Keywords

Pulmonary Hypertension

Pulmonary hypertension is a clinical condition defined by the elevation of mean pulmonary arterial pressure ≥25 mmHg with normal pulmonary wedge pressure (PWP ≤ 15 mmHg). The term “pulmonary hypertension” represents a collection of various diseases with this hemodynamic definition (Galiè et al. 2009a; Chemla et al. 2002). Pulmonary arterial hypertension (PAH) (Group 1) includes idiopathic PAH, heritable PAH, PAH induced by drugs and toxins, persistent pulmonary hypertension of the newborn, and PAH associated with various diseases such as connective tissue diseases, HIV infection, portal hypertension, congenital heart disease, schistosomiasis, and chronic hemolytic anemia. Pulmonary hypertension is also associated with hypoxic lung diseases such as chronic obstructive pulmonary disease and lung fibrosis (Group 3) and often worsens the prognosis of these primary diseases. Other classes of pulmonary hypertension include pulmonary hypertension due to left heart dysfunction (Group 2), chronic thromboembolic pulmonary hypertension (Group 4), pulmonary veno-occlusive disease and/or pulmonary capillary hemangiomatosis (Group 1), and diverse forms of pulmonary hypertension with unclear and/or multifactorial mechanisms (associated with myeloproliferative disorders, splenectomy, sarcoidosis, and other rare medical conditions) (Group 5) (Simonneau et al. 2004; Galiè et al. 2009a).

The increased pulmonary arterial pressure and resistance strain the right ventricle (RV), initially causing compensatory concentric RV hypertrophy, which in turn transitions to right-sided heart failure and death (Nauser and Stites 2003; Farber and Loscalzo 2004). The median survival for patients with PAH has been reported to be 2.8 years from the time of diagnosis without treatment (Runo and Loyd 2003). Even with currently available therapies, the prognosis remains poor, with a 3 year survival of PAH patients reported to be 58–75 % (Benza et al. 2010; Humbert et al. 2010; Thenappan et al. 2010).

Pulmonary hypertension is characterized by vasoconstriction within the pulmonary vasculature and histological abnormalities of the vascular wall, including medial hypertrophy, intimal proliferation, fibrosis, adventitial thickening, thrombotic lesions, and inflammatory infiltrates (Morrell et al. 2009; Humbert et al. 2004; Sweeney and Yuan 2000; Mandegar and Yuan 2002). In advanced stages of PAH, all of these characteristics are present in complex plexiform lesions.

Various biochemical pathways lead to vasoconstriction of pulmonary arteries and increased pulmonary vascular resistance, in part due to the abnormal production of vasoactive compounds. Increased vasoconstriction could also be due to abnormal expression and function of the potassium channels in pulmonary vascular smooth muscles cells (SMCs) (Burg et al. 2008). Endothelial dysfunction causes overexpression of vasoconstrictive and proliferative substances such as endothelin-1 and thromboxane A2 (Humbert et al. 2008). In addition, the levels of vasodilators and antiproliferative agents such as nitric oxide and prostacyclin are decreased. These abnormalities elevate the vascular tone and promote vascular thickening through the proliferation and hypertrophy of endothelial cells, SMCs, and fibroblasts. Inflammatory cells may also play an important role, since perivascular inflammatory infiltrates are present in lungs with pulmonary hypertension (Hassoun et al. 2009). However, the exact pathogenetic pathways that lead to these features are unknown.

Currently Available Therapy for Pulmonary Hypertension

Despite significant progress in treatment options in the last decade, pulmonary hypertension remains a serious lethal condition. Currently used drugs reduce symptoms, but mortality due to pulmonary hypertension remains high (Galiè et al. 2009b). Several groups of vasodilators are used as treatment strategies for pulmonary hypertension. Calcium channel blockers are the first group used in the therapy; however, only a small number of patients respond to this treatment (Sitbon et al. 2005). Three classes of drugs have been specifically approved to treat PAH (Group I). Among them, prostanoids are the most potent drugs in the treatment of PAH (Galiè et al. 2003; Safdar 2011). This class of agents, however, is not stable, and therefore, they often need to be applied continuously through an intravenous route. Prostanoids have also been used clinically and can be administered subcutaneously and through inhalation as well. However, the oral prostanoid, beraprost, does not provide a significant hemodynamic benefit (Galiè et al. 2002; Barst et al. 2003). Endothelin-1 receptor antagonists and phosphodiesterase-5 inhibitors are oral vasodilators used in PAH (Galiè et al. 2004; Ghofrani et al. 2004). There is still a need for new efficient therapies that reverse the features associated with advanced stages of this disease and decrease the mortality associated with PAH. Therapies to prevent and/or treat other forms of pulmonary hypertension are also needed. In addition, there are currently no specific therapies available to prevent and/or treat right heart failure.

In this chapter, evidence for the role of free radicals and the beneficial effects of antioxidants in pulmonary hypertension and right heart failure is presented.

Reactive Oxygen Species (ROS) and Antioxidants

Sequential electron reduction of molecular oxygen (O2) produces ROS. One-electron reduction produces superoxide anion radical (O2 •−), two-electron reduction produces hydrogen peroxide (H2O2), and three-electron reduction results in the formation of hydroxyl radical (HO.). O2 •− is formed through various means in the biological system including enzymes such as xanthine oxidase and NADPH oxidase, which perform one-electron reduction of O2 to form O2 •−. H2O2 is often formed through the dismutation reaction, in which two molecules of O2 •− interact with each other to donate/accept an electron. In this reaction, the O2 •− molecule, which receives an electron, becomes H2O2. In the biological system, the source of the electron that reduces H2O2 to HO. includes reduced metal irons and in particular iron ions in the Fenton reaction. Iron ions are normally present in the cytosol in oxidized form as ferric ion (Fe3+). Thus, reduction of ferric to ferrous ion (Fe2+) is required to elicit the Fenton reaction and HO. formation (Freeman and Crapo 1982; Halliwell and Gutteridge 1985; Suzuki and Ford 1994).

Traditionally, ROS have been thought to nonspecifically and indiscriminately react with biological molecules and cause damage to biologic events (Halliwell and Gutteridge 1985). However, during the early 1990s, it was observed that (1) ligand-receptor interactions produce ROS, (2) antioxidants block signal transduction, and (3) ROS can stimulate signaling events; therefore, ROS have been proposed to serve as second messengers (Schreck et al. 1991; Suzuki and Ford 1992; Sen and Packer 1996; Wolin 1996; Suzuki et al. 1997). Notably, Schreck et al. (1991) proposed that H2O2 was a second messenger for the activation of NF-κB transcription factor in T cells. In vascular smooth muscle cells of systemic circulation, Rao and Berk (1992) demonstrated that ROS promote cell growth. Subsequently, ROS were reported to mediate signal transduction induced by angiotensin II in these cells (Griendling et al. 1994) and platelet-derived growth factor (Sundaresan et al. 1995) through NADPH oxidase. The concept of the role of ROS in cell signal transduction has been supported by a large number of studies and is now widely accepted (Sen and Packer 1996; Wolin 1996; Suzuki et al. 1997; Rhee et al. 2000; Forman et al. 2010).

The term “antioxidant” can be broadly defined as any molecule that can inhibit the formation and actions of ROS. These molecules could (1) inhibit the production of ROS (e.g., inhibitors of oxidases), (2) directly scavenge ROS, (3) scavenge other reactive substances that may be produced in response to the actions and reactions of ROS, (4) chelate metal ions such as iron to inhibit Fenton reaction, and (5) promote the expression of antioxidant proteins.

Oxidative Stress and Antioxidant Status in Human Pulmonary Hypertension

In humans, several reports have provided evidence for increased oxidation in pulmonary hypertension. Patients with pulmonary hypertension were found to have increased lipid peroxidation compared to healthy control subjects as indicated by the gas chromatography/mass spectrometry measurements of urine isoprostane levels (Cracowski et al. 2001) and plasma malondialdehyde (Irodova et al. 2002). Bowers et al. (2004) reported that lungs from idiopathic PAH patients had increased DNA oxidation levels based on measurements of 8-hydroxyl guanosine levels as well as increased nitrotyrosine levels. Our laboratory has shown that plasma from patients with idiopathic PAH has increased protein carbonyl content (Wong et al. 2012) and reduced levels of lipophilic antioxidants, including alpha-tocopherol and beta-carotene (Preston et al. 2005), compared to control subjects. Plasma from idiopathic PAH patients was also found to contain malondialdehyde-modified albumin (Odhiambo et al. 2007). In lung tissues, superoxide dismutase 2 expression is lower in patients with PAH compared to control subjects (Bowers et al. 2004; Archer et al. 2010).

Role of ROS and the Effects of Antioxidants in Animal Models of Pulmonary Hypertension

Gillespie and coworkers in piglets provided early evidence for the role of ROS and the effectiveness of antioxidant molecules in pulmonary hypertension. Pulmonary hypertension induced by group B streptococcus was attenuated by a HO. scavenger dimethylthiourea (Pauly et al. 1988; Bowdy et al. 1990) and bilirubin (Pauly et al. 1991). Dimethylthiourea also reversed sepsis-induced pulmonary hypertension (Shook et al. 1990). In rats, pulmonary hypertension was found to be inhibited by compounds thought to have reactive oxygen scavenging activities including dimethylthiourea (Langleben et al. 1989; Lai et al. 1998), probucol (Irukayama-Tomobe et al. 2000), N-acetylcysteine (Hoshikawa et al. 2001; Lachmanová et al. 2005), tempol (Elmedal et al. 2004; Jankov et al. 2008), erdosteine (Uzun et al. 2006), allicin (Sun and Ku 2006), pyrrolidine dithiocarbamate (Sawada et al. 2007; Huang et al. 2008), sulfur dioxide (Jin et al. 2008), resveratrol (Csiszar et al. 2009; Yang et al. 2010), and EUK-134 (Redout et al. 2010). However, many of these antioxidants are weak and nonspecific.

More specific antioxidant enzymes, particularly superoxide dismutase (SOD), have also been shown to be effective as well. Abman and coworkers (Kinsella et al. 2005) demonstrated that SOD improves hemodynamics. Steinhorn and coworkers reported that SOD improves oxygenation and reduces oxidation (Lakshminrusimha et al. 2006), restores eNOS expression and function (Farrow et al. 2008), and normalizes phosphodiesterase 5 (Farrow et al. 2010) in neonatal lambs with persistent pulmonary hypertension. Gene transfer of extracellular SOD has also been shown to reduce pulmonary hypertension in rats (Kamezaki et al. 2008).

The inhibitors of the production of ROS are also effective in ameliorating pulmonary hypertension. Jankov et al. (2008) showed that a xanthine oxidase inhibitor, allopurinol, inhibits chronic hypoxic pulmonary hypertension in neonatal rats. In fetal lambs, the roles of uncoupled nitric oxide synthase-generated ROS in pulmonary hypertension were also documented (Konduri et al. 2007; Lakshminrusimha et al. 2007).

Accumulating evidence suggests that NADPH oxidase has a role in the development of pulmonary hypertension, and thus the inhibitors of NADPH oxidase may be promising as therapeutic agents. Black and coworkers (Brennan et al. 2003; Grobe et al. 2006) first proposed the role of NADPH oxidase in fetal lambs with pulmonary hypertension. Foltz and coworkers (Liu et al. 2006) reported that chronic hypoxia-induced pulmonary hypertension was inhibited in gp91phox knockout mice. The involvement of NADPH oxidase in pulmonary hypertension was also observed in newborn piglets (Dennis et al. 2009) and mice (Nisbet et al. 2009). More recently, NOX4 has specifically emerged as an important mediator of ROS generation and the development of various forms of pulmonary hypertension (Sturrock et al. 2006; Mittal et al. 2007; Gosemann et al. 2013; Wedgwood et al. 2013). In cultured pulmonary vascular cells, the NOX4 inhibitor GKT137831 attenuated hypoxia-induced cell proliferation (Green et al. 2012).

ROS in Endothelin-1 Signaling

Endothelin-1, a peptide composed of 21 amino acids, is a potent vasoconstrictor originally identified in vascular endothelial cells (Yanagisawa et al. 1988). Plasma endothelin-1 is elevated in patients with idiopathic PAH (Stewart et al. 1991; Giaid et al. 1993). Histological studies of lung tissues from patients with pulmonary hypertension demonstrated excess endothelin-1 production and increased expression of prepro-endothelin-1 (Giaid et al. 1993). Similarly, increased endothelin-1 expression was found in the lungs of fawn-hooded rats with pulmonary hypertension (Zamora et al. 1996). In secondary pulmonary hypertension, plasma levels of endothelin-1 are positively correlated with the severity of the disease and negatively correlated with prognosis (Yoshibayashi et al. 1991; Cody et al. 1992). In various animal models of secondary pulmonary hypertension, endothelin-receptor antagonists have been shown to block the progression of the disease (Miyauchi et al. 1993; Bonvallet et al. 1994; Okada et al. 1995; Eddahibi et al. 1995; Sakai et al. 1996; Ueno et al. 2000). Human studies have shown that an endothelin-receptor antagonist bosentan increased exercise capacity and improved hemodynamics in patients with pulmonary hypertension (Channick et al. 2001; Rubin et al. 2002). Bosentan has been approved for the treatment of human PAH, indicating the importance of understanding endothelin-1 signaling to develop better therapeutic agents to treat pulmonary hypertension. Other endothelin-receptor antagonists with different receptor specificities were also approved later and show similar treatment efficacy (Barst et al. 2004, 2006; Galiè et al. 2005).

Endothelin-1 is a mitogen of pulmonary artery SMCs (Hirata et al. 1989; Hassoun et al. 1992; Janakidevi et al. 1992). Activation of either ETA or ETB receptor can induce proliferation of human pulmonary artery SMCs (Davie et al. 2002). Endothelin-1 has also been shown to promote cell survival (antiapoptotic) signaling in pulmonary artery SMCs (Suzuki et al. 2007).

Wedgwood et al. (2001) reported that endothelin-1 induces the production of ROS in pulmonary artery SMCs of fetal sheep through NADPH oxidase and antioxidants block endothelin-1-induced cell proliferation. Our laboratory found that, in bovine and human pulmonary artery SMCs, endothelin-1, serotonin, and PDGF promote protein carbonylation, which may mediate oxidant signaling (Wong et al. 2008; Wong et al. 2012). Proteomic analysis revealed that one protein that is carbonylated in response to endothelin-1 is annexin A1 (Wong et al. 2008). This protein possesses antiproliferative and apoptotic activities and, upon oxidative modifications, is degraded by proteasomes. The loss of annexin A1 results in increased cell growth and survival (Fig. 74.1).

Fig. 74.1
figure 1

Protein carbonylation-dependent mechanism of ROS signaling in pulmonary artery smooth muscle cells proposed by Suzuki and coworkers. Mediators of pulmonary vascular remodeling such as endothelin-1, serotonin, and PDGF promote the production of reactive oxygen species. Metal-catalyzed production of hydroxyl radicals results in carbonylation of annexin A1, which normally functions as an antiproliferative and apoptotic protein. Carbonylated annexin A1 gets degraded by the proteasome, resulting in increased cell growth and survival

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ROS in Serotonin Signaling

Serotonin (5-hydroxytryptamine) is a potent vasoconstrictor and a mitogen of pulmonary artery SMCs. Evidence for the role of serotonin in the development of pulmonary hypertension was first recognized in fawn-hooded rats, in which a genetic deficit in serotonin platelet storage and high plasma levels of serotonin were associated with the development of pulmonary hypertension (Sato et al. 1992). Further studies showed that a continuous intravenous infusion of serotonin during the exposure of rats to hypoxia potentiated pulmonary hypertension (Eddahibi et al. 1997). Serotonin transporter-deficient mice also have been shown to develop less hypoxic pulmonary hypertension and vascular remodeling due to the inability of serotonin to promote SMC growth (Eddahibi et al. 2000). In patients with idiopathic PAH, high levels of plasma serotonin were detected (Herve et al. 1995). Eddahibi et al. (2001) reported that pulmonary artery SMCs from patients with pulmonary hypertension grow faster than cells from control subjects in response to serotonin. The mechanism of pulmonary artery SMC growth has been reported to involve both serotonin transporter (Lee et al. 1994) and serotonin receptors (Liu et al. 2004).

In bovine pulmonary artery SMCs, Fanburg and coworkers reported that serotonin uptake by serotonin transporter activates NADPH oxidase and the production of O2 •− (Lee et al. 1998, 1999) and H2O2 (Lee et al. 2001). They proposed that ROS are involved in serotonin-induced growth of pulmonary artery smooth muscle cells by activating the MEK/ERK pathway (Fig. 74.2). We have shown that the GATA4 transcription factor plays an important role in pulmonary artery SMC growth regulation and antioxidants inhibit serotonin-induced GATA4 activation (Suzuki et al. 2003). In human pulmonary artery SMCs, Lawrie et al. (2005) found that serotonin activates GATA4 through ROS produced by monoamine oxidase A.

Fig. 74.2
figure 2

The mechanism of serotonin signaling for the proliferation of pulmonary artery smooth muscle cells proposed by Fanburg and coworkers. Serotonin (5-HT) uptake by the serotonin transporter (SERT) activates NADPH oxidase and produces reactive oxygen species, which activate the MEK/ERK pathway for cell proliferation

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ROS and Right Heart Failure

In addition to the role of ROS in the pulmonary vasculature, these species may also mediate the development of RV hypertrophy and right heart failure. Thus, antioxidant-based therapies to prevent and/or treat pulmonary hypertension-induced heart failure may be possible. During the transition to heart failure, ROS are produced but RV is not able to activate antioxidant defenses (Ecarnot-Laubriet et al. 2003). RV antioxidant enzymes are increased during hypertrophy and decreased during right heart failure in rats (Farahmand et al. 2004). Redout et al. (2007, 2010) have shown that right heart failure due to monocrotaline-induced pulmonary hypertension increased the ROS production in rats and treatment with the antioxidant EUK-134 attenuated right heart failure. In the SU5416/hypoxia model of pulmonary hypertension and right heart failure in rats, Protandim, which is a plant extract that induces Nrf2-dependent promotion of endogenous antioxidant defenses, has been shown to prevent fibrosis and capillary loss in the RV and preserved RV function (Bogaard et al. 2009).

Our laboratory reported that ROS activate the CBF/NF-Y transcription factor, thereby triggering a right heart-specific activation of hypertrophic responses (Park et al. 2010). In addition, treating perfused isolated rat hearts with serotonin promoted protein carbonylation, specifically in the RV, but not in the left ventricle (Liu et al. 2008). These differential responses to serotonin between RV and left ventricle may be defined by the low expression of monoamine oxidase A in the RV compared to the left ventricle, which may preserve the cytosolic level of serotonin and the ability of serotonin to produce O2 •− through NADPH oxidase (Liu et al. 2008). These results suggest that ROS signaling regulates pulmonary hypertension-induced RV hypertrophy and right heart failure.

Conclusions

Evidence suggests that the production of ROS plays an important role in the pathogenesis of pulmonary hypertension as well as right heart failure. As described above, studies using various animal models have shown that compounds with antioxidant properties are effective in inhibiting pulmonary hypertension. Evidence for the benefit of antioxidant therapy in human pulmonary hypertension is, however, lacking. Since currently available treatment options for pulmonary hypertension do not bring satisfactory results on the prognosis of patients, new therapeutic strategies are needed. Many antioxidants have been proven to be safe in humans. Based on results from animal studies, effects of antioxidants in the treatment of pulmonary hypertension should be explored in humans. Since animal data show that antioxidants have antiproliferative actions on remodeled pulmonary arteries, the antioxidant therapy may inhibit the progression of the disease and possibly reverse pulmonary vascular remodeling. In addition, antioxidants may prevent the transition from compensatory RV hypertrophy to heart failure. Thus, the use of antioxidants may be a new reinforcement in the complex therapy for pulmonary hypertension that could improve the prognosis of patients (Fig. 74.3). Thus, further understanding of the mechanisms of ROS involvement in the pulmonary circulation and the right heart should guide more effective human clinical trials.

Fig. 74.3
figure 3

A simplistic view of the proposed roles of ROS and the effects of antioxidants. Various triggers promote the pathogenesis of pulmonary hypertension. These triggers activate the generation of ROS, which in turn contribute to the development of pulmonary vascular remodeling and pulmonary hypertension. Increased pulmonary vascular pressure also promotes the production of ROS, which contribute to RV remodeling and subsequent right heart failure

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