular reserve is an advantage as it is associated with a superior aerobic exercise capacity (VO2max). This observation strongly suggests that exercise capacity is modulated by the functional state of the pulmonary circulation. However, why or when pulmonary vascular reserve may be related to a higher aerobic exercise capacity remains incompletely understood. The present chapter will discuss the role of each component of the pulmonary vascular reserve during exercise and develop the factors able to influence the pulmonary vascular reserve in

**Keywords:** pulmonary circulation, VO2max, ventilation, diffusion capacity

During aerobic exercise, muscular contractions increase oxygen peripheral demand proportionally to exercise intensity until a maximal level or maximal oxygen consumption (VO2max). VO2max is widely used as a cardiorespiratory fitness indicator as the capacity of oxygen consumption increases with exercise training with values approaching 80–90 ml/min/kg in endurance athletes vs 20–30 ml/min/ kg in healthy sedentary subjects. The oxygen consumption is dependent on the interdependence of the different components of the convectional and diffusional oxygen transport systems from ambient air to the mitochondria. Every transport step is a potential VO2max determinant: ventilation, pulmonary diffusion, blood oxygen transport (depending on cardiac output and arterial oxygen content), muscular diffusion, and mitochondrial activity. The importance of each contribution step varies under different health or environmental conditions. Nevertheless, one can reasonably assume that cardiac output (Q ) is most important at sea level, while at higher altitudes, lung or/and muscle diffusion may become more critical [1, 2].

Aerobic Exercise Capacity

*Vitalie Faoro and Kevin Forton*

#### **Chapter 5**

## Pulmonary Vascular Reserve and Aerobic Exercise Capacity

*Vitalie Faoro and Kevin Forton*

#### **Abstract**

Pulmonary circulation has long been known to have specific proprieties of recruitment and distention to keep the hemodynamic pressure low even when facing very high blood flow. Aerobic exercise especially at high intensity has the particularity to increase considerably the cardiac output. The ability of the pulmonary circulation to face high blood flow with maintaining low pressures is considered as the pulmonary vascular reserve. Furthermore, high pulmonary vascular reserve has been shown to be characterized by low pulmonary vascular resistance, high pulmonary vascular distensibility, high pulmonary capillary volume, and high lung diffusing capacity allowing for lower ventilation at a same metabolic cost. The pulmonary vascular reserve thus reflects the capacity of the pulmonary circulation, including the capillary network, to adapt to high exercise levels. Interestingly, a high pulmonary vascular reserve is an advantage as it is associated with a superior aerobic exercise capacity (VO2max). This observation strongly suggests that exercise capacity is modulated by the functional state of the pulmonary circulation. However, why or when pulmonary vascular reserve may be related to a higher aerobic exercise capacity remains incompletely understood. The present chapter will discuss the role of each component of the pulmonary vascular reserve during exercise and develop the factors able to influence the pulmonary vascular reserve in heathy individuals.

**Keywords:** pulmonary circulation, VO2max, ventilation, diffusion capacity

#### **1. Introduction**

During aerobic exercise, muscular contractions increase oxygen peripheral demand proportionally to exercise intensity until a maximal level or maximal oxygen consumption (VO2max). VO2max is widely used as a cardiorespiratory fitness indicator as the capacity of oxygen consumption increases with exercise training with values approaching 80–90 ml/min/kg in endurance athletes vs 20–30 ml/min/ kg in healthy sedentary subjects. The oxygen consumption is dependent on the interdependence of the different components of the convectional and diffusional oxygen transport systems from ambient air to the mitochondria. Every transport step is a potential VO2max determinant: ventilation, pulmonary diffusion, blood oxygen transport (depending on cardiac output and arterial oxygen content), muscular diffusion, and mitochondrial activity. The importance of each contribution step varies under different health or environmental conditions. Nevertheless, one can reasonably assume that cardiac output (Q ) is most important at sea level, while at higher altitudes, lung or/and muscle diffusion may become more critical [1, 2].

For many years, exercise physiologists have focused on the left side of the heart and the systemic circulation to explain aerobic exercise performance and limitation. However, more recently, robust and growing studies suggest that the right ventricle (RV) might also be an important determinant of maximal cardiac output and VO2max [3, 4]. More broadly, the RV-pulmonary circulation unit, including the capillary network, has been identified as a potential factor modulating the aerobic exercise capacity in normoxia [5–8] and in hypoxia [7–10]. Indeed, pulmonary vascular reserve, or the ability of the pulmonary circulation to extend, recruit, and vasodilate to smooth an intravascular pressure increase, is critical in minimizing RV afterload and maximizing peak cardiac output at exercise [5, 6]. To discuss the potential importance of this phenomenon, the role of each physiological component of the RV-pulmonary circulation unit and interactions with gas exchange will be reviewed.

#### **2. Right ventricle**

Cardiac output increases with exercise intensity in order to ensure oxygen supply to the working muscles. Since the right and left heart are disposed in a series in the cardiovascular system, it is impossible for one ventricle to generate a blood flow exceeding that of the other. The maximal cardiac output is therefore depending on the "weakest" ventricle's performance. Increases in RV afterload may, thereby, possibly serve to limit overall cardiac output [3]. Additionally, the heart being constrained within a stiff pericardium, congestion in the RV may shift the interventricular septum to the left resulting in left ventricular diastolic volume restriction, further limiting maximal Q. This is observable in specific circumstances such as congestive heart failure or highly trained endurance athletes [3].

The normal right ventricle is a thin-walled flow generator perfectly adapted to face the low-pressure, high-compliant pulmonary circulation [3, 11]. However, RV anatomical and physiological properties are maybe not designed to face dramatical afterload increase at high levels of exercise. As compared to the left ventricle (LV), load increases are greater for the RV during exercise [12], and its contractile reserve may become insufficient for adequate blood supply to peripheral demand [12, 13]. Relative to the LV, the greater load that the RV faces during exercise is dominantly attributed to a larger exercise-induced increase in pulmonary artery pressure (PAP) relative to systemic vascular pressure [3].

Increased PAP during exercise is known to limit exercise capacity in pulmonary hypertension patients through a decreased maximum cardiac output by an overloaded right ventricle [11]. Recently it has also been suggested that the same phenomenon could appear in healthy individuals exercising at high workloads at sea level [5–8] but even more at altitude [7–10].

Invasive or noninvasive studies in healthy subjects described a ceiling level of the mean PAP approximating 40–50 mmHg when exercising at maximal workloads corresponding to the extreme afterload level which the RV can face while maintaining a high cardiac output [6, 14–17]. The RV is thus placed under great stress during intense exercise. This leads to the idea that RV outflow might become a limiting factor when the ventricular work demand is overwhelmed, particularly in cases of extreme cardiac outputs (high intensity exercise, endurance athletes) or increased PAP (pathological or hypoxic conditions). Recently, D'Alto et al. demonstrated that echocardiographic RV systolic function indices (tricuspid annular plane systolic excursion (TAPSE), S′, and TAPSE/PAP) correlate with maximal workload in healthy subjects. This finding illustrates that a higher RV contractility reserve, defined as the difference between peak exercise and rest, is an advantage to reach high exercise levels and suggests a potential role of the RV in exercise capacity limitation [13].

**61**

**Figure 1.**

*agreement with previous reports [8, 14, 23, 26].*

*Pulmonary Vascular Reserve and Aerobic Exercise Capacity*

Pulmonary circulation opposes resistances to the ejecting RV that can be quantified by the PAP at a given cardiac output [18]. According to Poiseuille's law, applicable for a Newtonian fluid flowing laminarly in a straight cylinder, flow and driving pressure are proportional. This would imply that with unchanged resistance every increase in flow would increase PAP. However, the pulmonary circulation has this specific property to reduce resistances by two possible mechanisms: (1) recruitment, enlistment of previously closed pulmonary capillary [19, 20], and (2) distension, expansion of already filled pulmonary capillaries when pressure increases [21]. It is generally accepted that the initial vascular recruitment at the onset of exercise followed by distension allows for the pulmonary circulation to face a high blood flow with limited increase in pressure and maintaining RV systolic function at minimal energy cost [22]. Indeed, low pressure in the pulmonary circulation is essential to prevent two potential exercise capacity limitation mechanisms: fluid leaking from the capillaries to the interstitial space with subsequent gas exchange

alterations and RV outflow and oxygen transport limitation [18, 22].

During exercise, PAP increases along with Q but not always with a one to one ratio [15, 23]. In exercising subjects, the PAP can be measured at different exercise intensities or Q allowing for the calculation of the PAP vs. Q slope, as illustrated in **Figure 1**. The PAP vs. Q slope is a more accurate estimation of pulmonary vascular resistance (PVR) as compared to a single measure at rest [14, 22, 24]. Invasive catheterization and noninvasive stress echocardiography studies showed that pulmonary vascular response to exercise varies considerably from one individual to another, with slopes of mean PAP/Q ranging from 0.5 or 1 mmHg/l/min in young adults to 2.5 mmHg/l/min in elderly [14, 23]. This means that with a 10 l/min exercise-induced Q increase, for example, normal PAP increase would range from 5 to 25 mmHg. This great interindividual variability of pulmonary vascular response

*Stress echocardiography multiple measurements of mean pulmonary arterial pressure (mPAP) at increasing flow (Q ) from rest until maximal exercise in one healthy subject. Pulmonary vascular resistance (PVR) is evaluated from the angular coefficient of the mPAP vs. Q linear regression line (ΔmPAP/ΔQ ). The PVR of 0.8 mmHg/l/min found in this example is in good agreement with limits of normal (gray background) [23]. From the curvilinearity of this PAP vs. Q relationship (dotted line), a mathematical distensible model relating mPAP, Q, left atrial pressure (LAP), and total PVR at rest (R0) allows for a calculation of a distensible factor: α (cfr formula). The present subject shows a distensibility (α) corresponding of 1.4% increase of the pulmonary vascular diameter per mmHg of pressure elevation during the entire exercise test and is in good* 

*DOI: http://dx.doi.org/10.5772/intechopen.88399*

**3. Pulmonary circulation**

**3.1 Pulmonary vascular resistance**

#### **3. Pulmonary circulation**

*Interventional Pulmonology and Pulmonary Hypertension - Updates on Specific Topics*

For many years, exercise physiologists have focused on the left side of the heart and the systemic circulation to explain aerobic exercise performance and limitation. However, more recently, robust and growing studies suggest that the right ventricle (RV) might also be an important determinant of maximal cardiac output and

VO2max [3, 4]. More broadly, the RV-pulmonary circulation unit, including the capillary network, has been identified as a potential factor modulating the aerobic exercise capacity in normoxia [5–8] and in hypoxia [7–10]. Indeed, pulmonary vascular reserve, or the ability of the pulmonary circulation to extend, recruit, and vasodilate to smooth an intravascular pressure increase, is critical in minimizing RV afterload and maximizing peak cardiac output at exercise [5, 6]. To discuss the potential importance of this phenomenon, the role of each physiological component of the RV-pulmonary circulation unit and interactions with gas exchange will be reviewed.

Cardiac output increases with exercise intensity in order to ensure oxygen supply to the working muscles. Since the right and left heart are disposed in a series in the cardiovascular system, it is impossible for one ventricle to generate a blood flow exceeding that of the other. The maximal cardiac output is therefore depending on the "weakest" ventricle's performance. Increases in RV afterload may, thereby, possibly serve to limit overall cardiac output [3]. Additionally, the heart being constrained within a stiff pericardium, congestion in the RV may shift the interventricular septum to the left resulting in left ventricular diastolic volume restriction, further limiting maximal Q. This is observable in specific circumstances such as

The normal right ventricle is a thin-walled flow generator perfectly adapted to face the low-pressure, high-compliant pulmonary circulation [3, 11]. However, RV anatomical and physiological properties are maybe not designed to face dramatical afterload increase at high levels of exercise. As compared to the left ventricle (LV), load increases are greater for the RV during exercise [12], and its contractile reserve may become insufficient for adequate blood supply to peripheral demand [12, 13]. Relative to the LV, the greater load that the RV faces during exercise is dominantly attributed to a larger exercise-induced increase in pulmonary artery pressure (PAP)

Increased PAP during exercise is known to limit exercise capacity in pulmonary hypertension patients through a decreased maximum cardiac output by an overloaded right ventricle [11]. Recently it has also been suggested that the same phenomenon could appear in healthy individuals exercising at high workloads at sea

Invasive or noninvasive studies in healthy subjects described a ceiling level of the mean PAP approximating 40–50 mmHg when exercising at maximal workloads corresponding to the extreme afterload level which the RV can face while maintaining a high cardiac output [6, 14–17]. The RV is thus placed under great stress during intense exercise. This leads to the idea that RV outflow might become a limiting factor when the ventricular work demand is overwhelmed, particularly in cases of extreme cardiac outputs (high intensity exercise, endurance athletes) or increased PAP (pathological or hypoxic conditions). Recently, D'Alto et al. demonstrated that echocardiographic RV systolic function indices (tricuspid annular plane systolic excursion (TAPSE), S′, and TAPSE/PAP) correlate with maximal workload in healthy subjects. This finding illustrates that a higher RV contractility reserve, defined as the difference between peak exercise and rest, is an advantage to reach high exercise levels and suggests a

congestive heart failure or highly trained endurance athletes [3].

relative to systemic vascular pressure [3].

level [5–8] but even more at altitude [7–10].

potential role of the RV in exercise capacity limitation [13].

**60**

**2. Right ventricle**

#### **3.1 Pulmonary vascular resistance**

Pulmonary circulation opposes resistances to the ejecting RV that can be quantified by the PAP at a given cardiac output [18]. According to Poiseuille's law, applicable for a Newtonian fluid flowing laminarly in a straight cylinder, flow and driving pressure are proportional. This would imply that with unchanged resistance every increase in flow would increase PAP. However, the pulmonary circulation has this specific property to reduce resistances by two possible mechanisms: (1) recruitment, enlistment of previously closed pulmonary capillary [19, 20], and (2) distension, expansion of already filled pulmonary capillaries when pressure increases [21]. It is generally accepted that the initial vascular recruitment at the onset of exercise followed by distension allows for the pulmonary circulation to face a high blood flow with limited increase in pressure and maintaining RV systolic function at minimal energy cost [22]. Indeed, low pressure in the pulmonary circulation is essential to prevent two potential exercise capacity limitation mechanisms: fluid leaking from the capillaries to the interstitial space with subsequent gas exchange alterations and RV outflow and oxygen transport limitation [18, 22].

During exercise, PAP increases along with Q but not always with a one to one ratio [15, 23]. In exercising subjects, the PAP can be measured at different exercise intensities or Q allowing for the calculation of the PAP vs. Q slope, as illustrated in **Figure 1**. The PAP vs. Q slope is a more accurate estimation of pulmonary vascular resistance (PVR) as compared to a single measure at rest [14, 22, 24]. Invasive catheterization and noninvasive stress echocardiography studies showed that pulmonary vascular response to exercise varies considerably from one individual to another, with slopes of mean PAP/Q ranging from 0.5 or 1 mmHg/l/min in young adults to 2.5 mmHg/l/min in elderly [14, 23]. This means that with a 10 l/min exercise-induced Q increase, for example, normal PAP increase would range from 5 to 25 mmHg. This great interindividual variability of pulmonary vascular response

#### **Figure 1.**

*Stress echocardiography multiple measurements of mean pulmonary arterial pressure (mPAP) at increasing flow (Q ) from rest until maximal exercise in one healthy subject. Pulmonary vascular resistance (PVR) is evaluated from the angular coefficient of the mPAP vs. Q linear regression line (ΔmPAP/ΔQ ). The PVR of 0.8 mmHg/l/min found in this example is in good agreement with limits of normal (gray background) [23]. From the curvilinearity of this PAP vs. Q relationship (dotted line), a mathematical distensible model relating mPAP, Q, left atrial pressure (LAP), and total PVR at rest (R0) allows for a calculation of a distensible factor: α (cfr formula). The present subject shows a distensibility (α) corresponding of 1.4% increase of the pulmonary vascular diameter per mmHg of pressure elevation during the entire exercise test and is in good agreement with previous reports [8, 14, 23, 26].*

during exercise also suggests a great interindividual variability of RV energy cost. Interestingly, lower PAP/Q slopes have been found in fittest subjects [6–8, 10]. This observation suggests that lower RV output resistance helps to reach higher exercise intensities. Conversely, in patients with pulmonary hypertension, exercise is associated with a sharp increase in PAP (high PAP/Q slopes), and a right ventricular limitation affects exercise capacity [11, 25].

#### **3.2 Pulmonary vascular distensibility**

When multiple PAP are measured at increasing exercise or Q levels, it is possible to show that the PAP/Q relationship is not strictly linear but becomes curvilinear with a smoothened pressure increase at higher exercise intensities [14, 16, 26, 27]. The curvilinearity of the PAP/Q relationship reflects the distension of the pulmonary resistive vessels in order to limit the flow-induced pressure increase during exercise. This pulmonary vascular distension participates in decreasing PVR and RV afterload during exercise. The pulmonary vascular distensibility can be quantified with a mathematical model applied to the PA-Q relationship allowing the calculation of a coefficient of distensibility; α (**Figure 1**). Alpha depends on the mechanical properties of the lung vascular walls and represents the percentage change in arteriolar diameter per mmHg of arteriolar pressure increase with exercise [16, 24, 26]. Direct in vitro or indirect in vivo measurements showed an average of 2% increase in diameter per mmHg of distending pressure in healthy pulmonary vessels [14, 26]. Higher alphas, representing a more distensible pulmonary circulation, have been shown to be associated to lower blood flow resistances (PAP/Q slopes) [6]. However, it is interesting to note that the distensibility of the pulmonary vasculature does not stay constant with the onset of exercise but tends to decrease with exercise intensity, indicating that pulmonary vascular compliance decreases along with increases in flow and intravascular distending pressures [6, 16, 23]. Argiento et al. described a mean distensibility *α* at rest of 2.2%/mmHg decreasing to 1.3%/mmHg at maximal exercise in 88 young heathy adults [23].

Fit subjects, with a high aerobic capacity, have been shown to have enhanced exercise-associated decrease in PVR and increase in pulmonary arterial compliance. This has been demonstrated recently with higher VO2max correlated to greater pulmonary arteriolar distensibility α [6, 8] associated with lower PVR at maximum exercise or lower PAP/Q slopes [6–8]. This observation was true at sea level but was even more pronounced at moderate or high altitude [7, 8, 10]. One could consider that a more distensible and low resistive pulmonary circulation is an advantage for aerobic exercise performance.

#### **4. Pulmonary capillaries, gas exchange, and ventilation**

It has previously been estimated that resistances in the pulmonary circulation are located for 60% at the precapillary level and for 40% at the capillary-venous level [28]. Pulmonary capillaries hemodynamic thus significantly contribute to changes in PVR during exercise and can therefore not be neglected.

#### **4.1 Pulmonary transit of agitated contrast**

The property of the pulmonary capillaries to distend during exercise can be studied by intravenous injection of an agitated contrast. Bubbles appearing in the right heart must transit through the pulmonary circulation to be observed in the left heart chambers with echocardiography. At rest, no bubble transit occurs from the

**63**

*Pulmonary Vascular Reserve and Aerobic Exercise Capacity*

opening of an arteriovenous shunt is still debated [31–33].

been described in some endurance-trained athletes [36].

**4.2 Lung diffusion capacity**

right to the left heart. However, during exercise in healthy individuals, pulmonary transit of agitated contrast (PTAC) occurs when contrast appears from the right to the left heart chamber after four to five heartbeats [29, 30]. Whether this exerciseinduced bubble transit is explained by pulmonary capillary distension or by the

In a recent study, La Gerche et al*.* used PTAC to assess pulmonary vascular reserve in exercising healthy individuals. They observed that subjects with no or minimal bubble transit through the pulmonary circulation also showed higher PVR assessed by steeper PAP/Q slopes, and individuals with high PTAC had lower exercise-induced increases in PAP and greater PVR reduction [5]. This observation suggests that a greater pulmonary vascular reserve can occur through a possible enhanced capillary distensibility. Moreover, this physiological advantage was associated with improved RV function and higher maximal Q. The authors of this study concluded that higher PTAC is advantageous to lower RV afterload and creating less RV fatigue during prolonged and intense exercise [5]. In support of this previous finding, in a similar study, Lalande et al. found that the amount of bubbles transiting through the pulmonary circulation was proportional to the increase in pulmonary capillary pressure and volume during exercise [6]. In this study positive PTAC occurred during exercise when a twofold increase in vascular pressure allowed for a 20–30% increase in capillary blood volume [6]. Capillary recruitment and dilation seem thus crucial to unload the RV at high levels of exercise but is also crucial to maintain capillary pressure low during intense exercise. In numerous studies, West et al. highlighted that an abnormal increase in PAP and subsequent capillary pressure elevation above a 20–25 mmHg threshold at exercise could possibly lead to a capillary stress failure known to elicit interstitial lung edema and altered ventilation/perfusion relationships [34, 35]. Capillary damages have indeed previously

Capillary blood volume can be estimated noninvasively from lung diffusing capacity measurements using double gas tracers: carbon monoxide (CO) and nitric oxide (NO) differing in their affinity for hemoglobin. The Roughton and Forster equation, 1/*D*LCO = 1/*D*m + 1/*θV*c, states that lung diffusion from the alveola to the erythrocyte's hemoglobin is the result of two resistances in series: the alveolocapillary membrane diffusion component and an intracapillary component. *D*LCO is the measured diffusing capacity of the lung for CO, *D*m the membrane component, *θ* the hemoglobin affinity for CO, and *V*c the capillary blood volume [37]. Transposing this equation for NO, which has particularly high hemoglobin affinity, two equations can be solved with two unknowns allowing for *V*c calculation [38]. Exercising at sea level is associated with an increase in DLCO, DLNO, Dm, and Vc linearly with the workload intensity without ceiling effect. This suggests that recruitment and distention of the pulmonary circulation does not reach a limit even at high exercise levels. Also, a predominant exercise-induced increase in *V*c relative to *D*m has been described suggesting a predominance of capillary distension rather than recruitment, whereas a recruitment would increase more Dm than Vc [39–41]. This is in keeping with the notion that exercise is associated with an increased diameter of pulmonary capillaries [6, 41–43]. Recent studies found indeed that the amount of blood in the pulmonary capillaries was a determinant of the aerobic exercise capacity [6, 8]. This observation is compatible with the hypothesis that exercise capacity is modulated by the functional state of the pulmonary circulation, including capillary vessels, and could be confirmed in more than 150 healthy adults tested in our laboratory (**Figure 2C**). Additionally, it also appeared that the

*DOI: http://dx.doi.org/10.5772/intechopen.88399*

*Interventional Pulmonology and Pulmonary Hypertension - Updates on Specific Topics*

limitation affects exercise capacity [11, 25].

**3.2 Pulmonary vascular distensibility**

aerobic exercise performance.

**4. Pulmonary capillaries, gas exchange, and ventilation**

changes in PVR during exercise and can therefore not be neglected.

**4.1 Pulmonary transit of agitated contrast**

It has previously been estimated that resistances in the pulmonary circulation are located for 60% at the precapillary level and for 40% at the capillary-venous level [28]. Pulmonary capillaries hemodynamic thus significantly contribute to

The property of the pulmonary capillaries to distend during exercise can be studied by intravenous injection of an agitated contrast. Bubbles appearing in the right heart must transit through the pulmonary circulation to be observed in the left heart chambers with echocardiography. At rest, no bubble transit occurs from the

during exercise also suggests a great interindividual variability of RV energy cost. Interestingly, lower PAP/Q slopes have been found in fittest subjects [6–8, 10]. This observation suggests that lower RV output resistance helps to reach higher exercise intensities. Conversely, in patients with pulmonary hypertension, exercise is associated with a sharp increase in PAP (high PAP/Q slopes), and a right ventricular

When multiple PAP are measured at increasing exercise or Q levels, it is possible to show that the PAP/Q relationship is not strictly linear but becomes curvilinear with a smoothened pressure increase at higher exercise intensities [14, 16, 26, 27]. The curvilinearity of the PAP/Q relationship reflects the distension of the pulmonary resistive vessels in order to limit the flow-induced pressure increase during exercise. This pulmonary vascular distension participates in decreasing PVR and RV afterload during exercise. The pulmonary vascular distensibility can be quantified with a mathematical model applied to the PA-Q relationship allowing the calculation of a coefficient of distensibility; α (**Figure 1**). Alpha depends on the mechanical properties of the lung vascular walls and represents the percentage change in arteriolar diameter per mmHg of arteriolar pressure increase with exercise [16, 24, 26]. Direct in vitro or indirect in vivo measurements showed an average of 2% increase in diameter per mmHg of distending pressure in healthy pulmonary vessels [14, 26]. Higher alphas, representing a more distensible pulmonary circulation, have been shown to be associated to lower blood flow resistances (PAP/Q slopes) [6]. However, it is interesting to note that the distensibility of the pulmonary vasculature does not stay constant with the onset of exercise but tends to decrease with exercise intensity, indicating that pulmonary vascular compliance decreases along with increases in flow and intravascular distending pressures [6, 16, 23]. Argiento et al. described a mean distensibility *α* at rest of 2.2%/mmHg decreasing to 1.3%/mmHg at maximal exercise in 88 young heathy adults [23]. Fit subjects, with a high aerobic capacity, have been shown to have enhanced exercise-associated decrease in PVR and increase in pulmonary arterial compliance. This has been demonstrated recently with higher VO2max correlated to greater pulmonary arteriolar distensibility α [6, 8] associated with lower PVR at maximum exercise or lower PAP/Q slopes [6–8]. This observation was true at sea level but was even more pronounced at moderate or high altitude [7, 8, 10]. One could consider that a more distensible and low resistive pulmonary circulation is an advantage for

**62**

right to the left heart. However, during exercise in healthy individuals, pulmonary transit of agitated contrast (PTAC) occurs when contrast appears from the right to the left heart chamber after four to five heartbeats [29, 30]. Whether this exerciseinduced bubble transit is explained by pulmonary capillary distension or by the opening of an arteriovenous shunt is still debated [31–33].

In a recent study, La Gerche et al*.* used PTAC to assess pulmonary vascular reserve in exercising healthy individuals. They observed that subjects with no or minimal bubble transit through the pulmonary circulation also showed higher PVR assessed by steeper PAP/Q slopes, and individuals with high PTAC had lower exercise-induced increases in PAP and greater PVR reduction [5]. This observation suggests that a greater pulmonary vascular reserve can occur through a possible enhanced capillary distensibility. Moreover, this physiological advantage was associated with improved RV function and higher maximal Q. The authors of this study concluded that higher PTAC is advantageous to lower RV afterload and creating less RV fatigue during prolonged and intense exercise [5]. In support of this previous finding, in a similar study, Lalande et al. found that the amount of bubbles transiting through the pulmonary circulation was proportional to the increase in pulmonary capillary pressure and volume during exercise [6]. In this study positive PTAC occurred during exercise when a twofold increase in vascular pressure allowed for a 20–30% increase in capillary blood volume [6]. Capillary recruitment and dilation seem thus crucial to unload the RV at high levels of exercise but is also crucial to maintain capillary pressure low during intense exercise. In numerous studies, West et al. highlighted that an abnormal increase in PAP and subsequent capillary pressure elevation above a 20–25 mmHg threshold at exercise could possibly lead to a capillary stress failure known to elicit interstitial lung edema and altered ventilation/perfusion relationships [34, 35]. Capillary damages have indeed previously been described in some endurance-trained athletes [36].

#### **4.2 Lung diffusion capacity**

Capillary blood volume can be estimated noninvasively from lung diffusing capacity measurements using double gas tracers: carbon monoxide (CO) and nitric oxide (NO) differing in their affinity for hemoglobin. The Roughton and Forster equation, 1/*D*LCO = 1/*D*m + 1/*θV*c, states that lung diffusion from the alveola to the erythrocyte's hemoglobin is the result of two resistances in series: the alveolocapillary membrane diffusion component and an intracapillary component. *D*LCO is the measured diffusing capacity of the lung for CO, *D*m the membrane component, *θ* the hemoglobin affinity for CO, and *V*c the capillary blood volume [37]. Transposing this equation for NO, which has particularly high hemoglobin affinity, two equations can be solved with two unknowns allowing for *V*c calculation [38].

Exercising at sea level is associated with an increase in DLCO, DLNO, Dm, and Vc linearly with the workload intensity without ceiling effect. This suggests that recruitment and distention of the pulmonary circulation does not reach a limit even at high exercise levels. Also, a predominant exercise-induced increase in *V*c relative to *D*m has been described suggesting a predominance of capillary distension rather than recruitment, whereas a recruitment would increase more Dm than Vc [39–41]. This is in keeping with the notion that exercise is associated with an increased diameter of pulmonary capillaries [6, 41–43]. Recent studies found indeed that the amount of blood in the pulmonary capillaries was a determinant of the aerobic exercise capacity [6, 8]. This observation is compatible with the hypothesis that exercise capacity is modulated by the functional state of the pulmonary circulation, including capillary vessels, and could be confirmed in more than 150 healthy adults tested in our laboratory (**Figure 2C**). Additionally, it also appeared that the

**Figure 2.**

*Correlations between lung capillary volume measured at rest (Vc), ventilatory equivalent for CO2 (VE/VCO2) measured at the ventilatory threshold (VT), and aerobic exercise capacity (VO2max). Larger blood capillary volume allows for better ventilation-perfusion adequacy decreasing ventilation at a given metabolic rate and higher aerobic capacity.*

blood volume of the pulmonary capillaries (Vc) measured by the DLCO and DLNO method was correlated to the ventilation at a given metabolic cost (VE/VCO2 ratio) measured during incremental cardiopulmonary exercise testing (**Figure 2A**). This founding, suggesting that better perfused lungs allows for lower ventilatory cost, is an advantage to reach higher exercise levels (**Figure 2B** and **C**).

The VE/VCO2 ratio represents the ventilation level needed to evacuate 1 L of CO2 for 1 minute and is therefore a good indicator of the ventilatory efficiency and represents the metabolic cost of ventilation. The VE/VCO2 ratio can be measured at the ventilatory threshold, when metabolic acidosis is not yet pronounced and does not substantially influence ventilation. However, the slope of the VE versus VCO2 relationship from rest until the respiratory compensation point might be more accurate to define the ventilatory chemosensibility [44].

Chronic heart failure and even more so pulmonary arterial hypertension increase the VE/VCO2 slope by a combination of increased dead space related to low cardiac output, early lactic acidosis, and increased chemosensitivity in the context of an increased sympathetic nervous system tone in relation with the severity of the pathology [45]. The VE/VCO2 slope has indeed been identified as a strong prognostic tool in patients with heart failure, and in some studies, its prognostic significance has outperformed the VO2max [44]. In the other hand, endurance athletes are known to have shallow VE/VCO2 slopes probably through a training-induced decrease in chemosensibility [46].

Interestingly, recent studies also showed a link between higher diffusion capacities (DLNO) and shallowest VE/VCO2 slopes [6, 8] in keeping with previous notion that higher lung diffusing capacity allows for preserved gas exchange at a lower ventilatory cost [47]. In those studies, the higher diffusion capacities and lower VE/ VCO2 slopes were associated to higher aerobic capacity [6, 8].

#### **5. Pulmonary vascular reserve**

The pulmonary vascular reserve is the ability of the pulmonary circulation to accommodate high flows by moderating pressure increase with vascular recruitment, dilatation, and/or distension and allows low hemodynamic pressures in the pulmonary circulation. The more the pulmonary circulation is able to face high blood flow with maintaining low pressures during exercise the greater the pulmonary vascular reserve. This is critical in minimizing RV afterload and maximizing cardiac output during exercise. When pulmonary vascular reserve is compromised, RV ejection may also be compromised, increasing right atrial pressure and limiting maximal cardiac output [18]. Pulmonary vascular reserve avoids abnormal increase

**65**

*Pulmonary Vascular Reserve and Aerobic Exercise Capacity*

in PAP and subsequently increases in pulmonary capillary pressure, protecting from an interstitial pulmonary edema [35, 48]. Finally, a better vascular reserve allows for a greater capillary distention increasing the lung capillary volume which has been shown to be associated with better ventilation-perfusion adequacy, better lung

A pioneer study by La Gerche et al. demonstrated that favorable changes in pulmonary vascular reserve provide a physiological advantage for RV function during exercise [5]. Indeed, subjects with the higher PTAC had the lowest PVR and lowest exercise-induced B-type natriuretic peptide blood levels (usually elevated with ventricular volume and pressure overload) associated with higher maximal Q [5]. Subsequently, Lalande et al. observed that the individuals with the highest VO2max had the greatest pulmonary vascular reserve, in this study defined as greater arteriolar distensibility α and capillary bed volume along with lowest PVR at maximum exercise [6]. Following these observations, Pavelescu et al. reviewed diffusion capacity measurements and echocardiographic measurements of the pulmonary circulation in a larger number of healthy subjects and confirmed that better aerobic exercise capacity is associated with lower PVR and higher lung diffusing capacity

Taken together, all those observations strongly suggest that exercise capacity is modulated by the functional state of the pulmonary circulation. A great pulmonary vascular reserve is therefore an advantage in endurance athletic performance especially when exercise is performed at extreme cardiac output levels. However, when or why pulmonary vascular reserve may allow for a higher aerobic exercise capacity

It is well-known that interindividual pulmonary vascular response to exercise varies considerably. Beyond that, different factors have been identified to influence the pulmonary vascular reserve such as body position, sex, race, age, and environ-

Pulmonary vascular response to exercise testing is either performed in a supine

Racial differences have been suspected to influence pulmonary vascular reserve as black African Americans compared to white Americans of European descent are

position during catheterization or in a semi-recumbent position during stress echocardiography, while exercise testing is usually performed in a sitting or upright position. Invasive studies previously reported a lower resting PVR in the recumbent position compared with upright position explained by a vascular recruitment when venous return is increased with gravity [49]. However, the authors observed that differences faded and disappeared with exercise-induced cardiac output increase, because of vascular recruitment with pulmonary blood flow elevation. Accordingly, those observations have been confirmed recently by Forton et al. who compared maximal exercise testing in supine, semi-recumbent, and upright positions and showed no body position effect on PAP/Q relationships, alpha, and VO2max [50]. Influence of posture [17] PAP rest supine (14.0 + −3.3 mmHg) versus upright

diffusion capacity, and lower ventilatory cost at a given metabolic rate [7, 8].

*DOI: http://dx.doi.org/10.5772/intechopen.88399*

allowing for lower exercise ventilation [7].

**6. Influences of pulmonary vascular reserve**

is still incompletely described.

mental factors.

**6.1 Body position**

(13.6 + −3.1 mmHg)

**6.2 Race and sex**

#### *Pulmonary Vascular Reserve and Aerobic Exercise Capacity DOI: http://dx.doi.org/10.5772/intechopen.88399*

*Interventional Pulmonology and Pulmonary Hypertension - Updates on Specific Topics*

blood volume of the pulmonary capillaries (Vc) measured by the DLCO and DLNO method was correlated to the ventilation at a given metabolic cost (VE/VCO2 ratio) measured during incremental cardiopulmonary exercise testing (**Figure 2A**). This founding, suggesting that better perfused lungs allows for lower ventilatory cost, is

*Correlations between lung capillary volume measured at rest (Vc), ventilatory equivalent for CO2 (VE/VCO2) measured at the ventilatory threshold (VT), and aerobic exercise capacity (VO2max). Larger blood capillary volume allows for better ventilation-perfusion adequacy decreasing ventilation at a given metabolic rate and* 

The VE/VCO2 ratio represents the ventilation level needed to evacuate 1 L of CO2 for 1 minute and is therefore a good indicator of the ventilatory efficiency and represents the metabolic cost of ventilation. The VE/VCO2 ratio can be measured at the ventilatory threshold, when metabolic acidosis is not yet pronounced and does not substantially influence ventilation. However, the slope of the VE versus VCO2 relationship from rest until the respiratory compensation point might be more

Chronic heart failure and even more so pulmonary arterial hypertension increase the VE/VCO2 slope by a combination of increased dead space related to low cardiac output, early lactic acidosis, and increased chemosensitivity in the context of an increased sympathetic nervous system tone in relation with the severity of the pathology [45]. The VE/VCO2 slope has indeed been identified as a strong prognostic tool in patients with heart failure, and in some studies, its prognostic significance has outperformed the VO2max [44]. In the other hand, endurance athletes are known to have shallow VE/VCO2 slopes probably through a training-induced

Interestingly, recent studies also showed a link between higher diffusion capacities (DLNO) and shallowest VE/VCO2 slopes [6, 8] in keeping with previous notion that higher lung diffusing capacity allows for preserved gas exchange at a lower ventilatory cost [47]. In those studies, the higher diffusion capacities and lower VE/

The pulmonary vascular reserve is the ability of the pulmonary circulation to accommodate high flows by moderating pressure increase with vascular recruitment, dilatation, and/or distension and allows low hemodynamic pressures in the pulmonary circulation. The more the pulmonary circulation is able to face high blood flow with maintaining low pressures during exercise the greater the pulmonary vascular reserve. This is critical in minimizing RV afterload and maximizing cardiac output during exercise. When pulmonary vascular reserve is compromised, RV ejection may also be compromised, increasing right atrial pressure and limiting maximal cardiac output [18]. Pulmonary vascular reserve avoids abnormal increase

an advantage to reach higher exercise levels (**Figure 2B** and **C**).

accurate to define the ventilatory chemosensibility [44].

VCO2 slopes were associated to higher aerobic capacity [6, 8].

decrease in chemosensibility [46].

**Figure 2.**

*higher aerobic capacity.*

**5. Pulmonary vascular reserve**

**64**

in PAP and subsequently increases in pulmonary capillary pressure, protecting from an interstitial pulmonary edema [35, 48]. Finally, a better vascular reserve allows for a greater capillary distention increasing the lung capillary volume which has been shown to be associated with better ventilation-perfusion adequacy, better lung diffusion capacity, and lower ventilatory cost at a given metabolic rate [7, 8].

A pioneer study by La Gerche et al. demonstrated that favorable changes in pulmonary vascular reserve provide a physiological advantage for RV function during exercise [5]. Indeed, subjects with the higher PTAC had the lowest PVR and lowest exercise-induced B-type natriuretic peptide blood levels (usually elevated with ventricular volume and pressure overload) associated with higher maximal Q [5]. Subsequently, Lalande et al. observed that the individuals with the highest VO2max had the greatest pulmonary vascular reserve, in this study defined as greater arteriolar distensibility α and capillary bed volume along with lowest PVR at maximum exercise [6]. Following these observations, Pavelescu et al. reviewed diffusion capacity measurements and echocardiographic measurements of the pulmonary circulation in a larger number of healthy subjects and confirmed that better aerobic exercise capacity is associated with lower PVR and higher lung diffusing capacity allowing for lower exercise ventilation [7].

Taken together, all those observations strongly suggest that exercise capacity is modulated by the functional state of the pulmonary circulation. A great pulmonary vascular reserve is therefore an advantage in endurance athletic performance especially when exercise is performed at extreme cardiac output levels. However, when or why pulmonary vascular reserve may allow for a higher aerobic exercise capacity is still incompletely described.

#### **6. Influences of pulmonary vascular reserve**

It is well-known that interindividual pulmonary vascular response to exercise varies considerably. Beyond that, different factors have been identified to influence the pulmonary vascular reserve such as body position, sex, race, age, and environmental factors.

#### **6.1 Body position**

Pulmonary vascular response to exercise testing is either performed in a supine position during catheterization or in a semi-recumbent position during stress echocardiography, while exercise testing is usually performed in a sitting or upright position. Invasive studies previously reported a lower resting PVR in the recumbent position compared with upright position explained by a vascular recruitment when venous return is increased with gravity [49]. However, the authors observed that differences faded and disappeared with exercise-induced cardiac output increase, because of vascular recruitment with pulmonary blood flow elevation. Accordingly, those observations have been confirmed recently by Forton et al. who compared maximal exercise testing in supine, semi-recumbent, and upright positions and showed no body position effect on PAP/Q relationships, alpha, and VO2max [50].

Influence of posture [17] PAP rest supine (14.0 + −3.3 mmHg) versus upright (13.6 + −3.1 mmHg)

#### **6.2 Race and sex**

Racial differences have been suspected to influence pulmonary vascular reserve as black African Americans compared to white Americans of European descent are

known to have higher prevalence of hypertension and higher mortality rates for most cardiovascular diseases [51]. Recently, Simaga et al. tested this hypothesis and showed an intrinsically less distensible pulmonary circulation in healthy black sub-Saharan African men as compared to healthy white Caucasians, and this was associated with a lower aerobic exercise capacity [52]. Lower DLNO and DLCO are also reported in Africans as compared to sex-, age-, and body size-matched Caucasians and are explained by racial-related smaller lungs proportionally to body size [53]. However, those racial differences in pulmonary vascular function at exercise did not appear when women were compared [52]. This latest observation is in keeping with previous studies showing that premenopausal women have a more distensible pulmonary circulation with a coefficient of distensibility α up to 45% higher compared to age-matched men [23]. The underlying explanation is not clearly established but might be related to the hormonal context.

#### **6.3 Aging**

Exercise capacity decreases with aging, as does the pulmonary vascular reserve. Invasive measurements have previously showed that a reduction in pulmonary microvascular distensibility occurs with age [17, 24]. Consistently, La Gerche et al. noticed that individuals with low PTAC were older than those with positive PTAC [5]. More recently, Argiento et al. confirmed this aging effect observation with noninvasive echocardiographic measurements and showed that while maximal cardiac output was reduced in fifties or older individuals, PAP and PVR were higher with a lower alpha at maximal exercise [23].

Influence of age [17] PAP <30 (12.8 + −3.1 mmHg) versus 30–50 (12.9 + −3.0 mmHg) versus > 50 years (14.7 + −4.0).

#### **6.4 Growth**

Aerobic capacity increases gradually with age during childhood and adolescence. The kinetics of this evolution differs in girls and boys related to pubertal hormonal changes reaching a peak in VO2max earlier in girls compared to boys. However, previous studies showed that VO2max is not so much a matter of age when VO2max is corrected by body weight [54, 55].

On the other hand, the maximal workload, endurance time, and maximum average running speed increase continuously with age attesting the complexity of the relations between VO2 at exercise, weight or body dimensions, and the mechanical performance of muscular work [56].

The progression of endurance time and load at a given VO2 with age is multifactorial and includes neuromuscular adaptations, movement technique, musculotendinous elastic energy storage, surface vs. weight ratio, body temperature, energy substrates use, and ventilatory response. Indeed, the VE/VCO2 slope decreases with age reflecting a more efficient ventilatory response during exercise. This has been attributed to chemosensitivity maturation with age [54, 57]. Concomitantly, it is also known that diffusion capacity of DLCO and DLNO increases during adolescence. The link between these two observations remains to be clarified as the DLCO and DLNO increase has previously been mainly attributed to increase in height [58].

Experiments from our exercise laboratory on heathy adolescents show that pulmonary arterial distensibility and chemosensitivity decrease with growth, while maximal Q, RV function and diffusion capacity increase in relation to increased aerobic exercise capacity.

Taken together, the aforementioned findings suggest that the different components of the RV- pulmonary circulation unit are mature at different times. Creating

**67**

*Pulmonary Vascular Reserve and Aerobic Exercise Capacity*

consequences of diesel exposure on exercise capacity.

tion during hypoxic exercise are sorely lacking [64].

limitation at high altitude is still a matter of debate.

a probable optimal pulmonary vascular reserve and exercise capacity at adulthood

Finally, some environmental factors have also been identified as potential pulmonary circulation stressors, namely, altitude and pollution. It has recently been shown that an acute exposure of 2 h to a dilute diesel exhaust increased the pulmonary vasomotor tone by decreasing the distensibility of pulmonary resistive vessels at high cardiac output or high exercise intensities [59]. Further studies are needed for a better understanding of this phenomenon and to evaluate the long-term

Increasing visitors and athletes are traveling to altitude but not without consequences on their physical condition. It has long been known that aerobic exercise capacity decreases exponentially with altitude ascent with a significant decline starting above 1000 m. Numerous studies have been conducted in the field, but the underlying mechanisms are until now not fully understood. Although causes might be multifactorial, decreased oxygen transport to the exercising muscle, with a decrease in arterial oxygenation (SpO2) and an altered maximum Q, is fingered

At high altitude, in resting conditions, signs of altered diastolic but preserved or enhanced systolic RV function have been described in chronic [61] or acute hypoxic conditions [62, 63]. RV seems thus to tolerate hypoxic conditions. However, a recent study showed inhomogeneous RV contraction in hypoxia but not during exercise, suggesting that hypoxic stress is not trivial [63]. How much this could account for altered RV maximal outflow remains unknown as studies on right ventricular func-

Since the pioneer study of Von Euler and Liljestrand in 1946 that when airways are exposed to hypoxic air, a local vasoconstrictive reflex modifies the lung perfusion in favor of better oxygenated alveoli [65]. This hypoxic pulmonary vasoconstriction (HPV) is a protective mechanism allowing for substantial improvement in arterial oxygenation [66]. However, at altitude, when the entire lung is hypoxic, a global arteriolar vasoconstriction reduces the pulmonary vascular distensibility and increases the PVR. The subsequent hypoxic pulmonary hypertension is generally mild to moderate [64]. However, during exercise, this substantial increased afterload on the right ventricle might become substantial [64]. Hypoxia may therefore affect the pulmonary vascular reserve with increased likelihood of RV function limitation and/or altered gas exchange by interstitial pulmonary edema or ventilation/perfusion mismatch. How much this accounts for aerobic exercise capacity

This last decade, a partial recovery of 10–25% of the hypoxia-induced decrease

in maximal oxygen uptake has been reported with intake-specific pulmonary vasodilating interventions [67–72]. Indeed, specific pulmonary vasodilating interventions have been reported to improve the decreased aerobic exercise capacity in

*DOI: http://dx.doi.org/10.5772/intechopen.88399*

but declining further with aging.

**6.5 Pollution**

**6.6 Hypoxia**

[1, 2, 60].

*6.6.1 Right ventricle*

*6.6.2 Pulmonary circulation*

a probable optimal pulmonary vascular reserve and exercise capacity at adulthood but declining further with aging.

#### **6.5 Pollution**

*Interventional Pulmonology and Pulmonary Hypertension - Updates on Specific Topics*

might be related to the hormonal context.

with a lower alpha at maximal exercise [23].

is corrected by body weight [54, 55].

performance of muscular work [56].

aerobic exercise capacity.

(12.9 + −3.0 mmHg) versus > 50 years (14.7 + −4.0).

**6.3 Aging**

**6.4 Growth**

known to have higher prevalence of hypertension and higher mortality rates for most cardiovascular diseases [51]. Recently, Simaga et al. tested this hypothesis and showed an intrinsically less distensible pulmonary circulation in healthy black sub-Saharan African men as compared to healthy white Caucasians, and this was associated with a lower aerobic exercise capacity [52]. Lower DLNO and DLCO are also reported in Africans as compared to sex-, age-, and body size-matched Caucasians and are explained by racial-related smaller lungs proportionally to body size [53]. However, those racial differences in pulmonary vascular function at exercise did not appear when women were compared [52]. This latest observation is in keeping with previous studies showing that premenopausal women have a more distensible pulmonary circulation with a coefficient of distensibility α up to 45% higher compared to age-matched men [23]. The underlying explanation is not clearly established but

Exercise capacity decreases with aging, as does the pulmonary vascular reserve.

Aerobic capacity increases gradually with age during childhood and adolescence. The kinetics of this evolution differs in girls and boys related to pubertal hormonal changes reaching a peak in VO2max earlier in girls compared to boys. However, previous studies showed that VO2max is not so much a matter of age when VO2max

On the other hand, the maximal workload, endurance time, and maximum average running speed increase continuously with age attesting the complexity of the relations between VO2 at exercise, weight or body dimensions, and the mechanical

The progression of endurance time and load at a given VO2 with age is multifactorial and includes neuromuscular adaptations, movement technique, musculotendinous elastic energy storage, surface vs. weight ratio, body temperature, energy substrates use, and ventilatory response. Indeed, the VE/VCO2 slope decreases with age reflecting a more efficient ventilatory response during exercise. This has been attributed to chemosensitivity maturation with age [54, 57]. Concomitantly, it is also known that diffusion capacity of DLCO and DLNO increases during adolescence. The link between these two observations remains to be clarified as the DLCO and DLNO increase has previously been mainly attributed to increase in height [58]. Experiments from our exercise laboratory on heathy adolescents show that pulmonary arterial distensibility and chemosensitivity decrease with growth, while maximal Q, RV function and diffusion capacity increase in relation to increased

Taken together, the aforementioned findings suggest that the different components of the RV- pulmonary circulation unit are mature at different times. Creating

Invasive measurements have previously showed that a reduction in pulmonary microvascular distensibility occurs with age [17, 24]. Consistently, La Gerche et al. noticed that individuals with low PTAC were older than those with positive PTAC [5]. More recently, Argiento et al. confirmed this aging effect observation with noninvasive echocardiographic measurements and showed that while maximal cardiac output was reduced in fifties or older individuals, PAP and PVR were higher

Influence of age [17] PAP <30 (12.8 + −3.1 mmHg) versus 30–50

**66**

Finally, some environmental factors have also been identified as potential pulmonary circulation stressors, namely, altitude and pollution. It has recently been shown that an acute exposure of 2 h to a dilute diesel exhaust increased the pulmonary vasomotor tone by decreasing the distensibility of pulmonary resistive vessels at high cardiac output or high exercise intensities [59]. Further studies are needed for a better understanding of this phenomenon and to evaluate the long-term consequences of diesel exposure on exercise capacity.

#### **6.6 Hypoxia**

Increasing visitors and athletes are traveling to altitude but not without consequences on their physical condition. It has long been known that aerobic exercise capacity decreases exponentially with altitude ascent with a significant decline starting above 1000 m. Numerous studies have been conducted in the field, but the underlying mechanisms are until now not fully understood. Although causes might be multifactorial, decreased oxygen transport to the exercising muscle, with a decrease in arterial oxygenation (SpO2) and an altered maximum Q, is fingered [1, 2, 60].

#### *6.6.1 Right ventricle*

At high altitude, in resting conditions, signs of altered diastolic but preserved or enhanced systolic RV function have been described in chronic [61] or acute hypoxic conditions [62, 63]. RV seems thus to tolerate hypoxic conditions. However, a recent study showed inhomogeneous RV contraction in hypoxia but not during exercise, suggesting that hypoxic stress is not trivial [63]. How much this could account for altered RV maximal outflow remains unknown as studies on right ventricular function during hypoxic exercise are sorely lacking [64].

#### *6.6.2 Pulmonary circulation*

Since the pioneer study of Von Euler and Liljestrand in 1946 that when airways are exposed to hypoxic air, a local vasoconstrictive reflex modifies the lung perfusion in favor of better oxygenated alveoli [65]. This hypoxic pulmonary vasoconstriction (HPV) is a protective mechanism allowing for substantial improvement in arterial oxygenation [66]. However, at altitude, when the entire lung is hypoxic, a global arteriolar vasoconstriction reduces the pulmonary vascular distensibility and increases the PVR. The subsequent hypoxic pulmonary hypertension is generally mild to moderate [64]. However, during exercise, this substantial increased afterload on the right ventricle might become substantial [64]. Hypoxia may therefore affect the pulmonary vascular reserve with increased likelihood of RV function limitation and/or altered gas exchange by interstitial pulmonary edema or ventilation/perfusion mismatch. How much this accounts for aerobic exercise capacity limitation at high altitude is still a matter of debate.

This last decade, a partial recovery of 10–25% of the hypoxia-induced decrease in maximal oxygen uptake has been reported with intake-specific pulmonary vasodilating interventions [67–72]. Indeed, specific pulmonary vasodilating interventions have been reported to improve the decreased aerobic exercise capacity in

hypoxia with little or no effect on normoxic exercise performance. Primary studies described an increase in maximal workload and VO2max after intake of sildenafil, a phosphodiesterase-5 inhibitor used to treat erectile dysfunction in healthy hypoxic subjects [9, 67–69]. It has been suggested that the underlying mechanism was an increase maximal Q due to a reduced RV afterload after HPV inhibition or pulmonary vasodilation. Similar results were reported after administration of dexamethasone [70] or endothelin receptor blockers [71, 72]. In most of these studies, pulmonary vasodilation effect was also associated with improved arterial oxygenation probably allowing improved oxygen transport to the exercising muscles [69, 73]. De Bisschop et al. showed that pharmacological pulmonary vasodilation improved lung diffusion capacity and also correlated to enhanced exercise capacity at high altitude [74]. The principal suggested underlying mechanisms was related to a pulmonary vasodilation associated decrease in capillary filtration pressure protecting from an interstitial lung edema [74].

#### *6.6.3 Lung diffusion capacity*

Acute or chronic hypoxic exposure is associated with enhanced pulmonary diffusion capacity at rest [7, 75, 76]. Moreover, the hypoxia-induced increase in the capillary component being more pronounced than the membrane component suggests a capillary distension in addition to recruitment. This observation has been attributed to increased pulmonary perfusion pressure caused by HPV associated with a venous component of hypoxic vasoconstriction both possibly contributing to increase effective capillary pressure [77].

Interestingly, Taylor et al. showed that recruitment of pulmonary capillaries in response to exercise at high altitude is limited and may therefore be a significant source of exercise limitation [78]. This is keeping with previous correlation found between lung diffusing capacity for nitric oxide (DLNO) and VO2max at altitude [7, 8, 74].

#### *6.6.4 Pulmonary vascular reserve*

The reviewing of data collected during four different high-altitude expeditions (>4350 m) highlighted that individuals with a larger increase in PVR and larger decreased ventilation efficacity with ascent to high altitude were the ones with the greater VO2max fall [7]. Higher aerobic capacity at high altitude was associated with more pronounced pulmonary vascular reserve as suggested by lower PVR, higher diffusion capacity, and lower VE/VCO2 [7]. Similarly, pulmonary vascular reserve has been described as an aerobic performance limiting factor in Andean or Himalayan highlanders [10, 79]. This observation has also been confirmed at moderate altitude, even though the overwhelming determinant of decreased VO2max and maximum workload is a decrease in arterial O2 content CaO2 [8].

#### **7. Conclusion**

In conclusion, aerobic exercise capacity is depending on the integrity of the different components of the oxygen transfer from ambient air to the mitochondrial cytochromes. The RV function coupling to the pulmonary circulation and the pulmonary capillary network is one of multiple determinants of aerobic exercise capacity. It becomes increasingly clear that a high pulmonary vascular reserve is an advantage for high-intensity exercise performance in heathy subjects. The pulmonary vascular reserve is characterized by lower exercise PAP and PVR and higher

**69**

**Author details**

Vitalie Faoro\* and Kevin Forton

\*Address all correspondence to: vfaoro@ulb.ac.be

provided the original work is properly cited.

Cardio-Pulmonary Exercise Laboratory, Université Libre de Bruxelles, Belgium

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

*Pulmonary Vascular Reserve and Aerobic Exercise Capacity*

pulmonary vascular distensibility associated with greater capillary volume and gas exchange allowing for a lower ventilatory cost at a given metabolic rate. When and how the pulmonary vascular reserve modulates aerobic capacity still need to be clarified. However, age, race, sex, and environmental factors such as pollution and

hypoxia have been identified as pulmonary vascular reserve influencers.

*DOI: http://dx.doi.org/10.5772/intechopen.88399*

*Interventional Pulmonology and Pulmonary Hypertension - Updates on Specific Topics*

protecting from an interstitial lung edema [74].

increase effective capillary pressure [77].

*6.6.4 Pulmonary vascular reserve*

*6.6.3 Lung diffusion capacity*

[7, 8, 74].

**7. Conclusion**

hypoxia with little or no effect on normoxic exercise performance. Primary studies described an increase in maximal workload and VO2max after intake of sildenafil, a phosphodiesterase-5 inhibitor used to treat erectile dysfunction in healthy hypoxic subjects [9, 67–69]. It has been suggested that the underlying mechanism was an increase maximal Q due to a reduced RV afterload after HPV inhibition or pulmonary vasodilation. Similar results were reported after administration of dexamethasone [70] or endothelin receptor blockers [71, 72]. In most of these studies, pulmonary vasodilation effect was also associated with improved arterial oxygenation probably allowing improved oxygen transport to the exercising muscles [69, 73]. De Bisschop et al. showed that pharmacological pulmonary vasodilation improved lung diffusion capacity and also correlated to enhanced exercise capacity at high altitude [74]. The principal suggested underlying mechanisms was related to a pulmonary vasodilation associated decrease in capillary filtration pressure

Acute or chronic hypoxic exposure is associated with enhanced pulmonary diffusion capacity at rest [7, 75, 76]. Moreover, the hypoxia-induced increase in the capillary component being more pronounced than the membrane component suggests a capillary distension in addition to recruitment. This observation has been attributed to increased pulmonary perfusion pressure caused by HPV associated with a venous component of hypoxic vasoconstriction both possibly contributing to

Interestingly, Taylor et al. showed that recruitment of pulmonary capillaries in response to exercise at high altitude is limited and may therefore be a significant source of exercise limitation [78]. This is keeping with previous correlation found between lung diffusing capacity for nitric oxide (DLNO) and VO2max at altitude

The reviewing of data collected during four different high-altitude expeditions (>4350 m) highlighted that individuals with a larger increase in PVR and larger decreased ventilation efficacity with ascent to high altitude were the ones with the greater VO2max fall [7]. Higher aerobic capacity at high altitude was associated with more pronounced pulmonary vascular reserve as suggested by lower PVR, higher diffusion capacity, and lower VE/VCO2 [7]. Similarly, pulmonary vascular reserve has been described as an aerobic performance limiting factor in Andean or Himalayan highlanders [10, 79]. This observation has also been confirmed at moderate altitude, even though the overwhelming determinant of decreased VO2max

and maximum workload is a decrease in arterial O2 content CaO2 [8].

In conclusion, aerobic exercise capacity is depending on the integrity of the different components of the oxygen transfer from ambient air to the mitochondrial cytochromes. The RV function coupling to the pulmonary circulation and the pulmonary capillary network is one of multiple determinants of aerobic exercise capacity. It becomes increasingly clear that a high pulmonary vascular reserve is an advantage for high-intensity exercise performance in heathy subjects. The pulmonary vascular reserve is characterized by lower exercise PAP and PVR and higher

**68**

pulmonary vascular distensibility associated with greater capillary volume and gas exchange allowing for a lower ventilatory cost at a given metabolic rate. When and how the pulmonary vascular reserve modulates aerobic capacity still need to be clarified. However, age, race, sex, and environmental factors such as pollution and hypoxia have been identified as pulmonary vascular reserve influencers.

### **Author details**

Vitalie Faoro\* and Kevin Forton Cardio-Pulmonary Exercise Laboratory, Université Libre de Bruxelles, Belgium

\*Address all correspondence to: vfaoro@ulb.ac.be

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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et al. Sildenafil inhibits altitudeinduced hypoxemia and pulmonary hypertension. American Journal of Respiratory and Critical Care Medicine.

[64] Naeije R, Dedobbeleer C. Pulmonary hypertension and the right ventricle in hypoxia. Experimental Physiology.

2018;**103**(10):1338-1346

2013;**98**(8):1247-1256

1946;**12**:301-320

2005;**171**:275-281

2007;**8**:155-163

2009;**180**:346-352

[68] Hsu AR, Barnholt KE,

cardiac output and exercise

**74**

[79] Groepenhoff H, Overbeek MJ, Mulè M, van der Plas M, Argiento P, Villafuerte FC, et al. Exercise pathophysiology in patients with chronic mountain sickness exercise in chronic mountain sickness. Chest. 2012;**142**(4):877-884

**77**

**Chapter 6**

**Abstract**

2-Methoxyestradiol in Pulmonary

Pulmonary arterial hypertension (PAH), a debilitating and incurable disease, predominantly develops in women. Estradiol metabolism leads to the production of numerous metabolites with different levels of estrogenic activity and very often opposing biological effects. Dysregulated estradiol metabolism was recently linked to the penetrance, progression, and prognosis of the disease. Ongoing clinical trials are examining the effects of estradiol synthesis/signaling inhibition in patients with PAH. In this chapter, the effects of sex, sex hormones, and estradiol metabolism on the healthy pulmonary circulation and vascular pathobiology are discussed in the light of estradiol metabolism as potential pharmacological target in PAH. The effects of estrogens and their metabolites on vascular pathobiology and disease progression, their involvement in PAH-associated diseases, and the pros and cons for interventions at different levels of estradiol metabolism are discussed. Finally, we propose that 2-methoxyestradiol (2ME), a major non-estrogenic metabolite of estradiol, mediates at least in part the beneficial effects of estradiol and that 2ME exhibits opposing effects to estradiol on several processes relevant to the underlying pathophysiology of PAH, including angiogenesis, metabolic reprograming, inflammation, and immunity. Based on cellular and in vivo effects, 2ME should be viewed

**Keywords:** pulmonary hypertension, estradiol metabolism, 2-methoxyestradiol,

Pulmonary arterial hypertension (PAH) is a progressive incurable disease of pulmonary vasculature that ultimately leads to failure of the right ventricle (RV) and death. Notably the disease predominantly develops in women. The first report in 1951 by Dr. David Dresdale and colleagues from Maimonides Hospital of Brooklyn on hemodynamic aspects of primary pulmonary hypertension (PH) included three 25- to 35-year-old women [1]. Similarly, in 1952 in the second seminal report on PAH, British cardiologist Paul Wood recognized that this is "...relatively rare disease, usually encountered in women between 20 and 30, but may be met at any age and in either sex..." [2]. These early observations of female preponderance of PAH were confirmed by epidemiological studies conducted in the last two decades, and the data from various registries worldwide report a female-to-male ratio (F:M) ranging

Arterial Hypertension: A New

Disease Modifier

as a disease modifier in women with PAH.

angiogenesis, inflammation

**1. Introduction**

*Stevan P. Tofovic and Edwin K. Jackson*

**Chapter 6**

## 2-Methoxyestradiol in Pulmonary Arterial Hypertension: A New Disease Modifier

*Stevan P. Tofovic and Edwin K. Jackson*

### **Abstract**

Pulmonary arterial hypertension (PAH), a debilitating and incurable disease, predominantly develops in women. Estradiol metabolism leads to the production of numerous metabolites with different levels of estrogenic activity and very often opposing biological effects. Dysregulated estradiol metabolism was recently linked to the penetrance, progression, and prognosis of the disease. Ongoing clinical trials are examining the effects of estradiol synthesis/signaling inhibition in patients with PAH. In this chapter, the effects of sex, sex hormones, and estradiol metabolism on the healthy pulmonary circulation and vascular pathobiology are discussed in the light of estradiol metabolism as potential pharmacological target in PAH. The effects of estrogens and their metabolites on vascular pathobiology and disease progression, their involvement in PAH-associated diseases, and the pros and cons for interventions at different levels of estradiol metabolism are discussed. Finally, we propose that 2-methoxyestradiol (2ME), a major non-estrogenic metabolite of estradiol, mediates at least in part the beneficial effects of estradiol and that 2ME exhibits opposing effects to estradiol on several processes relevant to the underlying pathophysiology of PAH, including angiogenesis, metabolic reprograming, inflammation, and immunity. Based on cellular and in vivo effects, 2ME should be viewed as a disease modifier in women with PAH.

**Keywords:** pulmonary hypertension, estradiol metabolism, 2-methoxyestradiol, angiogenesis, inflammation

#### **1. Introduction**

Pulmonary arterial hypertension (PAH) is a progressive incurable disease of pulmonary vasculature that ultimately leads to failure of the right ventricle (RV) and death. Notably the disease predominantly develops in women. The first report in 1951 by Dr. David Dresdale and colleagues from Maimonides Hospital of Brooklyn on hemodynamic aspects of primary pulmonary hypertension (PH) included three 25- to 35-year-old women [1]. Similarly, in 1952 in the second seminal report on PAH, British cardiologist Paul Wood recognized that this is "...relatively rare disease, usually encountered in women between 20 and 30, but may be met at any age and in either sex..." [2]. These early observations of female preponderance of PAH were confirmed by epidemiological studies conducted in the last two decades, and the data from various registries worldwide report a female-to-male ratio (F:M) ranging

from 2:1 to 4:1 [3–9] and up to 4:1 to 9:1 for connective tissue disease [10–12]. However, with aging the female preponderance of disease disappears, and an M:F of only 1.2:1.0 has been reported in elderly PAH patients [13]. The latter strongly suggests involvement of female sex hormones in the development of PAH.

#### **1.1 Vascular pathobiology in PAH**

Pulmonary vascular remodeling is a pathological hallmark of PAH. Vascular morphological manifestations of the disease include (i) concentric and asymmetric obliterative proliferation of endothelial cells (ECs) and distal formation of multicellular plexiform lesions (PLXL); (ii) the muscularization of distal non-musculinized precapillary vessels; (iii) adventitia remodeling in form of fibrosis, inflammation, and perivascular edema; and (iv) PLXL, dilation lesions, and arteritis classified as complex lesions [14]. Three-dimensional analysis of vascular lesions in patients with severe PAH reveals the existence of two major phenotypes of ECs. The normal quiescent apoptosis-sensitive ECs are located in the peripheral areas of the lesion, are negative for phosphorylated MAPK, and have a high expression of p27kip1 (a marker of slow proliferation). The highly proliferative apoptosis-resistant cells in the central core of the vascular lesion have elevated MAPK activity and increased expression of HIF-1α, VEGF protein, and VEGF-2 receptor and low expression of p27kip1 [15, 16].

Both inflammation and immune cell response are recognized as important pathogenic factors in PAH [17–19]. For example, in experimental PH perivascular inflammation, due to macrophages, mast cells, and T and B lymphocytes, precedes vascular remodeling and elevated pulmonary pressure [20], and in PAH patients, the degree of perivascular infiltration by immune cells correlates with vascular remodeling and pulmonary artery pressure [21]. As discussed below, inflammation may markedly influence estradiol metabolism, and vice versa, estrogens and their metabolites may instigate, perpetuate, or inhibit inflammation and modulate immune cell responses in PAH.

#### **2. Opposing effects of estradiol and 2-methoxyestradiol on estrogen metabolism**

Since our first report that 2ME, a major non-estrogenic metabolite of 17β-estradiol (E2), attenuates the development and progression of monocrotaline (MCT)-induced PH and that estrogens may be pathogenic in PAH [22], a growing body of evidence suggests the involvement of dysregulated estradiol metabolism and elevated estrogen levels in the development, progression, and prognosis of PAH.

#### **2.1 Increased estradiol production in PAH**

Formation and metabolism of estrogens are complex (**Figure 1**). The pivotal precursors for synthesis of both androgens and estrogens are dehydroepiandrosterone (DHEA) and its biologically inactive sulfate (DHEA-S). DHEA is produced in the adrenal gland of men and postmenopausal women and in the ovaries and placenta of premenopausal women. DHEA, the most abundant steroid in circulation, is also produced by peripheral conversion from circulating DHEA-S. DHEA is transported into cells by organic anion transporters (OATPs) that are expressed in various tissues including the endothelial and inflammatory cells and lungs [23–25]. The delicate balance between DHEA and DHEA-S is controlled by the relative activity of sulfotransferase (DHEA → DHEA-S) and sulfatase (DHEA-S → DHEA) [26]. Both DHEA and DHEA-S are protective in experimental models of pulmonary

**79**

**Figure 1.**

*2-Methoxyestradiol in Pulmonary Arterial Hypertension: A New Disease Modifier*

hypertension [27–31] including models of angioproliferative PH [32, 33]. Recently, lower DHEA-S and higher E2 levels have been linked to a greater risk of PAH and worse hemodynamics, functional status, and greater risk of death [34, 35]. Notably, DHEA improves PAH in patients with obstructive pulmonary disease [36], a finding supporting the potential therapeutic application of DHEA in PAH patients. DHEA is an over-the-counter supplement with no major side effects; however, its safety during chronic use in pharmacological doses is unknown. One potential adverse effect in this regard would be increased circulating/tissue E2 levels that may exacer-

*Dehydroepiandrosterone is the most abundant steroid in circulation that is also produced by peripheral conversion from its circulating inactive sulfated metabolite DHEA-S. The balance between inactive sulfated sex steroids and sex steroids and their biologically active metabolites and metabolic precursors is controlled by sulfatase (STS) and sulfotransferase (SULT). Aromatase is a key enzyme in estrogen production because this enzyme controls aromatization of androgenic precursors to estrogens and intracrine production of estrogens. 2-Hydroxylation/methylation pathway of estrogen metabolism produces non-estrogenic metabolites with opposite effects to maternal estrogens. Hydroxylation of estradiol (E2) at C4 and C16 position leads to production of highly estrogenic metabolites with proliferative, pro-inflammatory, and angiogenic properties.*

Aromatase (encoded by the CYP19A1 gene) is the rate-limiting enzyme catalyzing the conversion of upstream androgenic precursors to estrogens (androstenedione → estrone and testosterone → E2; **Figure 1**). In premenopausal women, estrogens are produced predominantly in the ovarian granulosa cells and are released into the bloodstream where they act primarily in an endocrine fashion. In postmenopausal women and in men, estrogen synthesis takes place in extra-gonadal

tissues (liver, heart, skin/fat tissue, and brain) where estrogens act mainly as paracrine or autocrine factors. Aromatase expression in these various sites is under the control of tissue-specific promoters regulated by different cohorts of transcription factors. Therefore, aromatase activity differs substantially in various tissues and organs in health and disease [37]. Notably, human endothelium expresses a complete aromatase-estrogen-E2 receptor system [38], and increased expression of aromatase has been reported in hPASMCs from female PAH patients and in the lungs of female rats and mice with angioproliferative PH [39]. Increased aromatase activity and plasma E2 levels seen in both men and women with advanced liver disease is associated with increased risk of portopulmonary PAH [40], and increased aromatization of androgens and elevated E2 levels have been reported in postmenopausal women and in men with PAH [34, 35]. Noteworthy, E2 stimulates aromatase activity and by increasing aromatization of androgen precursors may augment its own production as well as production of E1. Because of the importance of this enzyme in estrogen synthesis, blocking aromatase activity is an important pharmacological tool used for the treatment of estrogen-dependent diseases (breast cancer, endometriosis, and

bate endothelial remodeling and inflammation (infra vide).

*DOI: http://dx.doi.org/10.5772/intechopen.86812*

*2-Methoxyestradiol in Pulmonary Arterial Hypertension: A New Disease Modifier DOI: http://dx.doi.org/10.5772/intechopen.86812*

#### **Figure 1.**

*Interventional Pulmonology and Pulmonary Hypertension - Updates on Specific Topics*

**1.1 Vascular pathobiology in PAH**

immune cell responses in PAH.

**2.1 Increased estradiol production in PAH**

**metabolism**

from 2:1 to 4:1 [3–9] and up to 4:1 to 9:1 for connective tissue disease [10–12]. However, with aging the female preponderance of disease disappears, and an M:F of only 1.2:1.0 has been reported in elderly PAH patients [13]. The latter strongly suggests involvement of female sex hormones in the development of PAH.

Pulmonary vascular remodeling is a pathological hallmark of PAH. Vascular morphological manifestations of the disease include (i) concentric and asymmetric obliterative proliferation of endothelial cells (ECs) and distal formation of multicellular plexiform lesions (PLXL); (ii) the muscularization of distal non-musculinized precapillary vessels; (iii) adventitia remodeling in form of fibrosis, inflammation, and perivascular edema; and (iv) PLXL, dilation lesions, and arteritis classified as complex lesions [14]. Three-dimensional analysis of vascular lesions in patients with severe PAH reveals the existence of two major phenotypes of ECs. The normal quiescent apoptosis-sensitive ECs are located in the peripheral areas of the lesion, are negative for phosphorylated MAPK, and have a high expression of p27kip1 (a marker of slow proliferation). The highly proliferative apoptosis-resistant cells in the central core of the vascular lesion have elevated MAPK activity and increased expression of HIF-1α, VEGF protein, and VEGF-2 receptor and low expression of p27kip1 [15, 16]. Both inflammation and immune cell response are recognized as important pathogenic factors in PAH [17–19]. For example, in experimental PH perivascular inflammation, due to macrophages, mast cells, and T and B lymphocytes, precedes vascular remodeling and elevated pulmonary pressure [20], and in PAH patients, the degree of perivascular infiltration by immune cells correlates with vascular remodeling and pulmonary artery pressure [21]. As discussed below, inflammation may markedly influence estradiol metabolism, and vice versa, estrogens and their metabolites may instigate, perpetuate, or inhibit inflammation and modulate

**2. Opposing effects of estradiol and 2-methoxyestradiol on estrogen** 

Since our first report that 2ME, a major non-estrogenic metabolite of 17β-estradiol (E2), attenuates the development and progression of monocrotaline (MCT)-induced PH and that estrogens may be pathogenic in PAH [22], a growing body of evidence suggests the involvement of dysregulated estradiol metabolism and elevated estrogen levels in the development, progression, and prognosis of PAH.

Formation and metabolism of estrogens are complex (**Figure 1**). The pivotal precursors for synthesis of both androgens and estrogens are dehydroepiandrosterone (DHEA) and its biologically inactive sulfate (DHEA-S). DHEA is produced in the adrenal gland of men and postmenopausal women and in the ovaries and placenta of premenopausal women. DHEA, the most abundant steroid in circulation, is also produced by peripheral conversion from circulating DHEA-S. DHEA is transported into cells by organic anion transporters (OATPs) that are expressed in various tissues including the endothelial and inflammatory cells and lungs [23–25]. The delicate balance between DHEA and DHEA-S is controlled by the relative activity of sulfotransferase (DHEA → DHEA-S) and sulfatase (DHEA-S → DHEA) [26]. Both DHEA and DHEA-S are protective in experimental models of pulmonary

**78**

*Dehydroepiandrosterone is the most abundant steroid in circulation that is also produced by peripheral conversion from its circulating inactive sulfated metabolite DHEA-S. The balance between inactive sulfated sex steroids and sex steroids and their biologically active metabolites and metabolic precursors is controlled by sulfatase (STS) and sulfotransferase (SULT). Aromatase is a key enzyme in estrogen production because this enzyme controls aromatization of androgenic precursors to estrogens and intracrine production of estrogens. 2-Hydroxylation/methylation pathway of estrogen metabolism produces non-estrogenic metabolites with opposite effects to maternal estrogens. Hydroxylation of estradiol (E2) at C4 and C16 position leads to production of highly estrogenic metabolites with proliferative, pro-inflammatory, and angiogenic properties.*

hypertension [27–31] including models of angioproliferative PH [32, 33]. Recently, lower DHEA-S and higher E2 levels have been linked to a greater risk of PAH and worse hemodynamics, functional status, and greater risk of death [34, 35]. Notably, DHEA improves PAH in patients with obstructive pulmonary disease [36], a finding supporting the potential therapeutic application of DHEA in PAH patients. DHEA is an over-the-counter supplement with no major side effects; however, its safety during chronic use in pharmacological doses is unknown. One potential adverse effect in this regard would be increased circulating/tissue E2 levels that may exacerbate endothelial remodeling and inflammation (infra vide).

Aromatase (encoded by the CYP19A1 gene) is the rate-limiting enzyme catalyzing the conversion of upstream androgenic precursors to estrogens (androstenedione → estrone and testosterone → E2; **Figure 1**). In premenopausal women, estrogens are produced predominantly in the ovarian granulosa cells and are released into the bloodstream where they act primarily in an endocrine fashion. In postmenopausal women and in men, estrogen synthesis takes place in extra-gonadal tissues (liver, heart, skin/fat tissue, and brain) where estrogens act mainly as paracrine or autocrine factors. Aromatase expression in these various sites is under the control of tissue-specific promoters regulated by different cohorts of transcription factors. Therefore, aromatase activity differs substantially in various tissues and organs in health and disease [37]. Notably, human endothelium expresses a complete aromatase-estrogen-E2 receptor system [38], and increased expression of aromatase has been reported in hPASMCs from female PAH patients and in the lungs of female rats and mice with angioproliferative PH [39]. Increased aromatase activity and plasma E2 levels seen in both men and women with advanced liver disease is associated with increased risk of portopulmonary PAH [40], and increased aromatization of androgens and elevated E2 levels have been reported in postmenopausal women and in men with PAH [34, 35]. Noteworthy, E2 stimulates aromatase activity and by increasing aromatization of androgen precursors may augment its own production as well as production of E1. Because of the importance of this enzyme in estrogen synthesis, blocking aromatase activity is an important pharmacological tool used for the treatment of estrogen-dependent diseases (breast cancer, endometriosis, and

endometrial cancer). Anastrozole (a third-generation aromatase inhibitor) reduces E2 levels and attenuates PH in female mice exposed to hypoxia [39] and in Sugene 5416 + hypoxia rats with angioproliferative PH [39, 41]. Moreover, when combined with the selective estrogen receptor degrader fulvestrant, anastrozole reverses PH in BMPR2-mutant mice [42]. Likewise, in PAH patients treatment with anastrozole reduces elevated E2 levels by 40% and E1 levels by 70% and significantly increases functional capacity, i.e., 6-minute-walk distance [43]. Notably, E2 augments gonadal aromatase activity, and by increasing aromatization of androgens, E2 may augment its own production as well as that of other estrogens [44]. Inflammation and inflammatory cytokines upregulate aromatase activity, and TNFα is one of the most potent inducers of aromatase. In contrast to estrogens that do not have effect on TNFα induction of aromatase [45], 2ME inhibits both basal and TNFα-stimulated aromatase activity [45–47].

In addition to aromatase, another potential source of increased estrogen production in PAH is the "sulfatase pathway." In addition to DHEA-S, other substrates for STS are biologically inactive estrone sulfate (E1-S) and estradiol sulfate (E2-S), and sulfatase plays a key role in intracrine regeneration of biologically active E2 and E1 (**Figure 1**). Inflammatory cytokines increase STS activity. More importantly, STS expression is stimulated by estrogens via estrogen receptor alpha (ERα) signaling, and at least in breast cancer, STS is upregulated by the elevated local E2 levels [26]. Thereby, in an inflammatory environment, E2 through feed-forward mechanisms may increase its on production via both the sulfatase and aromatase pathways (**Figure 2**), as implicated by elevated aromatase activity and E2 levels in both experimental PH [39] and in men and women with PAH [34, 35, 40].

#### **2.2 Dysregulated estradiol metabolism in PAH**

Once formed, E2 is primarily metabolized by oxidation at C2, C4, and C16 positions and converted to metabolites with different estrogenic activities and diverse (often opposite) biological effects. In humans, E2 hydroxylation is mediated by

#### **Figure 2.**

*Opposing effects of E2 and 2ME on estrogens and arachidonic acid metabolism. Inflammation and dual metabolic activity of CYP1B1 instigate estradiol feed-forward mechanisms that involve sulfatase, aromatase, COMT, and CYP1B1 (red arrows). Thereby, the increased E2 and arachidonic acid pro-inflammatory metabolites may contribute to the development of inflammatory and angioproliferative phenotypes in women. In contrast, 2ME by inhibiting CYP1B1 activity, macrophage influx/activation, and proinflammatory cytokine induction of estrogen-producing enzymes (blue arrows) balances inflammation and E2 production and its metabolisms into mitogenic pro-inflammatory and angiogenic metabolites. CYP1B1 activation results in production of pro-inflammatory arachidonic acid metabolites (black arrows). COMT = catechol-O-methyltransferase; CYP = cytochrome p450 enzymes; EETs = epoxyeicosatrienoic acids; HETEs = hydroxyeicosatetraenoic acids; sEH = soluble epoxide hydrolase, degrades EETs.*

**81**

*2-Methoxyestradiol in Pulmonary Arterial Hypertension: A New Disease Modifier*

multiplying CYP450 enzymes (CYP1A1/1A2/3A4/1B1) with 2-hydroxyestradiol (2HE) being the main metabolite; however, 4-hydroxyestradiol (4HE; **Figures 1** and **2**) is formed to a lesser degree (~5%). This is followed by methylation of hydroxyl groups catalyzed by catechol-O-methyl transferase (COMT). The hydroxylation/methylation pathway is a major metabolic pathway that accounts for ~50% of E2 metabolism. It largely takes place in the liver and leads to production of 2ME, a major non-estrogenic metabolite with antiproliferative, anti-angiogenic, and anti-inflammatory effects [48]. In addition to hepatocytes and numerous cancer cell lines, conversion of E2 to downstream 2HE and 2ME takes place in cardiovascular and renal compartments [48], and a solid line of evidence suggests that 2ME mediates the antiproliferative

Notably, the protective effects of E2 in experimental PH are mediated, at least in part, by 2ME [50, 51]. Furthermore, it seems that in highly proliferative states, 2ME may oppose estrogen-driven proliferation. For example, in highly proliferative human leiomyoma cells (hLCs) characterized by doubled ERα signaling (inherently regulated by microtubule dynamics) [52], COMT overexpression or treatment with 2ME stabilizes microtubules, attenuates E2-induced proliferation, inhibits ERα signaling, and reduces HIF-1α and aromatase expression in hLCs [53, 54]. Unfortunately, it seems that elevated E2 levels seen in PAH may adversely affect both hepatic and extrahepatic 2ME production. In this regard, men have higher COMT activity than women [55, 56], and sex hormones regulate COMT activity that is highly expressed in human and rat lungs [57, 58]. The exposure to E2 reduces hepatic COMT activity in rats [59, 60]; in vitro E2 decreases COMT transcription, activity, and protein levels [61, 62]; and tamoxifen, by antagonizing E2, increases COMT activity in peripheral tissues [63]. Together, these findings suggest that reduced COMT activity by elevated E2 and subsequent decreased in 2ME production may render women more susceptible to the development of PAH. At present it is unknown whether there is reduced 2ME production in PAH. Yet, reduced 2ME production has been linked to the development of preeclampsia [64], increased

*DOI: http://dx.doi.org/10.5772/intechopen.86812*

effects of E2 in cardiovascular and renal cells [49].

sensitivity to angiotensin II [65], and insulin resistance [66].

**and estrogen and arachidonic acid metabolism**

**2.3 Opposing effects of estradiol and 2-methoxyestradiol on CYP1B1 activity** 

E2 and 2ME have opposing effects on CYP1B1, another E2 metabolizing enzyme implicated in pathogenesis of PAH. Human CYP1B1 mRNA and protein are constitutively expressed in the lung and in VSMCs and ECs [67]. CYP1B1 may facilitate E2 oxidation at C4 and C16, thus producing highly estrogenic and reactive metabolites 4-hydroxyestradiol (4HE) and 16α-hydroxyestrone (16αHE1). Experimental and human data suggest a major pathogenic role for CYP1B1 and 16αHE1 in PAH. In this regard, CYP1B1 increases the risk of PAH and RV dysfunction in humans and plays a pathogenic role in the 16αHE1-BMPR2 interaction in experimental PH [68–76]. Notably, E2 and 2ME have divergent effects on CYP1B1 activity. Estradiol is not only a substrate for CYP1B1, but also it is transcriptional activator of CYP1B1 [77]. In contrast, in vitro 2ME exerts feedback inhibition on CYP1B1 activity [78]. Moreover, 2ME inhibits aryl hydrocarbon receptor-mediated induction of CYP1B1 and reduces CYP1B1 production of reactive metabolites. In vivo, 2ME significantly inhibits CYP1B1 expression and attenuates pressure overload-induced cardiac remodeling [78, 79]. In vivo CYP1B1 inhibition by 2ME reduces biosynthesis of mid-chain hydroxyeicosatetraenoic acids (HETEs) [79], suggesting a significant role for CYP1B1 in arachidonic acid metabolism. Indeed, due to its lipoxygenase-like activity, CYP1B1 facilitates arachidonic acid metabolism into HETEs and epoxyeicosatrienoic acids (EETs) [80]. In the pulmonary vasculature, EETs and HETEs have

#### *2-Methoxyestradiol in Pulmonary Arterial Hypertension: A New Disease Modifier DOI: http://dx.doi.org/10.5772/intechopen.86812*

multiplying CYP450 enzymes (CYP1A1/1A2/3A4/1B1) with 2-hydroxyestradiol (2HE) being the main metabolite; however, 4-hydroxyestradiol (4HE; **Figures 1** and **2**) is formed to a lesser degree (~5%). This is followed by methylation of hydroxyl groups catalyzed by catechol-O-methyl transferase (COMT). The hydroxylation/methylation pathway is a major metabolic pathway that accounts for ~50% of E2 metabolism. It largely takes place in the liver and leads to production of 2ME, a major non-estrogenic metabolite with antiproliferative, anti-angiogenic, and anti-inflammatory effects [48]. In addition to hepatocytes and numerous cancer cell lines, conversion of E2 to downstream 2HE and 2ME takes place in cardiovascular and renal compartments [48], and a solid line of evidence suggests that 2ME mediates the antiproliferative effects of E2 in cardiovascular and renal cells [49].

Notably, the protective effects of E2 in experimental PH are mediated, at least in part, by 2ME [50, 51]. Furthermore, it seems that in highly proliferative states, 2ME may oppose estrogen-driven proliferation. For example, in highly proliferative human leiomyoma cells (hLCs) characterized by doubled ERα signaling (inherently regulated by microtubule dynamics) [52], COMT overexpression or treatment with 2ME stabilizes microtubules, attenuates E2-induced proliferation, inhibits ERα signaling, and reduces HIF-1α and aromatase expression in hLCs [53, 54]. Unfortunately, it seems that elevated E2 levels seen in PAH may adversely affect both hepatic and extrahepatic 2ME production. In this regard, men have higher COMT activity than women [55, 56], and sex hormones regulate COMT activity that is highly expressed in human and rat lungs [57, 58]. The exposure to E2 reduces hepatic COMT activity in rats [59, 60]; in vitro E2 decreases COMT transcription, activity, and protein levels [61, 62]; and tamoxifen, by antagonizing E2, increases COMT activity in peripheral tissues [63]. Together, these findings suggest that reduced COMT activity by elevated E2 and subsequent decreased in 2ME production may render women more susceptible to the development of PAH. At present it is unknown whether there is reduced 2ME production in PAH. Yet, reduced 2ME production has been linked to the development of preeclampsia [64], increased sensitivity to angiotensin II [65], and insulin resistance [66].

#### **2.3 Opposing effects of estradiol and 2-methoxyestradiol on CYP1B1 activity and estrogen and arachidonic acid metabolism**

E2 and 2ME have opposing effects on CYP1B1, another E2 metabolizing enzyme implicated in pathogenesis of PAH. Human CYP1B1 mRNA and protein are constitutively expressed in the lung and in VSMCs and ECs [67]. CYP1B1 may facilitate E2 oxidation at C4 and C16, thus producing highly estrogenic and reactive metabolites 4-hydroxyestradiol (4HE) and 16α-hydroxyestrone (16αHE1). Experimental and human data suggest a major pathogenic role for CYP1B1 and 16αHE1 in PAH. In this regard, CYP1B1 increases the risk of PAH and RV dysfunction in humans and plays a pathogenic role in the 16αHE1-BMPR2 interaction in experimental PH [68–76]. Notably, E2 and 2ME have divergent effects on CYP1B1 activity. Estradiol is not only a substrate for CYP1B1, but also it is transcriptional activator of CYP1B1 [77]. In contrast, in vitro 2ME exerts feedback inhibition on CYP1B1 activity [78]. Moreover, 2ME inhibits aryl hydrocarbon receptor-mediated induction of CYP1B1 and reduces CYP1B1 production of reactive metabolites. In vivo, 2ME significantly inhibits CYP1B1 expression and attenuates pressure overload-induced cardiac remodeling [78, 79]. In vivo CYP1B1 inhibition by 2ME reduces biosynthesis of mid-chain hydroxyeicosatetraenoic acids (HETEs) [79], suggesting a significant role for CYP1B1 in arachidonic acid metabolism. Indeed, due to its lipoxygenase-like activity, CYP1B1 facilitates arachidonic acid metabolism into HETEs and epoxyeicosatrienoic acids (EETs) [80]. In the pulmonary vasculature, EETs and HETEs have

*Interventional Pulmonology and Pulmonary Hypertension - Updates on Specific Topics*

endometrial cancer). Anastrozole (a third-generation aromatase inhibitor) reduces E2 levels and attenuates PH in female mice exposed to hypoxia [39] and in Sugene 5416 + hypoxia rats with angioproliferative PH [39, 41]. Moreover, when combined with the selective estrogen receptor degrader fulvestrant, anastrozole reverses PH in BMPR2-mutant mice [42]. Likewise, in PAH patients treatment with anastrozole reduces elevated E2 levels by 40% and E1 levels by 70% and significantly increases functional capacity, i.e., 6-minute-walk distance [43]. Notably, E2 augments gonadal aromatase activity, and by increasing aromatization of androgens, E2 may augment its own production as well as that of other estrogens [44]. Inflammation and inflammatory cytokines upregulate aromatase activity, and TNFα is one of the most potent inducers of aromatase. In contrast to estrogens that do not have effect on TNFα induction of aromatase [45], 2ME inhibits both basal and TNFα-stimulated aroma-

In addition to aromatase, another potential source of increased estrogen production in PAH is the "sulfatase pathway." In addition to DHEA-S, other substrates for STS are biologically inactive estrone sulfate (E1-S) and estradiol sulfate (E2-S), and sulfatase plays a key role in intracrine regeneration of biologically active E2 and E1 (**Figure 1**). Inflammatory cytokines increase STS activity. More importantly, STS expression is stimulated by estrogens via estrogen receptor alpha (ERα) signaling, and at least in breast cancer, STS is upregulated by the elevated local E2 levels [26]. Thereby, in an inflammatory environment, E2 through feed-forward mechanisms may increase its on production via both the sulfatase and aromatase pathways (**Figure 2**), as implicated by elevated aromatase activity and E2 levels in both

Once formed, E2 is primarily metabolized by oxidation at C2, C4, and C16 positions and converted to metabolites with different estrogenic activities and diverse (often opposite) biological effects. In humans, E2 hydroxylation is mediated by

*Opposing effects of E2 and 2ME on estrogens and arachidonic acid metabolism. Inflammation and dual metabolic activity of CYP1B1 instigate estradiol feed-forward mechanisms that involve sulfatase, aromatase, COMT, and CYP1B1 (red arrows). Thereby, the increased E2 and arachidonic acid pro-inflammatory metabolites may contribute to the development of inflammatory and angioproliferative phenotypes in women. In contrast, 2ME by inhibiting CYP1B1 activity, macrophage influx/activation, and proinflammatory cytokine induction of estrogen-producing enzymes (blue arrows) balances inflammation and E2 production and its metabolisms into mitogenic pro-inflammatory and angiogenic metabolites. CYP1B1 activation results in production of pro-inflammatory arachidonic acid metabolites (black arrows). COMT = catechol-O-methyltransferase; CYP = cytochrome p450 enzymes; EETs = epoxyeicosatrienoic acids;* 

*HETEs = hydroxyeicosatetraenoic acids; sEH = soluble epoxide hydrolase, degrades EETs.*

experimental PH [39] and in men and women with PAH [34, 35, 40].

**2.2 Dysregulated estradiol metabolism in PAH**

**80**

**Figure 2.**

tase activity [45–47].

vasoconstrictive, inflammatory, and mitogenic/angiogenic effects and have been implicated in the development of experimental hypoxic PH [81–84]. Noteworthy, in PAH patients increased production of HETEs correlates with a poor prognosis [85]. E2 not only stimulates production of HETEs and EETs but also by inhibiting expression/activity of soluble epoxide hydrolase (sEH) [86] suppresses the degradation of EETs. Several lines of evidence link low sEH activity to the pathophysiology of PH: (1) the lungs from PH patients express no/little sEH; (2) E2-, genetic-, and pharmacologically induced downregulation of sEH potentiates hypoxic vasoconstriction; (3) hypoxia downregulates sEH; and (3) sEH−/− mice have exacerbated pulmonary vascular remodeling when exposed to chronic hypoxia [87–89]. Therefore, elevated E2 levels in PAH through a feed-forward mechanism may shift both E2 and AA metabolism toward production of pro-inflammatory/angiogenic/mitogenic metabolites. Based on its inhibitory effects, 2ME should suppress the production of these pathogenic E2 and AA metabolites (**Figure 2**).

#### **3. Divergent effects of 2ME and estradiol in pulmonary endothelium in PAH**

Dysregulated angiogenesis with formation of occlusive and plexiform lesions is a hallmark of PAH. Although estrogens provide protection in healthy systemic vascular beds, they have opposite effects on malignant proangiogenic/highly proliferative vessels [90] that share many similarities with vascular changes in PAH. The highly proliferative apoptosis-resistant cells in the central core of vascular lesions in PAH have elevated MAPK activity; increased expression of HIF-1α, VEGF protein, and VEGF-2 receptor; and low expression of p27kip1 (marker of low cell growth) [15, 16]. In human pulmonary artery ECs (hPAECs) and at physiological concentrations (1–10 nM), E2 (1) stimulates cell proliferation [91]; (2) promotes the phosphorylation of p42/44 and p38 MAPK via ERs; (3) downregulates the cell cycle inhibitor p27Kip1; (4) stimulates cell migration; (5) induces HIF-1α expression and VEGF synthesis; and (6) protects against apoptosis [92–94]. Also, E2 stimulates proliferation of human pulmonary artery vascular smooth muscle cells (hPASMCs). In canine pulmonary arterial segments, E2 tends to inhibit proliferation of PASMCs in segments with intact endothelium but significantly enhances proliferation in segments stripped of endothelium [95], suggesting opposite effects of E2 in intact versus injured pulmonary vessels. Thereby, in the pulmonary vasculature exposed to known and unknown multiple hits, estrogens may potentiate pathological endothelial remodeling in PAH (**Figure 3**).

In contrast to E2, it is well established that 2ME has strong anti-angiogenic, antiproliferative, and pro-apoptotic effects [96] and thereby may prevent PAH or inhibit the progression of PAH. In this regard, of particular importance for PAH are the effects of 2ME on the HIF-1α/VEGF axis. One of the most consistently reported effects of 2ME is HIF-1α downregulation, and 2ME has been increasingly used as pharmacological tool to inhibit HIF-1α in numerous studies outside the PAH field. HIF-1α transcriptional activity regulates more than 40 genes and respective proteins, including those that play a key role in vascular reactivity and angiogenesis [97, 98]. The role of HIF-1α in PAH is supported by multiple findings including the following: (1) obliterative endothelial lesions in severe PH in humans overexpress HIF-1α [15]; (2) in experimental PH there is similar increase in HIF-1α that correlates with the development of PH and pulmonary vascular remodeling and RV hypertrophy; (3) heterozygous deficiency in HIF-1α protects against the development of PH [99, 100]; and (4) pathologic normoxic HIF-1α signaling activation leads to the glycolytic shift (the Warburg effect) in highly proliferative ECs [101].

**83**

**Figure 3.**

*2-Methoxyestradiol in Pulmonary Arterial Hypertension: A New Disease Modifier*

Hypoxia stimulates 2ME formation which inhibits the production of hypoxiadriven angiogenesis and angiogenic cytokines (VEGF and FGF-2) [31, 102]. Therefore, 2ME should be viewed as a local modulator that fine-tunes the rate of angiogenesis. Recent studies in experimental PH support the notion of 2ME as a local anti-angiogenic factor in PAH and E2 as promoter of angiogenesis. For example, (1) basal HIF-1α protein expression is higher in female hPASMCs than in males; (2) the antimitogenic effects of 2ME in hPASMCs are associated with reduced HIF-1α expression; (3) 2ME attenuates intermittent and chronic hypoxia-induced PH [22, 103, 104]; (4) in both male and female hypoxic PH rats, 2ME attenuates the disease while decreasing HIF-1α protein expression [103]; (5) female rats with Sugene 5416 + hypoxia (SU+Hx)-

*In the injured highly proliferative/angiogenic endothelium in pulmonary vasculature in PAH, 2ME behaves as biological antagonist of estradiol (E2). 2ME and E2 have opposite effects on key regulators of angioproliferation (p27Kip1, AKT, HIF1-α, VEGF), and 2ME is a more potent modulator of prostacyclin,* 

induced PH have more severe occlusive and plexiform lesions and sporadically develop grade 6 lesions (necrotizing arteritis); (6) E2 exacerbates angioproliferative lesions and perivascular inflammation in ovariectomized SU+Hx rats [105, 106]; and (7) in intact female SU+Hx rats, 2ME, but not E2, exhibits therapeutic effects [107]. The effects of 2ME in PAH patients are unknown. Yet, at least in experimental angioproliferative PH, 2ME could be viewed as biological antagonist of E2 in the endothelium and

The major metabolic changes that take place in PAH occur in the form of the shift from oxidative phosphorylation to glycolysis. Known as the Warburg effect, this event is frequently observed and has been systematically investigated in cancer tissue. Notably, the Warburg effect has also been reported in pulmonary vasculature cells in PAH patients [108] and linked to highly proliferative, angiogenic, and apoptosis-resistant cancer cells and vascular cells in PAH. Not surprisingly, the

as a modifier of "dysregulated angiogenesis."

*endothelin, and nitric oxide synthesis/release than E2.*

**4. Metabolic reprograming and 2ME in PAH**

*DOI: http://dx.doi.org/10.5772/intechopen.86812*

*2-Methoxyestradiol in Pulmonary Arterial Hypertension: A New Disease Modifier DOI: http://dx.doi.org/10.5772/intechopen.86812*

**Figure 3.**

*Interventional Pulmonology and Pulmonary Hypertension - Updates on Specific Topics*

pathogenic E2 and AA metabolites (**Figure 2**).

pathological endothelial remodeling in PAH (**Figure 3**).

**in PAH**

vasoconstrictive, inflammatory, and mitogenic/angiogenic effects and have been implicated in the development of experimental hypoxic PH [81–84]. Noteworthy, in PAH patients increased production of HETEs correlates with a poor prognosis [85]. E2 not only stimulates production of HETEs and EETs but also by inhibiting expression/activity of soluble epoxide hydrolase (sEH) [86] suppresses the degradation of EETs. Several lines of evidence link low sEH activity to the pathophysiology of PH: (1) the lungs from PH patients express no/little sEH; (2) E2-, genetic-, and pharmacologically induced downregulation of sEH potentiates hypoxic vasoconstriction; (3) hypoxia downregulates sEH; and (3) sEH−/− mice have exacerbated pulmonary vascular remodeling when exposed to chronic hypoxia [87–89]. Therefore, elevated E2 levels in PAH through a feed-forward mechanism may shift both E2 and AA metabolism toward production of pro-inflammatory/angiogenic/mitogenic metabolites. Based on its inhibitory effects, 2ME should suppress the production of these

**3. Divergent effects of 2ME and estradiol in pulmonary endothelium** 

Dysregulated angiogenesis with formation of occlusive and plexiform lesions is a hallmark of PAH. Although estrogens provide protection in healthy systemic vascular beds, they have opposite effects on malignant proangiogenic/highly proliferative vessels [90] that share many similarities with vascular changes in PAH. The highly proliferative apoptosis-resistant cells in the central core of vascular lesions in PAH have elevated MAPK activity; increased expression of HIF-1α, VEGF protein, and VEGF-2 receptor; and low expression of p27kip1 (marker of low cell growth) [15, 16]. In human pulmonary artery ECs (hPAECs) and at physiological concentrations (1–10 nM), E2 (1) stimulates cell proliferation [91]; (2) promotes the phosphorylation of p42/44 and p38 MAPK via ERs; (3) downregulates the cell cycle inhibitor p27Kip1; (4) stimulates cell migration; (5) induces HIF-1α expression and VEGF synthesis; and (6) protects against apoptosis [92–94]. Also, E2 stimulates proliferation of human pulmonary artery vascular smooth muscle cells (hPASMCs). In canine pulmonary arterial segments, E2 tends to inhibit proliferation of PASMCs in segments with intact endothelium but significantly enhances proliferation in segments stripped of endothelium [95], suggesting opposite effects of E2 in intact versus injured pulmonary vessels. Thereby, in the pulmonary vasculature exposed to known and unknown multiple hits, estrogens may potentiate

In contrast to E2, it is well established that 2ME has strong anti-angiogenic, antiproliferative, and pro-apoptotic effects [96] and thereby may prevent PAH or inhibit the progression of PAH. In this regard, of particular importance for PAH are the effects of 2ME on the HIF-1α/VEGF axis. One of the most consistently reported effects of 2ME is HIF-1α downregulation, and 2ME has been increasingly used as pharmacological tool to inhibit HIF-1α in numerous studies outside the PAH field. HIF-1α transcriptional activity regulates more than 40 genes and respective proteins, including those that play a key role in vascular reactivity and angiogenesis [97, 98]. The role of HIF-1α in PAH is supported by multiple findings including the following: (1) obliterative endothelial lesions in severe PH in humans overexpress HIF-1α [15]; (2) in experimental PH there is similar increase in HIF-1α that correlates with the development of PH and pulmonary vascular remodeling and RV hypertrophy; (3) heterozygous deficiency in HIF-1α protects against the development of PH [99, 100]; and (4) pathologic normoxic HIF-1α signaling activation leads to the glycolytic shift (the Warburg effect) in highly proliferative ECs [101].

**82**

*In the injured highly proliferative/angiogenic endothelium in pulmonary vasculature in PAH, 2ME behaves as biological antagonist of estradiol (E2). 2ME and E2 have opposite effects on key regulators of angioproliferation (p27Kip1, AKT, HIF1-α, VEGF), and 2ME is a more potent modulator of prostacyclin, endothelin, and nitric oxide synthesis/release than E2.*

Hypoxia stimulates 2ME formation which inhibits the production of hypoxiadriven angiogenesis and angiogenic cytokines (VEGF and FGF-2) [31, 102]. Therefore, 2ME should be viewed as a local modulator that fine-tunes the rate of angiogenesis. Recent studies in experimental PH support the notion of 2ME as a local anti-angiogenic factor in PAH and E2 as promoter of angiogenesis. For example, (1) basal HIF-1α protein expression is higher in female hPASMCs than in males; (2) the antimitogenic effects of 2ME in hPASMCs are associated with reduced HIF-1α expression; (3) 2ME attenuates intermittent and chronic hypoxia-induced PH [22, 103, 104]; (4) in both male and female hypoxic PH rats, 2ME attenuates the disease while decreasing HIF-1α protein expression [103]; (5) female rats with Sugene 5416 + hypoxia (SU+Hx) induced PH have more severe occlusive and plexiform lesions and sporadically develop grade 6 lesions (necrotizing arteritis); (6) E2 exacerbates angioproliferative lesions and perivascular inflammation in ovariectomized SU+Hx rats [105, 106]; and (7) in intact female SU+Hx rats, 2ME, but not E2, exhibits therapeutic effects [107]. The effects of 2ME in PAH patients are unknown. Yet, at least in experimental angioproliferative PH, 2ME could be viewed as biological antagonist of E2 in the endothelium and as a modifier of "dysregulated angiogenesis."

#### **4. Metabolic reprograming and 2ME in PAH**

The major metabolic changes that take place in PAH occur in the form of the shift from oxidative phosphorylation to glycolysis. Known as the Warburg effect, this event is frequently observed and has been systematically investigated in cancer tissue. Notably, the Warburg effect has also been reported in pulmonary vasculature cells in PAH patients [108] and linked to highly proliferative, angiogenic, and apoptosis-resistant cancer cells and vascular cells in PAH. Not surprisingly, the

**Figure 4.**

*Cellular effects of 2ME that contributes to the reduced E2 production, inflammation, angioproliferation, metabolic reprograming, and vascular and right ventricular remodeling in PAH.*

Warburg effect has been explored as a potential anti-angiogenic target in cancer and more recently in PAH. The HIF-1α transcription factor has been identified as a master hypoxic regulator responsible for the metabolic shift in PAH [101, 109]. Hypoxic induction of HIF-1α leads to overexpression of pyruvate dehydrogenase kinase (PDK) which results in inhibition of pyruvate dehydrogenase that shunts pyruvate into glycolysis and induces conversion of glucose to lactate [108]. Dichloroacetate (DCA), a PDK inhibitor, reverses the Warburg effect and exhibits therapeutic effects in several animal models of PH [110–112]. Because 2ME is a strong HIF-1α inhibitor, 2ME should induce metabolic reprograming in PAH. Presently, the effects of 2ME on metabolic reprograming in PAH are unknown. However, 2ME inhibits lactate-induced mitochondrial biogenesis in highly proliferative osteosarcoma cells, and in apoptosis-resistant melanoma cells, 2ME attenuates proliferation and glycolysis by inhibiting HIF-1α and PDK expression [113–115]. Therefore, 2ME could be viewed as modulator of metabolic reprograming. Further studies are warranted to investigate the effects of 2ME on the Warburg effect that in PAH is associated with highly proliferative, angiogenic, and apoptosis-resistant phenotypes (**Figure 4**).

#### **5. Anti-inflammatory and immunomodulatory effects of 2ME**

Inflammation and altered immunity, i.e., perivascular accumulation of inflammatory and immune cells in pulmonary circulation, have been increasingly recognized as pathogenic factors in PAH [17–19]. In this regard, at young age women have more robust immune responses than men. Although initially beneficial, with aging these aggressive immune responses may become detrimental [116]. This may explain why various immune diseases are remarkably more frequent in women and why many immune diseases, such as systemic sclerosis (SSc), lupus, and mixed connective tissue disease, are associated with increased risk of PAH [117]. Furthermore, recently distinct immune phenotypes have been reported in PAH patients [118]. In experimental PH, dysregulated immunity in the form of deficient regulatory T-cell (Treg) activity contributes to increased inflammation [20]. Both alveolar macrophages and immune cells express steroidogenic enzymes including sulfatase and aromatase [119–121]. Inflammatory cytokines, prostanoids, and growth factors regulate the expression and activity of steroidogenic enzymes, and in turn, sex hormones

**85**

*2-Methoxyestradiol in Pulmonary Arterial Hypertension: A New Disease Modifier*

may influence the production and release of these autocrine/paracrine mediators [122]. E2 upregulates CYP1B1, aromatase, and sulfatase activity and inhibits sEH activity. Therefore, in an inflammatory environment, E2 may boost its own production, and via a feed-forward mechanism, E2 may enhance the production of proinflammatory, angiogenic, and mitogenic estrogens and increase the accumulation of pro-inflammatory arachidonic acid metabolites (**Figure 2**). In contrast to E2, non-estrogenic 2ME exhibits significant anti-inflammatory effects, largely through suppression of tissue recruitment and activation of macrophages [123, 124]. This is one of the most consistent in vivo effects of 2ME seen in experimental models of cardiovascular and renal injury [125, 126] and in pulmonary hypertension [50, 51, 127–129]. 2ME, its metabolic precursor 2HE, and the synthetic analog 2-ethoxyestradiol inhibit influx and activation of macrophages in MCT- and bleomycin-induced PH, and this inhibition correlates with reduced PH, vascular remodeling, and fibrosis. 2ME and its metabolic precursor 2HE also inhibit the synthesis of leukotrienes [130]. Blocking of leukotriene production by macrophages prevents endothelial injury and reverses experimental PH [131]. In experimental autoimmune rheumatoid arthritis, 2ME slows down disease progression by inhibiting inflammatory cytokine mRNA (IL-1β, TNF-α, IL-6, and IL-17), leucocyte infiltration, and neovascularization [31]. In several models of autoimmune inflammatory disease, the beneficial effects of 2ME were ascribed to the inhibition of immune cell activation, proliferation, and pro-inflammatory cytokine release [31, 132–134]. Finally, in fibroblasts from SSc disease patients that are at high risk for developing PAH, 2ME reduces hypoxia-induced production of connective tissue growth factor and collagen I by inhibiting the PI3K/Akt/mTOR or HIFα signaling [135]. Collectively, these data in inflammatory and autoimmune diseases point toward 2ME as potential modulator of inflammation and immunity relevant to the development and progression of PAH.

**6. 2-Methoxyestradiol and the renin-angiotensin system (RAS) in PAH**

The metabolic syndrome (MS) is recognized as risk factor for PH [149, 150]. Deficiency in PPARγ (a downstream target of BMPR2) and deficiency in apolipoprotein E and adiponectin (downstream targets of PPARγ) have been linked to the development of PH in rodents [151–153]. Moreover, COMT, via methoxyestradiols,

induced RV and LV hypertrophy and fibrosis [148].

**7. Role of 2ME and the metabolic syndrome in PAH**

Evidence suggests that the renin-angiotensin system (RAS) contributes to the development of PAH [136, 137]. For example, (1) there is increased systemic RAS activity in patients with idiopathic PAH; (2) in experimental and human PH, ACE activity and expression are increased in PAECs, PVSMCs, plexiform lesions, and the RV; (3) increased Ang II type 1 receptor expression and signaling correlates with PAH progression and vascular remodeling [137–142]; and (4) inhibition of RAS slows down the progression of MCT-induced PH [143]. Cumulating data also suggests that 2ME may behave as biological antagonist of Ang II. 2ME downregulates Ang II type I receptors [144–146]. In COMT−/− mice that have reduced 2ME production, 2ME treatment abolishes hypersensitivity to and injury induced by Ang II [65]. Furthermore, in Cyp1B1−/− mice that also have reduced 2ME production, 2ME treatment abolishes Ang II-induced oxidative and vascular injury [147]. Finally, of relevance in PAH, in vivo in high RAS activity models, 2ME attenuates Ang II-induced cardiac and vascular remodeling and fibrosis and isoproterenol-

*DOI: http://dx.doi.org/10.5772/intechopen.86812*

*2-Methoxyestradiol in Pulmonary Arterial Hypertension: A New Disease Modifier DOI: http://dx.doi.org/10.5772/intechopen.86812*

may influence the production and release of these autocrine/paracrine mediators [122]. E2 upregulates CYP1B1, aromatase, and sulfatase activity and inhibits sEH activity. Therefore, in an inflammatory environment, E2 may boost its own production, and via a feed-forward mechanism, E2 may enhance the production of proinflammatory, angiogenic, and mitogenic estrogens and increase the accumulation of pro-inflammatory arachidonic acid metabolites (**Figure 2**). In contrast to E2, non-estrogenic 2ME exhibits significant anti-inflammatory effects, largely through suppression of tissue recruitment and activation of macrophages [123, 124]. This is one of the most consistent in vivo effects of 2ME seen in experimental models of cardiovascular and renal injury [125, 126] and in pulmonary hypertension [50, 51, 127–129]. 2ME, its metabolic precursor 2HE, and the synthetic analog 2-ethoxyestradiol inhibit influx and activation of macrophages in MCT- and bleomycin-induced PH, and this inhibition correlates with reduced PH, vascular remodeling, and fibrosis. 2ME and its metabolic precursor 2HE also inhibit the synthesis of leukotrienes [130]. Blocking of leukotriene production by macrophages prevents endothelial injury and reverses experimental PH [131]. In experimental autoimmune rheumatoid arthritis, 2ME slows down disease progression by inhibiting inflammatory cytokine mRNA (IL-1β, TNF-α, IL-6, and IL-17), leucocyte infiltration, and neovascularization [31]. In several models of autoimmune inflammatory disease, the beneficial effects of 2ME were ascribed to the inhibition of immune cell activation, proliferation, and pro-inflammatory cytokine release [31, 132–134]. Finally, in fibroblasts from SSc disease patients that are at high risk for developing PAH, 2ME reduces hypoxia-induced production of connective tissue growth factor and collagen I by inhibiting the PI3K/Akt/mTOR or HIFα signaling [135]. Collectively, these data in inflammatory and autoimmune diseases point toward 2ME as potential modulator of inflammation and immunity relevant to the development and progression of PAH.

#### **6. 2-Methoxyestradiol and the renin-angiotensin system (RAS) in PAH**

Evidence suggests that the renin-angiotensin system (RAS) contributes to the development of PAH [136, 137]. For example, (1) there is increased systemic RAS activity in patients with idiopathic PAH; (2) in experimental and human PH, ACE activity and expression are increased in PAECs, PVSMCs, plexiform lesions, and the RV; (3) increased Ang II type 1 receptor expression and signaling correlates with PAH progression and vascular remodeling [137–142]; and (4) inhibition of RAS slows down the progression of MCT-induced PH [143]. Cumulating data also suggests that 2ME may behave as biological antagonist of Ang II. 2ME downregulates Ang II type I receptors [144–146]. In COMT−/− mice that have reduced 2ME production, 2ME treatment abolishes hypersensitivity to and injury induced by Ang II [65]. Furthermore, in Cyp1B1−/− mice that also have reduced 2ME production, 2ME treatment abolishes Ang II-induced oxidative and vascular injury [147]. Finally, of relevance in PAH, in vivo in high RAS activity models, 2ME attenuates Ang II-induced cardiac and vascular remodeling and fibrosis and isoproterenolinduced RV and LV hypertrophy and fibrosis [148].

#### **7. Role of 2ME and the metabolic syndrome in PAH**

The metabolic syndrome (MS) is recognized as risk factor for PH [149, 150]. Deficiency in PPARγ (a downstream target of BMPR2) and deficiency in apolipoprotein E and adiponectin (downstream targets of PPARγ) have been linked to the development of PH in rodents [151–153]. Moreover, COMT, via methoxyestradiols,

*Interventional Pulmonology and Pulmonary Hypertension - Updates on Specific Topics*

Warburg effect has been explored as a potential anti-angiogenic target in cancer and more recently in PAH. The HIF-1α transcription factor has been identified as a master hypoxic regulator responsible for the metabolic shift in PAH [101, 109]. Hypoxic induction of HIF-1α leads to overexpression of pyruvate dehydrogenase kinase (PDK) which results in inhibition of pyruvate dehydrogenase that shunts pyruvate into glycolysis and induces conversion of glucose to lactate [108]. Dichloroacetate (DCA), a PDK inhibitor, reverses the Warburg effect and exhibits therapeutic effects in several animal models of PH [110–112]. Because 2ME is a strong HIF-1α inhibitor, 2ME should induce metabolic reprograming in PAH. Presently, the effects of 2ME on metabolic reprograming in PAH are unknown. However, 2ME inhibits lactate-induced mitochondrial biogenesis in highly proliferative osteosarcoma cells, and in apoptosis-resistant melanoma cells, 2ME attenuates proliferation and glycolysis by inhibiting HIF-1α and PDK expression [113–115]. Therefore, 2ME could be viewed as modulator of metabolic reprograming. Further studies are warranted to investigate the effects of 2ME on the Warburg effect that in PAH is associated with highly proliferative, angiogenic, and apoptosis-resistant phenotypes (**Figure 4**).

*Cellular effects of 2ME that contributes to the reduced E2 production, inflammation, angioproliferation,* 

*metabolic reprograming, and vascular and right ventricular remodeling in PAH.*

**5. Anti-inflammatory and immunomodulatory effects of 2ME**

Inflammation and altered immunity, i.e., perivascular accumulation of inflammatory and immune cells in pulmonary circulation, have been increasingly recognized as pathogenic factors in PAH [17–19]. In this regard, at young age women have more robust immune responses than men. Although initially beneficial, with aging these aggressive immune responses may become detrimental [116]. This may explain why various immune diseases are remarkably more frequent in women and why many immune diseases, such as systemic sclerosis (SSc), lupus, and mixed connective tissue disease, are associated with increased risk of PAH [117]. Furthermore, recently distinct immune phenotypes have been reported in PAH patients [118]. In experimental PH, dysregulated immunity in the form of deficient regulatory T-cell (Treg) activity contributes to increased inflammation [20]. Both alveolar macrophages and immune cells express steroidogenic enzymes including sulfatase and aromatase [119–121]. Inflammatory cytokines, prostanoids, and growth factors regulate the expression and activity of steroidogenic enzymes, and in turn, sex hormones

**84**

**Figure 4.**

has been identified as a major factor modulating insulin resistance. The low-activity COMT158Val-Met is linked to MS [154], whereas high-activity COMT rs4680 is associated with lower HbA1c levels and protection from type 2 diabetes [155]. COMT deficiency in mice leads to disrupted glucose homeostasis [66], and 2ME which shares structural similarity with PPARγ ligands and acts as a PPARγ agonist [65, 156] (**Figure 4**) induces AMPK phosphorylation and improves insulin sensitivity in COMT−/− mice [66]. 2HE, a metabolic precursor of 2ME and COMT substrate, activates AMPK in human skeletal muscle, attenuates experimental PH in lean rats, and reduces MS-induced endothelial dysfunction in obese rats. Moreover, in rats with polygenic obesity and MS, in both PH-free females and PH male ZDSD rats, treatment with 2HE reduces glycosylated hemoglobin, RVPSP, and RV-EDP and attenuates vascular remodeling in male PH rats [157]. These data warrant further investigation of 2ME in MS-induced PH and support the notion of 2ME as potential disease modifier in MS-related PH.

#### **8. 2-Methoxyestradiol and current pharmacotherapy of PAH**

Despite significant advances in pharmacotherapy of PAH, mortality of patients with PAH remains high. Therefore, there is still a significant unmet medical need for more effective therapies. Currently approved drugs for treatment of PAH include medications that correct for prostanoid deficiency (prostanoids and prostacyclin receptor agonists) and deficiency of nitric oxide (PDE5 inhibitors and soluble guanylate cyclase stimulators) or combat overproduction of endothelin (endothelin receptor antagonists). Compared to ECs in healthy vessels, the ECs in affected vessels in PH show reduced prostacyclin and nitric oxide synthesis and overexpression of ET-1 [158–160]. Noteworthy, compared to estradiol, 2ME is a more potent inhibitor of endothelin synthesis in endothelial cells [48, 161], and 2ME and its metabolic precursor 2HE inhibit endothelin-induced vasoconstriction [162]. Furthermore, in ECs 2ME is a more potent stimulator of prostacyclin synthesis than estradiol [163, 164]. 2ME also increases basal and potentiates stimulated NO production in male and OVX female rats, but has no effect in intact females, and in vitro these effects are abolished by the eNOS inhibitor L-NAME [165]. 2ME induces vasodilation by stimulating NO release via PPARγ/PI3K/ Akt pathway [166] and increases NO production in uterine artery ECs from pregnant sheep [167]. Moreover, in L-NAME-treated rats, 2ME attenuates severe hypertension and renal, cardiac, and vascular injury and inflammation and reduces mortality by 87% [125]. Likewise, 2ME exhibits beneficial effects in MCT-induced PH and efficacy comparable to that of bosentan and sildenafil. Importantly, in combination with bosentan or sildenafil, 2ME has synergistic therapeutic effects (further reduces vascular remodeling, inflammatory responses, and survival) [128]. Finally, none of the approved therapies for PAH affects endothelial remodeling and "dysregulated angiogenesis" in pulmonary vasculature; in contrast, as discussed above in Section 3, 2ME is a strong anti-angiogenic agent and inhibitor of HIF-VEGF axis that is critical for the metabolic shift and development of occlusive and complex vascular lesions. Altogether, these data clearly indicate 2ME as a promising pharmacological agent capable of providing additional benefit in PAH patients on standard single or combination therapy.

#### **9. Pharmacokinetic aspects of the development of 2ME as a disease modifier in PAH**

The safety and efficacy of 2ME in human PAH are unknown. However, over the past two decades, numerous phase I and II clinical trials have been conducted to test

**87**

*2-Methoxyestradiol in Pulmonary Arterial Hypertension: A New Disease Modifier*

the safety and antitumor efficacy of 2ME in patients with solid malignancies. These studies show that 2ME is well tolerated and safe in doses up to 3 g/day. Unfortunately, even high oral doses of 2ME achieve only low plasma 2ME concentrations due to high pre-systemic metabolism (glucuronidation) in the liver. In experimental PH, therapeutic effects of 2ME are achieved by much lower doses (240 μg/kg/day) delivered by subcutaneous micro-infusions that produce high physiological concentrations of 2ME (~3 ng/ml; equivalent to levels observed during the last trimester of pregnancy) [168]. At these concentrations, 2ME does not induce estrogenic effects. In rats with MCT-induced PH, although higher doses of 2ME do not additionally reduce PH and RV hypertrophy, higher doses do further inhibit media remodeling and inflammation [127]. Likewise, in contrast to oral administration in healthy volunteers and cancer patients, subcutaneous administration of a long-acting formulation of 2ME in doses up to 10 mg produced blood levels of 2ME >1 ng/ml over a 3-week period, with no estrogenic or other adverse effects reported [169]. Currently, various parenteral formulations of 2ME with supposedly high bioavailability are under investigation.

An expanding body of knowledge indicates that many of the beneficial cellular and systemic effects of E2 are due, at least in part, to its major and non-estrogenic metabolite 2ME. This underscores the importance of estradiol metabolism to 2ME in women's health and suggests that 2ME deficiency may contribute to many female predominant diseases, including PAH. 2ME should not be viewed only as partial mediator of E2 effects but in PAH should be considered a moderator of the harmful effects of estrogens related to several key events in PAH including altered estradiol and arachidonic acid metabolism, angiogenesis, inflammation, harmful immune responses, metabolic syndrome, and metabolic reprograming. The above discussion

hopefully makes the case for 2ME as unrecognized disease modifier in PAH.

The authors do not have any conflict of interest.

*DOI: http://dx.doi.org/10.5772/intechopen.86812*

**10. Conclusions and future directions**

**Conflict of interest**

#### *2-Methoxyestradiol in Pulmonary Arterial Hypertension: A New Disease Modifier DOI: http://dx.doi.org/10.5772/intechopen.86812*

the safety and antitumor efficacy of 2ME in patients with solid malignancies. These studies show that 2ME is well tolerated and safe in doses up to 3 g/day. Unfortunately, even high oral doses of 2ME achieve only low plasma 2ME concentrations due to high pre-systemic metabolism (glucuronidation) in the liver. In experimental PH, therapeutic effects of 2ME are achieved by much lower doses (240 μg/kg/day) delivered by subcutaneous micro-infusions that produce high physiological concentrations of 2ME (~3 ng/ml; equivalent to levels observed during the last trimester of pregnancy) [168]. At these concentrations, 2ME does not induce estrogenic effects. In rats with MCT-induced PH, although higher doses of 2ME do not additionally reduce PH and RV hypertrophy, higher doses do further inhibit media remodeling and inflammation [127]. Likewise, in contrast to oral administration in healthy volunteers and cancer patients, subcutaneous administration of a long-acting formulation of 2ME in doses up to 10 mg produced blood levels of 2ME >1 ng/ml over a 3-week period, with no estrogenic or other adverse effects reported [169]. Currently, various parenteral formulations of 2ME with supposedly high bioavailability are under investigation.

### **10. Conclusions and future directions**

An expanding body of knowledge indicates that many of the beneficial cellular and systemic effects of E2 are due, at least in part, to its major and non-estrogenic metabolite 2ME. This underscores the importance of estradiol metabolism to 2ME in women's health and suggests that 2ME deficiency may contribute to many female predominant diseases, including PAH. 2ME should not be viewed only as partial mediator of E2 effects but in PAH should be considered a moderator of the harmful effects of estrogens related to several key events in PAH including altered estradiol and arachidonic acid metabolism, angiogenesis, inflammation, harmful immune responses, metabolic syndrome, and metabolic reprograming. The above discussion hopefully makes the case for 2ME as unrecognized disease modifier in PAH.

### **Conflict of interest**

*Interventional Pulmonology and Pulmonary Hypertension - Updates on Specific Topics*

**8. 2-Methoxyestradiol and current pharmacotherapy of PAH**

**9. Pharmacokinetic aspects of the development of 2ME as a disease** 

The safety and efficacy of 2ME in human PAH are unknown. However, over the past two decades, numerous phase I and II clinical trials have been conducted to test

Despite significant advances in pharmacotherapy of PAH, mortality of patients with PAH remains high. Therefore, there is still a significant unmet medical need for more effective therapies. Currently approved drugs for treatment of PAH include medications that correct for prostanoid deficiency (prostanoids and prostacyclin receptor agonists) and deficiency of nitric oxide (PDE5 inhibitors and soluble guanylate cyclase stimulators) or combat overproduction of endothelin (endothelin receptor antagonists). Compared to ECs in healthy vessels, the ECs in affected vessels in PH show reduced prostacyclin and nitric oxide synthesis and overexpression of ET-1 [158–160]. Noteworthy, compared to estradiol, 2ME is a more potent inhibitor of endothelin synthesis in endothelial cells [48, 161], and 2ME and its metabolic precursor 2HE inhibit endothelin-induced vasoconstriction [162]. Furthermore, in ECs 2ME is a more potent stimulator of prostacyclin synthesis than estradiol [163, 164]. 2ME also increases basal and potentiates stimulated NO production in male and OVX female rats, but has no effect in intact females, and in vitro these effects are abolished by the eNOS inhibitor L-NAME [165]. 2ME induces vasodilation by stimulating NO release via PPARγ/PI3K/ Akt pathway [166] and increases NO production in uterine artery ECs from pregnant sheep [167]. Moreover, in L-NAME-treated rats, 2ME attenuates severe hypertension and renal, cardiac, and vascular injury and inflammation and reduces mortality by 87% [125]. Likewise, 2ME exhibits beneficial effects in MCT-induced PH and efficacy comparable to that of bosentan and sildenafil. Importantly, in combination with bosentan or sildenafil, 2ME has synergistic therapeutic effects (further reduces vascular remodeling, inflammatory responses, and survival) [128]. Finally, none of the approved therapies for PAH affects endothelial remodeling and "dysregulated angiogenesis" in pulmonary vasculature; in contrast, as discussed above in Section 3, 2ME is a strong anti-angiogenic agent and inhibitor of HIF-VEGF axis that is critical for the metabolic shift and development of occlusive and complex vascular lesions. Altogether, these data clearly indicate 2ME as a promising pharmacological agent capable of providing additional benefit in PAH patients on standard single or combination therapy.

disease modifier in MS-related PH.

has been identified as a major factor modulating insulin resistance. The low-activity COMT158Val-Met is linked to MS [154], whereas high-activity COMT rs4680 is associated with lower HbA1c levels and protection from type 2 diabetes [155]. COMT deficiency in mice leads to disrupted glucose homeostasis [66], and 2ME which shares structural similarity with PPARγ ligands and acts as a PPARγ agonist [65, 156] (**Figure 4**) induces AMPK phosphorylation and improves insulin sensitivity in COMT−/− mice [66]. 2HE, a metabolic precursor of 2ME and COMT substrate, activates AMPK in human skeletal muscle, attenuates experimental PH in lean rats, and reduces MS-induced endothelial dysfunction in obese rats. Moreover, in rats with polygenic obesity and MS, in both PH-free females and PH male ZDSD rats, treatment with 2HE reduces glycosylated hemoglobin, RVPSP, and RV-EDP and attenuates vascular remodeling in male PH rats [157]. These data warrant further investigation of 2ME in MS-induced PH and support the notion of 2ME as potential

**86**

**modifier in PAH**

The authors do not have any conflict of interest.

#### **Author details**

Stevan P. Tofovic1,2,3\* and Edwin K. Jackson2

1 Heart, Lung, Blood and Vascular Medicine Institute, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA

2 Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA

3 Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA

\*Address all correspondence to: tofovic@pitt.edu

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**89**

*2-Methoxyestradiol in Pulmonary Arterial Hypertension: A New Disease Modifier*

Respiratory Journal. 2010;**36**:549-555. DOI: 10.1183/09031936.00057010

[8] Jing ZC, Xu XQ, Han ZY, Wu Y, Deng KW, Wang H, et al. Registry and survival study in Chinese patients with idiopathic and familial pulmonary arterial hypertension. Chest. 2007;**132**:373-379. DOI: 10.1378/

[9] Tamura Y, Kumamaru H, Satoh T, Miyata H, Ogawa A, Tanabe N, et al. Effectiveness and outcome of pulmonary arterial hypertensionspecific therapy in Japanese patients with pulmonary arterial hypertension. Circulation Journal: Official Journal of the Japanese Circulation Society. 2017;**82**:275-282. DOI: 10.1253/circj.

[10] Humbert M, Sitbon O, Chaouat A, Bertocchi M, Habib G, Gressin V, et al. Pulmonary arterial hypertension in France: Results from a national registry. American Journal of

Respiratory and Critical Care Medicine. 2006;**173**:1023-1030. DOI: 10.1164/

[11] McGoon MD, Miller DP. REVEAL: A contemporary US pulmonary arterial hypertension registry. European Respiratory Review: An Official Journal of the European Respiratory Society. 2012;**21**:8-18. DOI:

[12] Zhang R, Dai LZ, Xie WP, Yu ZX, Wu BX, Pan L, et al. Survival of Chinese

[13] Hoeper MM, Huscher D, Ghofrani HA, Delcroix M, Distler O, Schweiger C,

et al. Elderly patients diagnosed with idiopathic pulmonary arterial hypertension: Results from the

patients with pulmonary arterial hypertension in the modern treatment era. Chest. 2011;**140**:301-309. DOI:

rccm.200510-1668OC

10.1183/09059180.00008211

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chest.06-2913

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[2] Wood P. Pulmonary hypertension. British Medical Bulletin. 1952;**8**:348-353

registry. Chest. 2012;**142**:448-456. DOI:

[4] Benza RL, Miller DP, Gomberg-Maitland M, Frantz RP, Foreman AJ, Coffey CS, et al. Predicting survival in pulmonary arterial hypertension: Insights from the registry to evaluate early and long-term pulmonary arterial hypertension disease

management (REVEAL). Circulation. 2010;**122**:164-172. DOI: 10.1161/ CIRCULATIONAHA.109.898122

[5] Escribano-Subias P, Blanco I, Lopez-

[6] Humbert M, Sitbon O, Chaouat A, Bertocchi M, Habib G, Gressin V, et al. Survival in patients with idiopathic, familial, and anorexigen-associated pulmonary arterial hypertension in the modern management era. Circulation. 2010;**122**:156-163. DOI: 10.1161/ CIRCULATIONAHA.109.911818

[7] Humbert M, Sitbon O, Yaici A, Montani D, O'Callaghan DS, Jais X, et al. Survival in incident and prevalent cohorts of patients with pulmonary arterial hypertension. The European

Meseguer M, Lopez-Guarch CJ, Roman A, Morales P, et al. Survival in pulmonary hypertension in Spain: Insights from the spanish registry. The European Respiratory Journal. 2012;**40**:596-603. DOI: 10.1183/09031936.00101211

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**Author details**

Stevan P. Tofovic1,2,3\* and Edwin K. Jackson2

Pittsburgh, Pennsylvania, USA

School of Medicine, Pittsburgh, Pennsylvania, USA

School of Medicine, Pittsburgh, Pennsylvania, USA

\*Address all correspondence to: tofovic@pitt.edu

provided the original work is properly cited.

1 Heart, Lung, Blood and Vascular Medicine Institute, University of Pittsburgh

2 Department of Pharmacology and Chemical Biology, University of Pittsburgh

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

3 Department of Medicine, University of Pittsburgh School of Medicine,

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rccm.201201-0164OC

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[156] Barchiesi F, Jackson EK, Fingerle J, Gillespie DG, Odermatt B, Dubey RK. 2-methoxyestradiol, an estradiol metabolite, inhibits neointima formation and smooth muscle cell growth via double blockade of the cell cycle. Circulation Research. 2006;**99**:266-274. DOI: 10.1161/01. RES.0000233318.85181.2e

[157] Tofovic SP, Hu J, Jackson EK. Schneider F 2-hydroxyestradiol attenuates metabolic syndrome-induced pulmonary hypertension. American Journal of Respiratory and Critical Care Medicine. 2015;**191**:A4096

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[161] Dubey RK, Jackson EK, Keller PJ, Imthurn B, Rosselli M. Estradiol metabolites inhibit endothelin synthesis by an estrogen receptorindependent mechanism. Hypertension. 2001;**37**:640-644

[162] Hill BJ, Gebre S, Schlicker B, Jordan R, Necessary S. Nongenomic inhibition of coronary constriction by 17ss-estradiol, 2-hydroxyestradiol, and 2-methoxyestradiol. Canadian Journal of Physiology and Pharmacology. 2010;**88**:147-152. DOI: 10.1139/Y09-120

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*Interventional Pulmonology and Pulmonary Hypertension - Updates on Specific Topics*

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2009;**105**:107

**102**

## *Edited by Theodoros Aslanidis*

This book, published by IntechOpen, focuses on interesting aspects of pulmonary medicine. The first section of the book is dedicated to interventional pulmonology, and includes updates on bronchial thermoplasty, virtual bronchoscopy, and endobronchial ultrasound. The second section highlights special aspects of pulmonary circulation and pulmonary hypertension. Throughout the book, the authors offer us not only a "vigorous" review of the current literature but also a research path to further advancement.

Published in London, UK © 2020 IntechOpen © vitanovski / iStock

Interventional Pulmonology and Pulmonary Hypertension - Updates on Specific Topics

Interventional Pulmonology

and Pulmonary Hypertension

Updates on Specific Topics

*Edited by Theodoros Aslanidis*