Pulmonary vascular remodeling: a target for therapeutic intervention in pulmonary hypertension

Pharmacology & Therapeutics 92 (2001) 1 – 20

Associate editor: R.M. Wadsworth

Pulmonary vascular remodeling: a target for therapeutic intervention in pulmonary hypertension Trina K. Jeffery, Janet C. Wanstall* Department of Physiology and Pharmacology, The University of Queensland, St Lucia, Brisbane, Queensland, 4072, Australia

Abstract Pulmonary vascular remodeling is an important pathological feature of pulmonary hypertension, leading to increased pulmonary vascular resistance and reduced compliance. It involves thickening of all three layers of the blood vessel wall (due to hypertrophy and/or hyperplasia of the predominant cell type within each layer), as well as extracellular matrix deposition. Neomuscularisation of non-muscular arteries and formation of plexiform and neointimal lesions also occur. Stimuli responsible for remodeling involve transmural pressure, stretch, shear stress, hypoxia, various mediators [angiotensin II, endothelin (ET)-1, 5-hydroxytryptamine, growth factors, and inflammatory cytokines], increased serine elastase activity, and tenascin-C. In addition, there are reductions in the endothelium-derived antimitogenic substances, nitric oxide, and prostacyclin. Intracellular signalling mechanisms involved in pulmonary vascular remodeling include elevations in intracellular Ca2 + and activation of the phosphatidylinositol pathway, protein kinase C, and mitogen-activated protein kinase. In animal models of pulmonary hypertension, various drugs have been shown to attenuate pulmonary vascular remodeling. These include angiotensin-converting enzyme inhibitors, angiotensin receptor antagonists, ET receptor antagonists, ET-converting enzyme inhibitors, nitric oxide, phosphodiesterase 5 inhibitors, prostacyclin, Ca2 + -channel antagonists, heparin, and serine elastase inhibitors. Inhibition of remodeling is generally accompanied by reductions in pulmonary artery pressure. The efficacy of some of the drugs varies, depending on the animal model of the disease. In view of the complexity of the remodeling process and the diverse aetiology of pulmonary hypertension in humans, it is to be anticipated that successful anti-remodeling therapy in the clinic will require a range of different drug options. D 2001 Elsevier Science Inc. All rights reserved. Keywords: Pulmonary vascular remodeling; Pulmonary hypertension; Anti-remodeling drugs; Pulmonary blood vessels Abbreviations: ACE, angiotensin-converting enzyme; ANP, atrial natriuretic peptide; AT1 and AT2 receptors, angiotensin II Types I and II receptors; bFGF, basic fibroblast growth factor; cAMP, cyclic AMP; cGMP, cyclic GMP; ECE, endothelin-converting enzyme; EGF, epidermal growth factor; E.L., elastic lamina; ET, endothelin; 5-HT, 5-hydroxytryptamine; IGF, insulin-like growth factor; MAPK, mitogen-activated protein kinase; NEP, neutral endopeptidase; NO, nitric oxide; o.d., outer diameter; PDE, phosphodiesterase; PDGF, platelet-derived growth factor; PKC, protein kinase C; TGF, transforming growth factor; VEGF, vascular endothelial growth factor.

Contents 1. 2. 3. 4.

Introduction . . . . . . . . . . . . . . . . . . . . . . . Characteristics of pulmonary vascular remodeling . . . Methods used to assess pulmonary vascular remodeling Mechanisms of pulmonary vascular remodeling . . . . 4.1. Mechanical factors . . . . . . . . . . . . . . . . 4.2. Hypoxia . . . . . . . . . . . . . . . . . . . . . 4.3. Mediators . . . . . . . . . . . . . . . . . . . . 4.3.1. Angiotensin II . . . . . . . . . . . . . 4.3.2. Endothelin-1 . . . . . . . . . . . . . .

* Corresponding author. Tel.: +61-7-3365-3113; fax: +61-7-3365-1766. E-mail address: [email protected] (J.C. Wanstall). 0163-7258/01/$ – see front matter D 2001 Elsevier Science Inc. All rights reserved. PII: S 0 1 6 3 - 7 2 5 8 ( 0 1 ) 0 0 1 5 7 - 7

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5. 6.

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4.3.3. 5-Hydroxytryptamine . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.4. Growth factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.5. Inflammatory cytokines . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. Tenascin-C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5. Role of the endothelium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.6. Role of serine elastase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.7. Intracellular signalling mechanisms . . . . . . . . . . . . . . . . . . . . . . . Consequences of pulmonary vascular remodeling. . . . . . . . . . . . . . . . . . . . Therapeutic interventions for the inhibition of pulmonary vascular remodeling . . . . 6.1. Angiotensin-converting enzyme inhibitors and angiotensin receptor antagonists 6.2. Endothelin receptor antagonists and endothelin-converting enzyme inhibitors . 6.3. Nitric oxide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4. Phosphodiesterase inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.5. Prostacyclin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.6. Ca2+-channel antagonists . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.7. Heparin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.8. Drugs affecting the extracellular matrix . . . . . . . . . . . . . . . . . . . . . 6.9. Miscellaneous drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Pulmonary hypertension is characterised by elevations in pulmonary artery pressure and pulmonary vascular resistance. There are multiple aetiologies of pulmonary hypertension (some of which are listed in Table 1) and also a variety of animal models of the disease (Table 2). However, the two pathological features that are common to most, if not all, forms of pulmonary hypertension are abnormal pulmonary vasoconstriction and alterations in pulmonary vascular structure (pulmonary vascular remodeling); both of these features contribute to the elevations in pressure and resistance. Drugs in current use for the treatment of pulmonary hypertension are mainly vasodilators, including Table 1 Classification and some causes of pulmonary hypertension Classification

Example

Pulmonary arterial hypertension

Primary pulmonary hypertension Drug-induced (e.g., anorexigens) Persistent pulmonary hypertension of the newborn Chronic obstructive pulmonary disease High altitude Adult respiratory distress syndrome Cystic fibrosis Pulmonary veno-occlusive disease Left-sided heart disease Pulmonary embolism

Hypoxic pulmonary hypertension

Pulmonary venous hypertension

Thromboembolic pulmonary hypertension Disorders of the pulmonary vasculature

Pulmonary capillary hemangiomatosis

Classification based on, and examples selected from, the World Health Organisation (1998) classification of pulmonary hypertension.

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Ca2 + -channel antagonists, prostacyclin (prostaglandin I2), and nitric oxide (NO) gas (for a review, see Wanstall & Jeffery, 1998). As vasodilators, each of these drug types acts by opposing any abnormal vasoconstriction. Since abnormal vasoconstriction becomes progressively less important and vascular remodeling progressively more important as the disease advances (Reeves et al., 1986), an alternative, and possibly more fruitful, approach may be to target pulmonary vascular remodeling.

2. Characteristics of pulmonary vascular remodeling Pulmonary vascular remodeling is characterised by thickening of all three layers of the blood vessel wall, viz., the adventitia, the media, and the intima. The thickening is due to hypertrophy (cell growth) and/or hyperplasia (prolifera-

Table 2 Some animal models of pulmonary hypertension Species

Model

Rats

Monocrotaline ± pneumonectomy Chronic hypoxia Balloon endarterectomy Genetic (Fawn-Hooded rat) Myocardial infarction Ligation of ductus arteriosus, in utero (model for persistent pulmonary hypertension of the newborn) High altitude (hypoxia) Thromboembolism Chronic hypoxia Chronic hypoxia

Sheep

Calves Dogs Mice Guinea pigs

This table includes those animal models of pulmonary hypertension that are referred to in the text.

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tion) of the predominant cell type within each of the layers (i.e., fibroblasts, smooth muscle cells, and endothelial cells), as well as increased deposition of extracellular matrix components (e.g., collagen, elastin, and fibronectin). Thickening of the media occurs consistently in arteries, at all levels of the pulmonary arterial tree, and less frequently in veins (Dingemanns & Wagenvoort, 1978). In addition, there is extension of new smooth muscle into the partially muscular and non-muscular peripheral arteries. This is due to the differentiation of precursor cells (viz., pericytes and ‘‘intermediate’’ cells, the latter being intermediate between pericytes and muscle cells in structure) into smooth muscle cells (Meyrick & Reid, 1980a), and is termed neomuscularisation. These various alterations in vascular structure are seen in both human pulmonary hypertension and animal models of the disease, and take place more rapidly than the remodeling of systemic arteries in systemic hypertension (Zhao & Winter, 1996). Remodeling of pulmonary arteries from rats with chronic hypoxic pulmonary hypertension is illustrated in Fig. 1 (main pulmonary artery) and Fig. 2 (intralobar pulmonary artery). An additional feature that is seen in some forms of pulmonary hypertension in humans (notably pulmonary arterial hypertension, Table 1) is a complex vascular struc-

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ture known as a plexiform lesion (Voelkel et al., 1997). Plexiform lesions typically appear in small pulmonary artery branches, immediately after their origin from a parent artery. The plexiform lesion comprises a dilated ‘‘sac’’ containing a plexus of capillary-like channels separated by proliferating cells (e.g., myofibroblasts) and atypical endothelial (or fibrillary) cells, which are embedded in an acellular matrix (Harris & Heath, 1986; Stewart, 1995; Cool et al., 1999). Another abnormality seen in some forms of pulmonary hypertension is the development of neointimal lesions, comprising smooth muscle cells and extracellular matrix, which are located on the luminal side of the internal elastic lamina (E.L.). In patients with pulmonary hypertension (Rabinovitch et al., 1980; Jones & Reid, 1995) and in pulmonary hypertensive rats [hypoxic and monocrotalinetreated rats (Hislop & Reid, 1974, 1976)], a decrease in arterial density has been reported. However, other studies in these rat models have not confirmed this observation, and it has been suggested that inappropriate fixation and perfusion conditions and measurement techniques may have given rise to false estimates of total vessel number (Kay et al., 1982; Finlay et al., 1986). The nature of these various structural abnormalities has been known for some time, but the mechanisms responsible

Fig. 1. Images of transverse sections of the vessel walls of (a) a normal main pulmonary artery from a control rat and (b) a remodelled main pulmonary artery from a chronically hypoxic rat (rat exposed to 10% oxygen for 4 weeks). The internal and external E.L. mark the boundaries of the media. Note that in the remodelled artery, there is thickening of both the adventitia (increased deposition of collagen) and the media (increases in smooth muscle and the number of E.L.). Scale =
100 mm. The vessels were viewed under a light microscope, and images were captured via a video camera linked to a computer. Unpublished data of T. K. Jeffery and J. C. Wanstall.

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Fig. 2. Images of transverse sections of (a) a normal intralobar pulmonary artery from the lung of a control rat and (b) a remodelled intralobar pulmonary artery from the lung of a chronically hypoxic rat (rat exposed to 10% oxygen for 4 weeks). The internal and external E.L. mark the boundaries of the media. Note that in the remodelled artery, there is marked thickening of the media due to an increase in smooth muscle. The adventitia is also thickened, due to increased deposition of collagen. Scale =
50 mm. The vessels were viewed under a light microscope, and images were captured via a video camera linked to a computer. Unpublished data of T. K. Jeffery and J. C. Wanstall.

for remodeling of the pulmonary vasculature have been the subject of more recent investigations and are discussed in Section 4.

3. Methods used to assess pulmonary vascular remodeling A variety of methods have been used to assess the different aspects of pulmonary vascular remodeling. Medial thickening of pulmonary arteries and neomuscularisation of normally non-muscular arteries can be assessed histologically in lung sections stained for smooth muscle (e.g., van Gieson’s stain) and elastin (e.g., Miller’s stain or Verhoeff’s stain), or immunolabelled with antibodies for smooth muscle a-actin. The conditions under which the lungs are fixed vary between laboratories. The lungs are generally distended with formalin (via the trachea, under a defined head of pressure) and are then immersed, while in an inflated state, in formalin for several days. Most, but not all, investigators perfuse the pulmonary circulation before inflating the lungs with formalin; physiological pressures are not always used for this arterial perfusion and the

perfusates vary, e.g., physiological salt solution (Jeffery & Wanstall, 1999), formalin (Di Carlo et al., 1995), or a warm barium /gelatin mixture (Hislop & Reid, 1976). Medial thickness of arteries in the histological sections is quantified by measuring wall thickness, as delineated by the internal and external E.L., and expressing it as a percentage of vessel diameter (Morrell et al., 1995b; Jeffery & Wanstall, 1999, 2001). In some studies, the percentage of smooth muscle cells within the media is quantified by labelling for smooth muscle a-actin (Takahashi et al., 1996a). The histological assessment of neomuscularisation involves counting all of the vessels with diameters < 50 mm in a complete lung section and determining the percentages of vessels that are fully muscularised, partially muscularised, and nonmuscularised (Hu et al., 1989; Eddahibi et al., 1998). Histological lung sections have also provided information on the total number of arteries in a given area of alveolar tissue and the ratio between numbers of arteries and alveoli (Hislop & Reid, 1976). An alternative, ‘‘functional’’ approach to assessing neomuscularisation is to obtain values for critical closing pressure from pressure /flow plots in perfused lungs (in situ or in vitro) (Bee & Wach, 1984; Jeffery & Wanstall, 1999).

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The intercept of the pressure /flow plot with the pressure axis (obtained by extrapolation) provides the critical closing pressure, i.e., the minimum intraluminal pressure required to keep the vessels in the alveolar region open in the face of opposing alveolar pressure together with any vascular tone (Wach et al., 1987). In normal lungs, vascular tone in the small arteries in this region is virtually non-existent due to the absence of vascular smooth muscle, and, hence, critical closing pressure is equivalent to alveolar pressure alone. However, if there is neomuscularisation (i.e., extension of new smooth muscle into the small, peripheral arteries), critical closing pressure is increased because it now represents the sum of alveolar pressure plus the pressure due to vascular tone (Bee & Wach, 1984). Both histological and biochemical techniques have been used to assess increases in extracellular matrix proteins. Histological methods include (1) qualitative assessment of lung sections specifically stained for collagen and elastin (Stenmark et al., 1987) and (2) quantification of the number of elastic lamellae in the vessel walls (Meyrick & Reid, 1980a). Biochemical approaches include determination of the hydroxyproline content of arteries or lungs as a measure of collagen; quantification of the incorporation of radiolabelled proline and valine into collagen and elastin, respectively; and assessment of collagen mRNA levels (Tozzi et al., 1989, 1994; Poiani et al., 1990; Kerr et al., 1984, 1987). Techniques that have been used to assess other aspects of pulmonary vascular remodeling include electron microscopy [used to detect alterations in endothelial cells, as well as other cell types (Rabinovitch et al., 1986; Meyrick & Reid, 1980a, 1980b)] and immunohistochemistry [e.g., 5-bromo-20-deoxyuridine labelling as an index of cell proliferation (Jones & Rabinovitch, 1996) and labelling with antibodies for von Willebrand factor as a marker for endothelial cells (Quinlan et al., 2000)].

4. Mechanisms of pulmonary vascular remodeling Pulmonary vascular remodeling occurs in response to a wide variety of stimuli, both physical (e.g., mechanical stretch, shear stress) and chemical (e.g., hypoxia, vasoactive substances, growth factors). Moreover, within any population of pulmonary vascular cells there is heterogeneity in their response, not only to the various growth promoting and proliferative stimuli, but also to mediators that inhibit growth (Wohrley et al., 1995; Dempsey et al., 1997; Frid et al., 1997; Wharton et al., 2000). For example, a greater proliferative response to platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) was observed in bovine smooth muscle cells from the outer media compared with those cells isolated from within the middle of the media (Dempsey et al., 1997). Also, prostacyclin inhibited proliferation of human smooth muscle cells from distal pulmonary arteries, but not from cells from proximal pulmonary arteries (Wharton et al., 2000). Cell

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heterogeneity, in relation to the responses of cells to remodeling stimuli, is discussed in detail in a review by Stenmark and Mecham (1997). The various physical and chemical stimuli for remodeling are reviewed individually in the following sections. 4.1. Mechanical factors It has long been considered that mechanical factors, such as increased transmural pressure, stretch, and shear stress (due to increased flow), contribute to the remodeling process. Changes that have been reported to occur in response to mechanical stimuli include increased extracellular matrix production [e.g., collagen and elastin (Tozzi et al., 1989)], smooth muscle cell hypertrophy (Kolpakov et al., 1995), and smooth muscle cell and fibroblast proliferation (Kolpakov et al., 1995). These changes may involve direct activation of stretch-sensitive ion channels (Kirber et al., 1992), Ca2 + influx (Bialecki et al., 1992) and /or increases in phosphatidylinositol metabolism (Kulik et al., 1991). Indirect mechanisms, via growth factors, may also be involved. For example, a recent study in pulmonary artery smooth muscle cells found increases in mRNA for insulin-like growth factor (IGF)-1 in response to mechanical stretch (Chaqour et al., 1999). 4.2. Hypoxia Hypoxia, which is particularly important in hypoxic pulmonary hypertension (Tables 1 and 2), causes pulmonary vasoconstriction. This leads to increases in pressure, tension, and shear stress, which are, in themselves, stimuli for remodeling (see Section 4.1). In addition, hypoxia inhibits the release of antimitogenic factors [e.g., prostacyclin (Madden et al., 1986)] and increases the production and /or release of mitogenic factors from pulmonary artery smooth muscle cells [e.g., interleukin-1 (Cooper & Beasley, 1999) and vascular endothelial growth factor (VEGF) and bFGF (Ambalavanan et al., 1999a)] and endothelial cells [e.g., endothelin (ET)-1 and PDGF (Dawes et al., 1994)]. The increased production of mitogenic factors by hypoxia has been proposed to occur by activation of a specific oxygen sensor (probably a haem-containing protein), leading to induction of various transcription factors (e.g., hypoxia-inducible transcription factor-1, activating protein-1, and nuclear factor-kB) that control the transcriptional activation of genes encoding for growth factors and other mediators of mitogenesis (for a review, see Faller, 1999). 4.3. Mediators Mediators that have been implicated in inducing pulmonary vascular remodeling include various vasoactive substances better known for their vasoconstrictor / vasodilator effects [e.g., angiotensin II, ET-1, 5-hydroxytryptamine

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(5-HT), as well as specific growth factors, inflammatory mediators, and components of the extracellular matrix. 4.3.1. Angiotensin II Direct evidence for the ability of angiotensin II to cause growth /proliferation of pulmonary artery smooth muscle cells has been obtained from in vitro experiments where angiotensin II was found to cause hypertrophy of cells from large conduit pulmonary arteries [humans (Rothman et al., 1994; Morrell et al., 1999)] and also proliferation of cells from small, resistance pulmonary arteries (Rothman et al., 1994). This effect of angiotensin II is mediated via angiotensin II Type I (AT1), rather than AT2, receptors (Morrell et al., 1999). The concept that angiotensin II has a role in pulmonary hypertension is supported by findings that the expression of angiotensin-converting enzyme (ACE) is increased in the endothelial layer of small, as well as elastic, pulmonary arteries (Morrell et al., 1995a; Schuster et al., 1996; Orte et al., 2000). Furthermore, in hypoxic, but not monocrotaline-treated (Cassis et al., 1997), pulmonary hypertensive rats, both angiotensin II binding and the number of AT1 receptors are increased (Zhao et al., 1996; Chassagne et al., 2000). Indirect support for angiotensin II-mediating pulmonary vascular remodeling comes from in vivo studies in which treatment of animals with either an ACE inhibitor or an angiotensin receptor antagonist leads to inhibition of pulmonary vascular remodeling (see Section 6.1). 4.3.2. Endothelin-1 In vitro, ET-1 is weakly mitogenic for pulmonary vascular smooth muscle cells, but in the presence of other growth factors or serum, a more pronounced effect is seen. Thus, ET-1 can be regarded as a co-mitogen (Hassoun et al., 1992; Janakidevi et al., 1992; Zamora et al., 1993). This proliferative effect of ET-1 is considered to be mediated by ETA, rather than ETB, receptors (Zamora et al., 1993). ET-1 has also been shown to stimulate proliferation of pulmonary fibroblasts (Peacock et al., 1992) and to induce pulmonary artery smooth muscle cells to produce Type V collagen, which has been shown to be increased in hypoxic rats (Mansoor et al., 1995). Thus, ET-1 has properties relevant to the remodeling process. Moreover, there is considerable circumstantial evidence implicating this mitogenic peptide in the pathology of pulmonary hypertension. In humans with the disease, there are increases in (1) plasma levels of ET-1 (e.g., Yoshibayashi et al., 1991; Stewart et al., 1991; Cody et al., 1992; Rosenberg et al., 1993; Tutar et al., 1999), (2) ET-1 peptide in lung or pulmonary artery endothelial cells (Giaid et al., 1993; Cacoub et al., 1993), and (3) expression of ET-converting enzyme (ECE) in lungs (Giaid, 1998). Comparable changes in all of these parameters have also been described in animal models of the disease

(Zamora et al., 1996; Takahashi et al., 1998; Bialecki et al., 1999; Aguirre et al., 2000; Kim et al., 2000). In addition, in animals, there are reports of increases in mRNA for preproET-1 and ET-1 in lungs (Elton et al., 1992; Stelzner et al., 1992; Ivy et al., 1998; Nakanishi et al., 1999) and for ETA and ETB receptors in pulmonary arteries (Soma et al., 1999) and lung homogenates (Li et al., 1994). Indirect evidence for a role for ET-1 in pulmonary vascular remodeling has been derived from experiments in which ET receptor antagonists or ECE inhibitors have been administered to pulmonary hypertensive animals (see Section 6.2). 4.3.3. 5-Hydroxytryptamine Although 5-HT is best known for its vasoconstrictor / vasodilator properties, it is becoming increasingly apparent that it also has a role in vascular smooth muscle cell hyperplasia and hypertrophy (Fanburg & Lee, 1997). The mitogenic effect of 5-HT has been demonstrated in bovine and rat pulmonary artery smooth muscle cells, but not endothelial cells or fibroblasts (Lee et al., 1994; Pitt et al., 1994). 5-HT not only has a direct mitogenic effect, but it also acts synergistically with other growth factors, including PDGF, epidermal growth factor (EGF), and fibroblast growth factor (Lee et al., 1991). It now appears that the mitogenic effect is dependent on prior uptake of 5-HT into the cell by its specific transporter (Eddahibi et al., 1999, 2000a). Whether or not activation of cell surface receptors also mediates mitogenesis appears to depend on the species; i.e., in rats, activation of 5-HT2A receptors are involved (Pitt et al., 1994), but in bovine pulmonary artery smooth muscle cells, mitogenesis is not receptor-mediated (Lee et al., 1991). A pathophysiological role for 5-HT in pulmonary hypertension is indicated by the fact that platelet and plasma levels of 5-HT are elevated in primary pulmonary hypertension in humans (Herve et al., 1990, 1995). Also, various appetite suppressant drugs such as fenfluramine, which have been associated with epidemics of pulmonary hypertension, cause elevated plasma levels of 5-HT (Brenot et al., 1995). Furthermore, Fawn Hooded rats, which have a deficiency in the storage of 5-HT by platelets, have a genetic predisposition to pulmonary hypertension (Gonzalez et al., 1998). These rats rapidly develop severe pulmonary hypertension if bred in the conditions of mild, high-altitude hypoxia existing in Denver, Colorado, USA (altitude, 5200 feet) (Sato et al., 1992), but they also become pulmonary hypertensive if bred in normoxic conditions (Kentera et al., 1988). 4.3.4. Growth factors Numerous growth factors are important in cell division and replication, although there are relatively few studies in which this has been demonstrated in cells specifically from the pulmonary vasculature. PDGF and bFGF cause prolif-

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eration, but not hypertrophy, of rat pulmonary artery smooth muscle cells (Rothman et al., 1994; Wang et al., 2000). IGF-1 also causes proliferation of bovine pulmonary vascular smooth muscle cells (Dempsey et al., 1990). Elevations in a variety of growth factors and/or their mRNA have been reported in pulmonary hypertension, including PDGF-A or -B (Arcot et al., 1993; Berg et al., 1998; Katayose et al., 1993), VEGF (Tuder et al., 1995; Christou et al., 1998; Shehata et al., 1999; Eddahibi et al., 2000b), transforming growth factor (TGF)-b (Perkett et al., 1990; Botney et al., 1992; Arcot et al., 1993), bFGF (Arcot et al., 1995), IGF-1 (Perkett et al., 1992), and EGF (Gillespie et al., 1989). These findings suggest a likely role for each of these growth factors in the remodeling of pulmonary arteries in pulmonary hypertension. 4.3.5. Inflammatory cytokines It has been suggested that inflammation, with the associated increase in inflammatory cytokines (e.g., interleukins, tumour necrosis factor), is an important factor in the development of pulmonary vascular remodeling (Voelkel & Tuder, 1995). In patients with primary pulmonary hypertension, the presence of perivascular inflammatory cells has been noted in association with plexiform lesions (Tuder et al., 1994), and serum concentrations of the inflammatory cytokines, interleukin-1 and interleukin-6, were found to be increased (Humbert et al., 1995). Interleukin-1 has been shown to be mitogenic in human and rat vascular smooth muscle cells, including those from the pulmonary artery (Libby et al., 1988; Cooper & Beasley, 1999; Wang et al., 2000). 4.4. Tenascin-C The extracellular matrix component tenascin-C (a glycoprotein) may contribute to proliferation by amplifying the proliferative effects of growth factors such as bFGF and EGF (Jones & Rabinovitch, 1996; Cowan et al., 2000b). Moreover, the expression of tenascin-C has been suggested to be increased by other factors that stimulate remodeling, viz., mechanical stress and serine elastase (Jones et al., 1997), and has been shown to be elevated in animal (Cowan et al., 1999) and clinical (Jones et al., 1997) pulmonary hypertension. Further evidence supporting a role for tenascin-C in pulmonary vascular remodeling comes from a recent study in which the further progression of pulmonary artery thickening was prevented with transfection of tenascin-C antisense to hypertrophied rat pulmonary arteries in culture (Cowan et al., 2000b). 4.5. Role of the endothelium The endothelium plays a critical role in pulmonary vascular remodeling. First, it acts as a physical barrier, protecting the underlying smooth muscle from blood-borne

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mediators (Dawes et al., 1995). Second, it detects both alterations in mechanical forces (Section 4.1; Tozzi et al., 1989) and reductions in oxygen tension [hypoxia (see Section 4.2; for a review, see Faller, 1999)]. Third, many of the mediators of remodeling described in Section 4.3 are produced and released from the endothelium (Vender et al., 1987; Vender, 1992; Dawes et al., 1994). In addition to being the source of growth factors /mediators, two important antimitogenic substances are derived from the endothelium; namely, NO and prostacyclin. There is evidence that both NO and prostacyclin have antimitogenic effects in pulmonary artery smooth muscle cells, i.e., they inhibit DNA synthesis and proliferation (Thomae et al., 1995; Ambalavanan et al., 1999b; Wharton et al., 2000). These antimitogenic effects of NO and prostacyclin may be via the production of cyclic GMP (cGMP) (Thomae et al., 1995; Ambalavanan et al., 1999b) and cyclic AMP (cAMP) (Wharton et al., 2000), respectively. Also, both NO and prostacyclin have been reported to inhibit the production of other growth factors (e.g., ET-1, PDGF) from endothelial or smooth muscle cells (Kourembanas et al., 1993; Wort et al., 2000). It is, therefore, significant that pulmonary hypertension is associated with impaired production of these two important endothelium-derived antimitogenic substances (prostacyclin: Badesch et al., 1989; Christman et al., 1992; Tuder et al., 1999; NO: Cremona et al., 1994; Dollberg et al., 1995). The decrease in NO represents a reduction in stimulated, but not basal, NO (Cremona et al., 1994). 4.6. Role of serine elastase Fragmentation of the internal E.L. has been observed in animal models of pulmonary hypertension (TodorovichHunter et al., 1992) and also in lung biopsies from pulmonary hypertensive patients (Rabinovitch et al., 1986). Elastase activity has been shown to be increased in both hypoxic and monocrotaline rat models of pulmonary hypertension (Maruyama et al., 1991; Todorovich-Hunter et al., 1992). An association between this heightened elastase activity and the increased number of breaks in the internal E.L. has been observed, suggesting a cause and effect relationship (Todorovich-Hunter et al., 1992). The increase in elastase activity has been shown to lead to the release of extracellular matrix-bound growth factors, such as bFGF, and it is proposed that this is the mechanism whereby increased elastase activity contributes to vascular remodeling (Thompson & Rabinovitch, 1996). 4.7. Intracellular signalling mechanisms A number of intracellular signalling mechanisms, including activation of the phosphatidylinositol pathway, elevations in intracellular Ca2 + , and activation of protein kinase C (PKC) and mitogen-activated protein kinases (MAPK) have been implicated in remodeling, although data specifically in pulmonary vascular cells are limited. In pulmonary

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artery smooth muscle cells, activation of PKC has been reported to cause proliferation (Dempsey et al., 1991) and to directly mediate the effects of some growth factors (e.g., PDGF) (Dempsey et al., 1990). For other growth factors / stimuli that do not directly activate PKC, it has been shown that a background of PKC activation is either a prerequisite for the remodeling effect (e.g., hypoxia) (Dempsey et al., 1991) or greatly augments this effect (e.g., IGF-1, i.e., acts synergistically) (Dempsey et al., 1990). PKC activation is also important in the proliferation of fibroblasts. In these cells, in contrast to smooth muscle cells, hypoxia causes proliferation in the absence of activators of PKC (e.g., other growth factors) (Das et al., 2000). However, activation of PKC magnifies the response. It is possible that in fibroblasts there is already a basal level of PKC activation. Also, the isoforms of PKC that are important may differ in smooth muscle cells and fibroblasts (Xu et al., 1997; Das et al., 2000). Another intracellular transduction mechanism that has been implicated is the MAPK pathway. There is evidence in human pulmonary artery smooth muscle cells linking the growth-promoting effects of angiotensin II with MAPK activation (Morrell et al., 1999). Also, it has been shown in canine pulmonary artery smooth muscle cells that ET-1 activates various forms of MAPK, viz. extracellularregulated kinases 1 and 2, p38, and Jun N-terminal kinase-1, and that the activation of Jun N-terminal kinase-1 MAPK leads to phosphorylation of the nuclear transcription factor c-Jun (Yamboliev et al., 1998).

comparable smooth muscle, but a larger lumen-to-wall ratio. This can be predicted from the mathematical models developed by Folkow in relation to systemic hypertension (Folkow et al., 1970; Folkow, 1971). Third, the alterations in the extracellular matrix, particularly the deposition of collagen in the media and adventitia, result in reduced arterial compliance (distensibility) (Tozzi et al., 1994). The high compliance of the normal pulmonary vascular bed (together with vessel recruitment) contributes to the ability of the pulmonary circulation to absorb considerable increases in cardiac output (e.g., on exercise), with only moderate increases in pulmonary artery pressure. However, this unique property of the pulmonary circulation may be compromised when compliance is reduced due to pulmonary vascular remodeling. Also when the compliance of conduit pulmonary arteries is decreased, right ventricular pressure increases (Zuckerman et al., 1991). Furthermore, a larger percentage of the stroke volume is delivered to the capillaries during systole, and this gives rise to increases in downstream shear stress (Reeves et al., 1995), a stimulus for further remodeling. Finally, the presence of significant structural remodeling reduces the effectiveness of pulmonary vasodilators in reducing pulmonary vascular resistance and pressure (Reeves et al., 1986), thus emphasising the importance of finding new therapies that target remodeling.

5. Consequences of pulmonary vascular remodeling

There are various animal studies in which different drug types have been investigated for their effects on pulmonary vascular remodeling in pulmonary hypertension. Some of these drug types have also been used in clinical trials. From the clinical trials, information on pulmonary artery pressure and /or survival is available, but data specifically on pulmonary vascular remodeling are lacking. One exception to this generalisation is the study of Wilkinson et al. (1988) in which pulmonary vascular structure was described in post mortem lung samples from patients with hypoxic pulmonary hypertension (hypoxic cor pulmonale), some of whom had received oxygen therapy. The results of the study were negative, in as much as supplemental oxygen was found to have had no beneficial effect on pulmonary vascular structure. The advent of intravascular ultrasound techniques, which can detect pulmonary vascular thickness, may allow studies of the effects of therapeutic interventions on pulmonary vascular remodeling in humans to be carried out in the future. However, in light of the current lack of relevant information in humans, the following review of the literature on drug therapy is confined to studies in pulmonary hypertensive animals. Moreover, this review excludes those animal studies in which pulmonary vascular remodeling was not specifically examined.

There are several physiological consequences of pulmonary vascular remodeling. First, the increase in smooth muscle in the media of muscular arteries, and also neomuscularisation of distal arteries, results in exaggerated increases in wall tension in response to contractile stimuli (i.e., vasoconstrictor agents, hypoxia). This is a nonspecific effect that simply reflects the increased amount of smooth muscle in the vessel wall, and is independent of any specific changes in sensitivity to individual constrictor agents (Emery et al., 1981; Twarog et al., 1988; Jeffery & Wanstall, 1999). Second, medial thickening and intimal proliferation result in narrowing of the lumen of individual pulmonary arteries (Rounds & Hill, 1984). According to Poiseuille’s law, even quite small decreases in overall arterial luminal diameter caused by pulmonary vascular remodeling will lead to pronounced increases in pulmonary vascular resistance and corresponding increases in pulmonary artery pressure, even under basal conditions. Moreover, in a remodelled vessel where there is a decrease in the lumen-to-wall ratio, any active shortening of the smooth muscle (induced by a vasoconstrictor) will cause a greater increase in vascular resistance than that caused by the same degree of smooth muscle shortening in an artery with

6. Therapeutic interventions for the inhibition of pulmonary vascular remodeling

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6.1. Angiotensin-converting enzyme inhibitors and angiotensin receptor antagonists Drugs that prevent either the production or the action of the potent mitogen /growth factor angiotensin II, i.e., ACE inhibitors and angiotensin receptor antagonists, respectively, have been examined in both hypoxic and monocrotalinetreated rats. In chronically hypoxic rats (2 – 4 weeks of hypoxic exposure), various ACE inhibitors have been shown to inhibit pulmonary vascular remodeling associated with the development of pulmonary hypertension. In these studies, medial thickening and muscularisation of vessels associated with alveolar ducts and alveolar walls were attenuated when rats were treated for the entire hypoxic exposure (Zakheim et al., 1975; Morrell et al., 1995b; Nong et al., 1996; van Suylen et al., 1998; Jeffery & Wanstall, 1999). However, in one study with the ACE inhibitor cilazapril, medial thickening of vessels was totally prevented (Clozel et al., 1991). Furthermore, quinapril was shown to reduce bromodeoxyuridine staining (an indicator of cell proliferation) in the media of pulmonary arteries of hypoxic rats (Nong et al., 1996). The anti-remodeling effects of ACE inhibitors may vary, depending on the size /location of the pulmonary vessel examined. For example, perindopril reduced medial thickening of intralobar vessels 30– 500 mm outer diameter (o.d.), but had no effect on thickening of the main pulmonary artery (2– 3 mm o.d.) (Jeffery & Wanstall, 1999). The beneficial effect of ACE inhibitor therapy in pulmonary hypertension is due to a decrease in the amount of angiotensin II rather than an increase in the amount of bradykinin, since treatment with a bradykinin receptor antagonist (CP 0597) did not prevent the beneficial effects of captopril (Morrell et al., 1995b). Attenuation of the changes in pulmonary vascular structure was associated with a corresponding reduction in pulmonary artery pressure of ~ 20%. It appears that doses of ACE inhibitors that have a beneficial effect on pulmonary vascular remodeling are much higher than those used in diseases of the systemic circulation. For example, in pulmonary hypertension, the minimum effective dose of perindopril required to inhibit pulmonary vascular remodeling was 10 mg /kg /day, whereas medial thickening of descending thoracic aorta from rats with experimental systemic hypertension was reduced at a dose of 1 mg /kg / day (Levy et al., 1989). The effect of ACE inhibitor treatment has also been examined in intervention studies, where treatment with perindopril or quinapril was started 7 or 12 days after the commencement of hypoxic exposure. In these studies, the progression of pulmonary vascular remodeling was attenuated, and this was associated with a corresponding reduction in pulmonary artery pressure (Nong et al., 1996; T. K. Jeffery & J. C. Wanstall, unpublished). In monocrotaline-treated rats, there are conflicting reports on the effects of ACE inhibitor therapy. van Suylen

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et al. (1998) reported that treatment of rats with captopril (12 mg /kg /day) for 4 weeks had no effect on medial thickening of arteries 30 –200 mm o.d. or neomuscularisation of distal pulmonary arteries or on pulmonary artery pressure, whereas Molteni et al. (1985) reported that treatment with captopril (60 mg /kg /day) for 6 weeks led to reductions in the degree of neomuscularisation of peripheral arteries. These contrasting effects of captopril may reflect the different doses of captopril used, and suggest that the dose of captopril used in the study of van Suylen et al. (1998) was possibly too low for a beneficial effect. The nonthiol ACE inhibitors CGS 13945 and CGS 16617 have also been effective in inhibiting pulmonary vascular remodeling when given to monocrotaline-treated rats for 6 weeks (Molteni et al., 1988). In another model of pulmonary hypertension, monocrotaline plus pneumonectomy, quinapril treatment, either for the entire period or after pulmonary hypertension had developed, reduced the formation of neointimal lesions, but had no effect on medial or adventitial hypertrophy (Okada et al., 1998). Pulmonary artery pressure was also reduced. The effect of angiotensin receptor antagonists has also been examined in pulmonary hypertension. The AT1 receptor antagonists losartan and GR138950C inhibited medial hypertrophy and neomuscularisation in hypoxic animals (Morrell et al., 1995b; Zhao et al., 1996), and in the monocrotaline-plus-pneumonectomy model of pulmonary hypertension, losartan also reduced neointimal formation (Okada et al., 1998). However, in monocrotaline-treated rats, with or without pneumonectomy, losartan had no beneficial effect on pulmonary vascular remodeling (Cassis et al., 1992; Okada et al., 1998), even at doses higher than that used in hypoxic animals (40 mg /kg /day vs. 20 mg /kg /day) (Morrell et al., 1995b; Okada et al., 1998). The AT2 receptor antagonist PD123319, when administered to hypoxic rats, had no effect on the development of pulmonary hypertension (Morrell et al., 1995b). 6.2. Endothelin antagonists and endothelin-converting enzyme inhibitors BQ-123, a selective ETA receptor antagonist, was the first ET receptor antagonist to be examined in pulmonary hypertensive animals (Miyauchi et al., 1993). Medial thickening and neomuscularisation were successfully inhibited with continuous infusion of this drug (1 –14.3 mg /day), and this has been shown in at least three different models of pulmonary hypertension, viz., in hypoxic (Bonvallet et al., 1994; Dicarlo et al., 1995) and monocrotaline-treated (Miyauchi et al., 1993) rats and in sheep in which the ductus arteriosus had been ligated (model of persistent pulmonary hypertension of the newborn) (Ivy et al., 1997). Inhibition of pulmonary vascular remodeling by BQ-123 was associated with concomitant reductions in pulmonary artery pressure. In hypoxic rats, the rise in pulmonary artery pressure was completely prevented, but

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only with the highest dose of BQ-123 studied (9.6 mg /day) (Bonvallet et al., 1994; Dicarlo et al., 1995). Since BQ-123 is a peptide that is not orally active and has a short duration of action requiring continuous intravenous infusion, other ETA receptor antagonists (nonpeptide) that are orally active and that can be administered once daily have also been examined in pulmonary hypertension (Table 3). Medial thickening of pulmonary arteries < 100 mm o.d. was attenuated or prevented with all of these drugs, with the exception of LU135252 (Prie et al., 1997; Nguyen et al., 2000). The combined ETA /ETB receptor antagonist bosentan has also been examined in pulmonary hypertension. Like the ETA receptor antagonists, treatment of hypoxic and monocrotaline rats with bosentan had beneficial effects on medial thickening and muscularisation of distal pulmonary arteries, as well as reducing pulmonary artery pressure (Chen et al., 1995; Eddahibi et al., 1995; Hill et al., 1997). Recently, Kim et al. (2000) demonstrated in a model of chronic thromboembolic pulmonary hypertension (dogs administered ceramic beads) that treatment with bosentan not only reduced medial thickening of pulmonary arteries, but also reduced adventitial thickening and prevented intimal fibrosis and peripheral neomuscularisation. In light of the results with ETA-selective antagonists, it is likely that the efficacy of bosentan on pulmonary vascular remodeling resides in its effect on ETA receptors. ET receptor antagonists have also been examined using an intervention strategy in which the drug was commenced once pulmonary hypertension had developed. Encouragingly, medial thickening of pulmonary arteries from hypoxic rats was not only attenuated, but actually was reversed toward values seen in normoxic animals when rats were treated with A127722, sitaxsentan, or bosentan (Chen et al., 1995, 1997; Tilton et al., 2000). In addition, any further rise in pulmonary artery pressure was either prevented or reversed. Close examination of these various studies with ET receptor antagonists suggests that their effectiveness may vary, depending on the model of pulmonary hypertension studied. With BQ-123, medial thickening of pulmonary arteries (50 – 100 mm o.d.) was completely prevented in

Table 3 Orally active ETA receptor antagonists used in various rat models of pulmonary hypertension ETA receptor antagonist

Pulmonary hypertension model

Reference

A127722 CI 1020 LU135252

Hypoxia Hypoxia Monocrotaline Myocardial infarction Hypoxia Hypoxia

Chen et al., 1997 Sheedy et al., 1998 Prie et al., 1997 Nguyen et al., 2000 Underwood et al., 1998 Tilton et al., 2000

Monocrotaline Hypoxia

Tilton et al., 2000 Bialecki et al., 1999

SB 217242 Sitaxsentan (TBC11251) ZD 1611

hypoxic rats, but only attenuated in monocrotaline-treated rats and in the sheep model of persistent pulmonary hypertension of the newborn. Furthermore, muscularisation of distal pulmonary vessels adjacent to alveolar ducts and alveolar walls of pulmonary arteries was reduced in both hypoxic and monocrotaline-treated rats, but this was not seen in the sheep model. Higher doses of bosentan and sitaxsentan were required to inhibit remodeling in monocrotaline-treated, compared with hypoxic, rats (Chen et al., 1995; Eddahibi et al., 1995; Hill et al., 1997; Tilton et al., 2000). These findings suggest that ET receptor antagonists most readily inhibit remodeling associated with hypoxic exposure. It is interesting to note that studies on LU135252, the only drug to be described as ineffective, were confined to monocrotaline (Prie et al., 1997) and myocardial infarction (Nguyen et al., 2000) models of pulmonary hypertension, i.e., hypoxic rats were not studied. An ECE inhibitor, FR901533, has also been examined for its effects in monocrotaline-induced pulmonary hypertensive rats (Takahashi et al., 1998). In those pulmonary vessels that were examined ( < 100 mm o.d.), medial thickening was attenuated, as was the rise in right ventricular systolic pressure (correlates to pulmonary artery pressure). 6.3. Nitric oxide The effects of NO have been studied using (1) inhaled NO gas, (2) NO donor drugs, (3) L-arginine (the precursor of NO), and (4) gene transfer of NO synthase. Inhalation of NO by rats exposed to chronic hypoxia has been demonstrated to inhibit medial thickening of pulmonary arteries (15 – 1000 mm o.d.), as well as to attenuate muscularisation of distal pulmonary arteries (i.e., those adjacent to alveolar ducts and alveolar walls) (Kouyoumdjian et al., 1994; Roberts et al., 1995; Roos et al., 1996; Horstman et al., 1998). These anti-remodeling effects of NO occurred in both newborn and adult rats, with a greater effect seen in newborns. In a dose-response study carried out by Horstman et al. (1998), a dose of NO as low as 50 ppb was able to attenuate remodeling, although higher doses were more effective. Interestingly, in those studies in which pulmonary artery pressure was measured, the beneficial effects on remodeling were not accompanied by reductions in pressure when recorded after the cessation of NO administration (Kouyoumdjian et al., 1994; Roos et al., 1996). However, if recorded during inhalation of NO, reductions in pulmonary artery pressure were seen (Kouyoumdjian et al., 1994), indicative of a vasodilator effect of NO. It is possible, therefore, that on cessation of NO administration, rebound pulmonary vasoconstriction occurred, masking any reduction in pressure that would have resulted from the attenuation of remodeling. As with other anti-remodeling drugs, the effectiveness of NO seems to vary, depending on the model of pulmonary hypertension examined. In monocrotaline-treated rats, inhalation of NO had no effect on remodeling (Maruyama

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et al., 1997; Horstman et al., 1998). Even with doses as high as 40 ppm [i.e., 3 orders of magnitude higher than those used in hypoxic rats (see above)], the only effect was a small reduction in neomuscularisation of alveolar duct vessels (Maruyama et al., 1997). In contrast to these findings in adult rats, in newborn rats treated with monocrotaline, vascular remodeling was inhibited with administration of NO (Roberts et al., 2000). NO donor drugs provide an alternative source of NO. The NO donor molsidomine has been found to attenuate medial thickening of pulmonary arteries 15– 150 mm o.d., as well as to prevent the increase in pulmonary artery systolic pressure when administered to hypoxic rats (Mathew et al., 1997). Treatment of both hypoxic and monocrotaline-treated rats with the NO precursor L-arginine (500 mg /kg /day) inhibited medial thickening and neomuscularisation, and this was associated with a reduction in pulmonary artery pressure (Mitani et al., 1997). It should be noted that no measures of NO production were incorporated in the protocol; the assumption that L-arginine was acting by modifying endogenous NO production was based on the observation that D-arginine was ineffective. The beneficial effect of L-arginine observed in monocrotaline-treated rats is surprising in light of the lack of effect of NO gas in adult rats in this model of pulmonary hypertension (see above). In the same study, when monocrotaline-treated rats with existing pulmonary hypertension were treated with L-arginine, i.e., when an intervention rather than a prevention regimen was used, neomuscularisation was reduced, but there was no effect on medial thickening or pulmonary artery pressure. A novel approach for increasing NO in the pulmonary vasculature is via gene transfer of NO synthase, the enzyme responsible for the production of NO from L-arginine. In hypoxic rats, the transfer by aerosol of an adenoviral vector containing the gene for inducible NO synthase was found to decrease neomuscularisation of small pulmonary arteries and to reduce both pulmonary vascular resistance and pulmonary artery pressure (Budts et al., 2000). The mechanism whereby NO inhibits pulmonary vascular remodeling is unclear. There are at least two theoretical possibilities; namely, (1) reductions in pressure /shear stress, reflecting the vasodilator properties of the drug, and / or (2) a direct antimitogenic effect. The latter possibility is supported by in vitro studies, where NO inhibited pulmonary artery smooth muscle cell proliferation (Thomae et al., 1995; Ambalavanan et al., 1999b). This possibility is further supported by the finding that in newborn rats exposed to hypoxia, where there was pulmonary vascular remodeling without any elevation in pulmonary artery pressure, the remodeling was inhibited by NO (Roberts et al., 2000). Any direct effect on smooth muscle proliferation may be via inhibition of the expression /production of ET-1 and /or PDGF (Kourembanas et al., 1993; Junbao et al., 1999). Another possible mechanism is inhibition of serine elastase

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(Mitani et al., 2000). These processes probably involve cGMP, since it has been shown that this cyclic nucleotide can inhibit pulmonary artery smooth muscle cell proliferation (Thomae et al., 1995; Ambalavanan et al., 1999b) and that inhibition of elastase activity by NO is cGMP-dependent (Mitani et al., 2000). 6.4. Phosphodiesterase inhibitors Since cGMP has antimitogenic properties (see Section 6.3), drugs that prevent the breakdown of this cyclic nucleotide by phosphodiesterase (PDE) should theoretically inhibit pulmonary vascular remodeling. Of the many different isozymes of PDE, PDE 5 is specific for cGMP (i.e., it has no action on cAMP) and is found in the pulmonary vasculature. Moreover, increases in PDE 5 activity have been reported in lungs from hypoxic pulmonary hypertensive rats (MacLean et al., 1997). Administration of the selective PDE 5 inhibitors E-4010 and 1,3-dimethyl-6(2-propoxy-5-methane sulphonylamidophenyl)-pyrazolo[3, 4-d]pyrimidin-4-(5H)-one to hypoxic and /or monocrotaline-treated rats led to attenuation of pulmonary artery medial thickening and neomuscularisation (Takahashi et al., 1996b; Eddahibi et al., 1998; Hanasato et al., 1999; Kodama & Adachi, 1999). These drugs produced no adverse effects on systemic artery pressure, even though they were administered orally or by i.v. infusion (i.e., not directly into the lung). Thus, these drugs, at the doses used, did not cause general vasodilation. Both of these PDE 5 inhibitors reduced pulmonary artery pressure, and in monocrotaline-treated rats, the survival rate was increased following treatment with E4010 (Kodama & Adachi, 1999). It is possible that treatment with a combination of NO and a PDE 5 inhibitor (which both increase cGMP, but by different mechanisms) may have a greater effect on remodeling than treatment with either drug alone, particularly as NO and a PDE 5 inhibitor have been shown to act synergistically as pulmonary vasodilators in vivo (Nagamine et al., 2000). 6.5. Prostacyclin In humans, the introduction of therapy with prostacyclin (i.v. or inhaled) represented a major breakthrough in the treatment of pulmonary hypertension (for reviews, see McLaughlin & Rich, 1998; Wanstall & Jeffery, 1998). Prostacyclin initially was used for its vasodilator properties, but there is the suggestion that the beneficial effects in humans also reflect an anti-remodeling action (McLaughlin et al., 1998). Two major disadvantages of prostacyclin are its short half-life, necessitating continuous infusion, and the high cost of the drug. This has led to the search for alternative approaches such as increasing endogenous production of prostacyclin by augmenting the enzyme responsible for its synthesis. Recently, Geraci et al. (1999) found that in transgenic mice with over-expression of pulmonary prostacyclin synthase, the development of pulmonary hyper-

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tension following 5 weeks of exposure to hypoxia was prevented; i.e., there was reduced medial thickening of pulmonary arteries and reduced right ventricular systolic pressure compared with the data in hypoxic wild-type mice. Nagaya et al. (2000) found that in monocrotaline-treated rats, intratracheal transfer of prostacyclin synthase via a hemagglutinating virus of Japan-liposome led to attenuation of medial thickening of pulmonary arteries (25 –100 mm o.d.), with reductions in pulmonary artery pressure (12%) and pulmonary vascular resistance (23%). 6.6. Ca2+-channel antagonists In both hypoxic and monocrotaline-treated rats, Ca 2 + -channel antagonist therapy has had beneficial effects on pulmonary vascular remodeling, with reductions in medial thickening and neomuscularisation observed (Stanbrook et al., 1984; Michael et al., 1986; Inoue et al., 1993; Takahashi et al., 1996a; Jeffery & Wanstall, 2001). The particular Ca2 + -channel antagonists that have been tested are the dihydropyridines nifedipine, nitrendipine and amlodipine. In hypoxic rats, there was no reduction in pulmonary artery pressure (Stanbrook et al., 1984; Michael et al., 1986). In contrast, in monocrotaline-treated rats, pulmonary artery pressure was reduced (Takahashi et al., 1996a). The reason for this difference is not known, but it may reflect the fact that in the absence of drug treatment, pulmonary artery pressure was much higher in monocrotaline-treated rats than in hypoxic rats. The mechanism of the anti-remodeling effect of Ca2 + -channel antagonists could arguably be secondary to reductions in pressure/stress, due to their vasodilator properties. However, it is thought that these drugs also inhibit the mitogenic effects of various growth factors. There is considerable evidence, particularly from in vitro studies in cells from systemic vessels, to support this view. In both rat and human vascular smooth muscle cells, as well as fibroblasts, DNA synthesis and smooth muscle cell proliferation in response to a variety of growth factors (e.g., bFGF, EGF, PDGF, and angiotensin II) is prevented by Ca2 + -channel antagonists (Roe et al., 1989; Block et al., 1989; Ko et al., 1993; Herembert et al., 1995; Kataoka et al., 1997; Stepien et al., 1997, 1998). Although the mitogenic action of some growth factors is dependent on the influx of extracellular Ca2 + , it has been proposed that the antimitogenic properties of Ca2 + -channel antagonists are, at least in part, independent of their action on voltage-operated Ca2 + channels. For example, it has been demonstrated that various Ca2 + -channel antagonists inhibit (1) intracellular release of Ca2 + by a direct action on phosphoinositide turnover (Roe et al., 1989; Block et al., 1989), (2) PKC (Kataoka et al., 1997), (3) the expression of early growth response genes (Stepien et al., 1997), and (4) mRNA expression of growth factors (e.g., TGF-b) (Kim et al., 1995).

6.7. Heparin Administration of heparin to hypoxic animals of various species (i.e., mice, rats, guinea pigs, and calves) has been shown to inhibit pulmonary vascular remodeling. Medial thickening of vessels was reduced in all animals (Hales et al., 1983; Fasules et al., 1987a; Hassoun et al., 1989; Thompson et al., 1994), except in rats, where neomuscularisation rather than medial thickening was inhibited (Hu et al., 1989). The effects of heparin on pulmonary artery pressure are also variable, depending on the species of animal examined, i.e., a reduction in pulmonary artery pressure was observed in mice and guinea pigs (Hales et al., 1983; Hassoun et al., 1989), but not in newborn calves and rats (Fasules et al., 1987a; Hu et al., 1989). In contrast to the studies in hypoxic animals, heparin had no beneficial effect on pulmonary vascular remodeling or pulmonary artery pressure in monocrotaline-treated rats (Fasules et al., 1987b), again highlighting differences between different models of pulmonary hypertension. The mechanism by which heparin inhibits changes in pulmonary vascular structure is not precisely known. It is evident that the anti-remodeling effects are not related to its anticoagulant properties, since another anticoagulant drug, warfarin, exhibited no anti-remodeling effects in hypoxic guinea pigs (Hassoun et al., 1989). In addition, Thompson et al. (1994) showed that the antiproliferative action of heparin did not correlate with its anticoagulating properties. Evidence that heparin has a direct antiproliferative action comes from in vitro studies, where proliferation of cultured bovine and rat pulmonary artery smooth muscle cells (Thompson et al., 1994), as well as rat pulmonary artery pericytes (precursors to smooth muscle cells found in distal pulmonary arteries) (Khoury & Langleben, 2000), was inhibited when heparin was added to the medium. The inhibition by heparin of pericyte proliferation occurred via halting cell cycle progression at the G0 /G1 phase through induction of the p21 gene, a potent inhibitor of cyclindependent protein kinases, which are the principal regulators of the G1 phase of the cell cycle (Khoury & Langleben, 2000). Other possible mechanisms are (1) inhibition of the action of 5-HT (Lee et al., 1997) and /or (2) activation of MAPK kinase-1 (Daum et al., 1997). 6.8. Drugs affecting the extracellular matrix In view of the evidence that an increase in serine elastase activity contributes to remodeling (see Section 4.6), drugs that are serine elastase inhibitors have been investigated for their anti-remodeling effects. In monocrotaline-treated and / or hypoxic rats, these drugs, which include SC-39026, a1-proteinase inhibitor, and SC-37698, have been successful in reducing neomuscularisation of distal pulmonary arteries and/or medial thickening (Ilkiw et al., 1989; Maruyama et al., 1991; Ye & Rabinovitch, 1991). More recently, two other serine elastase inhibitors, ZD0892 and

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M249314, when given to rats for 1 or 2 weeks 21 days after monocrotaline injection, reversed pulmonary vas-

13

cular remodeling, normalised pulmonary artery pressure, and dramatically increased survival (Cowan et al., 2000a).

Fig. 3. Some of the proposed mechanisms of pulmonary vascular remodeling in pulmonary hypertension, drug groups that inhibit the remodeling process, and some possible sites of action of these drug groups. a Includes pressure, stretch, and shear stress. b Includes bFGF, EGF, IGF-1, PDGF, TGF-b, and VEGF. c The drug groups in this numbered list are identified in the diagram by the corresponding numbers in the circles. Arrowheads with solid lines indicate a ‘‘direct’’ effect on pulmonary vascular remodeling. Arrowheads with broken lines indicate interdependence between different remodeling stimuli. AII, angiotensin II; HIF-1, hypoxia inducible factor-1; HPV, hypoxic pulmonary vasoconstriction; IP3, inositol trisphosphate.

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Other studies have examined agents that interfere with collagen synthesis. The two anti-fibrotic agents b-aminoproprionitrile and cis-hydroxyproline have both been shown to reduce the amount of collagen in the main pulmonary arteries from hypoxic pulmonary hypertensive rats (Kerr et al., 1984, 1987). In these studies, both agents were effective in the main pulmonary arteries, and, in addition, b-aminoproprionitrile reduced adventitial and perivascular connective tissue matrix in hilar pulmonary arteries (Kerr et al., 1984). The effect of cis-hydroxyproline was associated with attenuation of medial thickening of pulmonary arteries (20 – 400 mm o.d.) (Kerr et al., 1987). Both agents reduced pulmonary artery pressure. 6.9. Miscellaneous drugs Neutral endopeptidase (NEP) inhibitors, which inhibit the breakdown of various peptides including atrial natriuretic peptide (ANP), have been shown to inhibit medial thickening and/or neomuscularisation of pulmonary arteries, as well as reducing pulmonary hypertension in hypoxic rats (Winter et al., 1991; Stewart et al., 1992; Klinger et al., 1993). However, it is uncertain whether the beneficial effect of NEP inhibitors relates to impaired breakdown of ANP, since in one of these studies, plasma levels of this peptide were not increased, despite a beneficial effect on remodeling (Klinger et al., 1993). Moreover, in another study, continuous infusion of ANP resulted in only a very small reduction in medial thickening of pulmonary arteries (Jin et al., 1990). Therefore, possible benefits of NEP inhibitors require further study. The platelet-activating factor antagonist WEB 2170 inhibited medial thickening of pulmonary arteries (50 – 200 mm o.d.) and reduced pulmonary artery pressure when given to hypoxic or monocrotaline-treated rats for 3 weeks (Ono et al., 1992; Ono & Voelkel, 1992). In addition, in monocrotalinetreated rats, the amount of total lung collagen was reduced (Ono et al., 1992). Oestradiol, when administered to monocrotaline-treated rats or to newborn sheep with ligation of the ductus arteriosus, inhibited medial thickening of pulmonary arteries and neomuscularisation, respectively (Farhat et al., 1993; Parker et al., 2000). A reduction in pulmonary artery pressure was seen in rats, but not in sheep. The mechanism of action of oestradiol is unclear, but it may involve an increase in endothelial NO synthase in vascular smooth muscle cells, leading to increased cGMP, or reduced ET-1 (Parker et al., 2000). An interleukin receptor antagonist was found to be effective in inhibiting pulmonary vascular remodeling in monocrotaline-treated, but not hypoxic, rats (Voelkel et al., 1994). It is to be expected that interleukin receptor antagonists would be effective only in situations where the disease has an inflammatory component. Heme oxygenase catalyzes the breakdown of heme. One of the products is carbon monoxide, which increases cGMP

and, therefore, has vasodilator and antiproliferative properties. Treatment of hypoxic rats for 1 week with agents that induce heme oxygenase, namely NiCl2 and hemin, prevented medial thickening of pulmonary arterioles and also reduced pulmonary artery pressure (Christou et al., 2000). It should be noted that hypoxia, in itself, can increase heme oxygenase activity. However, this apparent paradox is explained by the fact that this is only a transient effect (Morita et al., 1995). Adrenomedullin, given by chronic infusion to monocrotaline-treated rats for 21 days, led to a small reduction in medial thickening of pulmonary arteries (25 – 100 mm o.d.), as well as a reduction in right ventricular systolic pressure (Yoshihara et al., 1998). The 5-HT2A receptor antagonist MI-9042 caused small reductions in medial thickening and neomuscularisation when administered to monocrotaline-treated rats for 28 days (Miyata et al., 2000).

7. Conclusion Pulmonary vascular remodeling in pulmonary hypertension is a complex, multi-factorial process, with many of the physical and chemical stimuli for remodeling acting synergistically and /or interdependently (Fig. 3). In view of the complexity of remodeling, drug therapy that targets more than one aspect of the process is likely to be more effective than any drug that interferes at only one point in the cascade of events. This could be achieved with drugs possessing more than one action (e.g., a drug that inhibits both ACE and ECE), a combination of drugs (e.g., a Ca2 + -channel antagonist plus an ACE inhibitor), or drugs that act at points in the pathway that are common to a number of stimuli/ mediators (e.g., a MAPK inhibitor). The additive effect on remodeling obtained with a combination of a Ca2 + -channel antagonist and an ACE inhibitor recently has been demonstrated in hypoxic pulmonary hypertensive rats (Jeffery & Wanstall, 2001). Experiments in animals support the view that pulmonary vascular remodeling is an appropriate target for therapy in pulmonary hypertension since attenuation of remodeling was almost invariably accompanied by reductions in pulmonary artery pressure and /or pulmonary vascular resistance. However, the literature has shown that in animals, the efficacy of drugs that attenuate remodeling can vary, depending on the model of pulmonary hypertension. This suggests that the relative importance of the different factors and pathways depicted in Fig. 3 also may differ, depending on the particular animal model. Similarly, in humans, the relative importance of the different mechanisms of remodeling is almost certainly a function of the underlying cause of the disease. Hence, it is to be anticipated that successful anti-remodeling therapy for clinical pulmonary hypertension, which has such diverse aetiologies (Table 1), will require a range of different drug options.

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Acknowledgements The financial support of the National Health and Medical Research Council of Australia is gratefully acknowledged.

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