Endothelial cell dysfunction and cross talk between endothelium and smooth muscle cells in pulmonary arterial hypertension

Vascular Pharmacology 49 (2008) 113–118

Contents lists available at ScienceDirect

Vascular Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / v p h

Review

Endothelial cell dysfunction and cross talk between endothelium and smooth muscle cells in pulmonary arterial hypertension Marc Humbert a,⁎, David Montani a, Frédéric Perros a, Peter Dorfmüller a, Serge Adnot b, Saadia Eddahibi b a

Université Paris-Sud 11, Centre National de Référence de l'Hypertension Artérielle Pulmonaire, Service de Pneumologie et Réanimation Respiratoire, Hôpital Antoine-Béclère, Assistance Publique des Hôpitaux de Paris, Clamart, France INSERM U841, Hôpital Henri-Mondor, Assistance Publique des Hôpitaux de Paris, Université Paris 12, Créteil, France

b

A R T I C L E

I N F O

Article history: Received 15 January 2008 Received in revised form 20 March 2008 Accepted 13 June 2008 Keywords: Chemokines Endothelial cells Endothelium Pulmonary arterial hypertension Serotonin Smooth muscle cells

A B S T R A C T The pathogenesis of pulmonary arterial hypertension (PAH) involves a complex and multifactorial process in which endothelial cell dysfunction appears to play an integral role in mediating the structural changes in the pulmonary vasculature. Disordered endothelial cell proliferation along with concurrent neoangiogenesis, when exuberant, results in the formation of glomeruloid structures known as the plexiform lesions, which are common pathological features of the pulmonary vessels of patients with PAH. In addition, an altered production of various endothelial vasoactive mediators, such as nitric oxide, prostacyclin, endothelin-1, serotonin, chemokines and thromboxane, has been increasingly recognized in patients with PAH. Because most of these mediators affect the growth of the smooth muscle cells, an alteration in their production may facilitate the development of pulmonary vascular hypertrophy and structural remodeling characteristic of PAH. It is conceivable that the beneficial effects of many of the treatments currently available for PAH, such as the use of prostacyclin, nitric oxide, and endothelin receptor antagonists, result at least in part from restoring the balance between these mediators. A greater understanding of the role of the endothelium in PAH will presumably facilitate the evolution of newer, targeted therapies. © 2008 Elsevier Inc. All rights reserved.

Contents 1. Pulmonary vascular remodeling in pulmonary arterial hypertension . 2. Serotonin in pulmonary arterial hypertension . . . . . . . . . . . 3. Chemokines in pulmonary arterial hypertension . . . . . . . . . . 4. Platelet-derived growth factor in pulmonary arterial hypertension . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Pulmonary arterial hypertension (PAH) is characterized by a progressive increase in pulmonary vascular resistance leading to right ventricular failure and ultimately death (Humbert et al., 2004). Vasoconstriction, remodeling of the pulmonary vessel wall, and thrombosis contribute to increased pulmonary vascular resistance in PAH (Humbert et al., 2004). However, remodeling of small pulmonary arteries represents the main pathological finding related to PAH with marked proliferation of pulmonary artery smooth muscle cells (PA-SMC) and pulmonary endothelial cells (P-EC) resulting in the obstruction of resistance pulmonary arteries (Humbert et al., 2004). ⁎ Corresponding author. Service de Pneumologie et Réanimation Respiratoire, Hôpital Antoine-Béclère, 157 rue de la Porte de Trivaux, 92140 Clamart, France. Tel.: +33 1 45 37 47 72; fax: +33 1 46 30 38 24. E-mail address: [email protected] (M. Humbert). 1537-1891/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.vph.2008.06.003

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

113 114 115 116 117

1. Pulmonary vascular remodeling in pulmonary arterial hypertension The process of pulmonary vascular remodeling involves all layers of the vessel wall and each cell type (endothelial, smooth muscle, and fibroblast) in the pulmonary vascular wall plays a specific role in the pathogenesis (Humbert et al., 2004). A feature common to all forms of PAH remodeling is the distal extension of smooth muscle into small peripheral, normally nonmuscular, pulmonary arteries within the respiratory acinus (Humbert et al., 2004). The cellular processes underlying muscularization of this distal part of the pulmonary arterial tree are incompletely understood. In addition, a hallmark of severe pulmonary hypertension is the formation of a layer of myofibroblasts and extracellular matrix between the endothelium and the internal elastic lamina, termed the neointima (Humbert et al., 2004).

114

M. Humbert et al. / Vascular Pharmacology 49 (2008) 113–118

The mechanisms that enable the adventitial fibroblast to migrate into the media (and ultimately the intima) are currently unclear, but there is good evidence to suggest that upregulation of matrix metalloproteinases (MMP2 and MMP9) occurs and that these molecules are involved in migration (Humbert et al., 2004). In many forms of pulmonary hypertension, as the vessel wall thickens, a concomitant increase occurs in neovascularization of the vasa vasorum. This neovascularization occurs primarily in the adventitia, although it extends into the outer parts of the media. This adventitial vessel formation could help circulating progenitor cells to access the vessel wall from the adventitial side. It is unknown whether circulating progenitor cells derived from the bone marrow contribute directly to the adventitial thickening and perhaps medial thickening, or whether bone marrowderived progenitor cells simply enhance the proliferative and migratory activity of the local adventitial fibroblasts (Humbert et al., 2004). Disorganized endothelial cell proliferation leading to formation of plexiform lesions is described in PAH (Budhiraja et al., 2004; Humbert et al., 2004). The initiating stimulus or injury that results in abnormal endothelial proliferation is unknown, but may include hypoxia, shear stress, inflammation, or response to drugs or toxins on a background of genetic susceptibility. However, although many animal models of pulmonary hypertension exist, none recapitulate the histology of PAH, particularly the presence of plexiform lesions (Budhiraja et al., 2004; Humbert et al., 2004). Endothelial cells may respond to injury in various ways affecting the process of vascular remodeling. Injury can alter not only cell proliferation and apoptosis but also homeostatic functions of the endothelium (including coagulation pathways, and production of growth factors and vasoactive agents) (Budhiraja et al., 2004; Humbert et al., 2004). The cells comprising plexiform lesions are endothelial channels supported by a stroma containing matrix proteins and myofibroblasts (Budhiraja et al., 2004; Humbert et al., 2004). Endothelial cells express markers of angiogenesis, such as vascular endothelial growth factor (VEGF) and its receptors (Budhiraja et al., 2004; Humbert et al., 2004). In addition, cells comprising plexiform lesions of idiopathic PAH are monoclonal in origin (Humbert et al., 2004). Therefore, although the lesions themselves are probably hemodynamically irrelevant, they may represent more than simply the result of severe elevation of intravascular pressures. It has been suggested that the endothelial proliferation seen in these lesions may be a marker of a fundamental endothelial abnormality in idiopathic PAH, possibly playing a key role in the pathogenesis of the condition. Intriguing defects in growth suppressive genes have been reported in plexiform lesions of patients with idiopathic PAH, including transforming growth factor-beta (TGF-β) receptor-2 and the apoptosisrelated gene, Bax (Humbert et al., 2004). It has been proposed that somatic mutations in growth regulatory genes allow clonal expansion of endothelial cells, that contribute to the formation of plexiform lesions and vascular obliteration (Humbert et al., 2004). Human herpesvirus-8 infection may also contribute to the growth of monoclonal endothelial cells in plexiform lesions from patients with idiopathic PAH (Humbert et al., 2004). These findings suggest that triggers, including vasculotropic viruses, can encourage the growth of endothelial cells by dysregulating cell growth or growth factor signalling (Humbert et al., 2004). The nature of the primary abnormality that triggers and perpetuates the proliferation of PA-SMC in PAH is unclear. Whether smooth muscle hyperplasia results from inherent characteristics of PA-SMC or from dysregulation of molecular events that govern PA-SMC growth, such as signals originating from P-EC, remains an open question (Adnot, 2005). P-EC may be involved in pulmonary vascular remodeling not only through their ability to control vascular tone, but also through the production and release of growth factors. P-EC dysfunction in PAH has been shown to be associated with decreased production of nitric oxide (NO) and prostacyclin and increased production of endothelin (ET-1) (Eddahibi et al., 2006; Giaid et al., 1993; Humbert

et al., 2004). In addition, a critical aspect of P-EC dysfunction in PAH is excessive release of paracrine factors that act either as growth factors to induce PA-SMC proliferation or as chemokines to recruit circulating inflammatory cells (Eddahibi et al., 2006; Sanchez et al., 2007). Indeed, exposure of PA-SMC to culture medium from P-EC induced PA-SMC proliferation and this effect was stronger when P-EC from patients with PAH were used (Eddahibi et al., 2006). Moreover, monocyte migration was markedly increased in the presence of P-EC, and the increase was larger with P-EC from patients with PAH, compared to controls (Sanchez et al., 2007). These observations indicate that, during PAH progression, P-EC constitutively produce and release excessive amounts of soluble factors that act on PA-SMC and inflammatory circulating cells to initiate or enhance pulmonary vascular remodeling and inflammation. Although alterations of this cross talk between P-EC and PA-SMC on the one hand, and between P-EC and inflammatory cells on the other hand, appear critical to pulmonary vessel remodeling in PAH, many questions remain unanswered. In particular, most of the paracrine growth factors released by P-EC from individuals with or without PAH remain unidentified; moreover, the mechanisms underlying overexpression of these factors are still unknown. Whether the molecular mechanisms explaining the abnormal phenotype of P-EC in PAH and alterations in cross talk between P-EC and PA-SMC are linked to alterations in the BMP or TGFβ/Smad pathways remains to be elucidated. Evidence that several growth factors contribute to the abnormal cross talk between P-EC and PA-SMC in PAH provides a rationale for combining drugs that act selectively on these pathways. 2. Serotonin in pulmonary arterial hypertension The role of endothelial cells in angiogenesis and remodeling is now better understood (Armulik et al., 2005; Jain, 2003). A critical aspect of vessel development is maturation, during which endothelial cells no longer proliferate or migrate but instead promote vessel stabilization by recruiting peri-endothelial support cells, which differentiate into smooth muscle cells (Folkman and D'Amore, 1996; Hanahan, 1997). Interactions between endothelial cells and mural cells (pericytes and vascular smooth muscle cells) in the blood vessel wall have recently emerged as central processes in the regulation of vascular formation, stabilization, remodeling, and function (Armulik et al., 2005). Failure of interactions between the two cell types, as seen in numerous genetic mouse models, results in severe and often lethal cardiovascular defects. Abnormal interactions between the two cell types are also implicated in a number of human diseases, including tumor angiogenesis, diabetic microangiopathy, ectopic tissue calcification, and vascular diseases (Armulik et al., 2005; Jain, 2003). Thus, endothelial cells constitutively produce and release growth factors that act on PA-SMC and whose physiological function may consist in recruiting PA-SMC and maintaining a smooth muscle coat around the pulmonary vessels (Folkman and D'Amore, 1996; Hanahan, 1997). Abnormal activation of this process may lead to pathological vascular remodeling. Deficiencies in this process may lead to abnormal dilation of resistance vessels such as that seen in hereditary hemorrhagic telangectasia. Recent studies provided evidence that dysregulation of the molecular pathways governing vessel maturation was involved in the pulmonary vascular remodeling process during PAH. In support of this concept, we showed that exposing PA-SMC to culture medium from PEC induced PA-SMC proliferation and that this effect was considerably stronger when the P-EC came from patients with PAH (Eddahibi et al., 2006). Interestingly, these results were obtained using nonstimulated quiescent cultured cells from adult patients, suggesting that P-EC may constitutively produce and release growth factors that act on PA-SMC. The stimulating effect of P-EC media on PA-SMC growth was greater with P-EC from patients with PAH than from controls, and with PASMC from patients than from controls, indicating that alterations in

M. Humbert et al. / Vascular Pharmacology 49 (2008) 113–118

cross talk between the two cell types were critical to pulmonary vessel remodeling. Serotonin (5-HT) is an endogenous vasoactive indoleamine substance found primarily in the gut, brain, and blood platelets. The neutral amino acid L-tryptophan is the primary precursor of serotonin, the synthesis of which is involved in first hydroxylation of the benzene ring of tryptophan by the rate-limiting monooxygenase, tryptophan hydroxylase (Tph), to form 5-hydroxytryptophan, and second, decarboxylation of its aminocarboxy terminal group by an aromatic L-amino acid decarboxylase, to form 5-HT. Tph activity therefore serves as a marker for 5-HT synthesis. Tph, which was initially thought to be derived from a single gene, exists in two isoforms, Tph1 and Tph2, encoded by separate genes. Tph1 is expressed mainly in the gut and in the pineal gland (Darmon et al., 1988). The recently identified Tph2 gene appears exclusively expressed in neurons and is known as the neuronal isoform (Walther et al., 2003). Thus, 5-HT synthesis is thought to be controlled chiefly by Tph2 in the central nervous system and Tph1 in peripheral organs (Cote et al., 2003; Darmon et al.,1988; Sakowski et al., 2006; Walther et al., 2003). Although peripherally produced 5-HT is synthesized chiefly by the enterochromaffin cells in the gut (Gershon, 1999), small amounts of 5-HT may also be formed in other organs. Human pulmonary vascular endothelial cells can produce 5-HT and express Tph1 and both 5-HT synthesis and Tph1 expression are increased in cells from patients with idiopathic PAH compared to cells from controls (Eddahibi et al., 2006). In addition to its vasoactive effects, 5-HT exerts mitogenic and comitogenic effects on PA-SMC. In contrast to the constricting action of 5-HT on smooth muscle cells, which is mainly mediated by 5-HT receptors (5-HT 1B/D, 2A, and 2B) (MacLean et al., 2000), the mitogenic and co-mitogenic effects of 5-HT require internalization of indoleamine by the 5-HT transporter (5-HTT) (Eddahibi et al., 1999; Lee et al., 1991). Accordingly, drugs that competitively inhibit 5-HTT also block the mitogenic effects of 5-HT on smooth muscle cells (Eddahibi and Adnot, 2002). 5-HTT is abundantly expressed in the lung, where it is predominantly located on PA-SMC (Eddahibi et al., 2001; Fanburg and Lee, 1997). It is encoded by a single gene expressed in several cell types such as neurons, platelets, and pulmonary vascular endothelial and smooth muscle cells. The level of 5-HTT expression seems considerably higher in human lung than in human brain, suggesting that altered 5-HTT expression may have direct consequences on PA-SMC function. The requirement for 5-HTT as a mediator for the mitogenic activity of 5-HT appears specific of PA-SMC, since no such effect has been reported with other SMC types. Direct evidence that 5-HTT plays a key role in pulmonary vascular remodeling was recently provided by studies showing that mice with targeted 5-HTT gene disruption developed less severe hypoxic PH than did wild-type controls and that selective 5-HTT inhibitors attenuated hypoxia- and monocrotaline-induced PH (Eddahibi et al., 2000; Guignabert et al., 2005). Conversely, increased 5-HTT expression was associated with increased severity of hypoxic PH (MacLean et al., 2004). To investigate whether 5-HTT overexpression in PA-SMC is sufficient to produce PH, we generated transgenic mice overexpressing 5-HTT under the control of the SM22 promoter. We found that these transgenic mice with selective overexpression of 5-HTT in smooth muscle cells spontaneously developed PH (Guignabert et al., 2005). One major point is that PH in SM22-5-HTT+ mice developed without any alterations in 5-HT bioavailability, and therefore as a sole consequence of the increased expression of the 5-HTT protein in smooth muscle cells. Taken together, these observations suggest a close correlation between 5-HTT expression and/or activity and the extent of pulmonary vascular remodeling during experimental PH. Evidence that the 5-HTT plays an important role in the pathogenesis of human idiopathic PAH was obtained more recently. 5-HTT expression was shown to be increased in platelets and lungs from patients with idiopathic PAH, where it predominated in the media of thickened pulmonary arteries and in onion-bulb lesions (Eddahibi et al., 2001). Interestingly, the higher level of 5-HTT protein and activity

115

persisted in cultured smooth muscle cells isolated from pulmonary arteries of patients with idiopathic PAH, as compared to cells from controls (Eddahibi et al., 2001). Moreover, PA-SMC from patients with idiopathic PAH grew faster than PA-SMC from controls when stimulated by serotonin or serum, as a consequence of increased expression of the serotonin transporter (Eddahibi et al., 2001). In the presence of 5-HTT inhibitors, the growth-stimulating effects of serum and serotonin were markedly reduced, and the difference between growth of PA-SMC from patients and controls was abolished. The proliferative response of PA-SMC to various growth factors such as PDGF, EGF, TGFβ, FGFα, and IGF did not differ between patients with idiopathic PAH and controls (Eddahibi et al., 2001). It follows that 5-HTT overexpression and/or activity in PA-SMC from patients with idiopathic PAH is responsible for the increased mitogenic response to serotonin and to serum (which contains micromolar concentrations of serotonin). To question whether 5-HT was involved in the abnormal cross talk between P-EC and PA-SMC during PAH progression, we added serumfree media derived from cultured P-EC collected from the lungs of patients with idiopathic PAH to cultured PA-SMC from the same individuals. Fluoxetine, which blocks the growth-promoting effect of 5-HT on PA-SMC by inhibiting 5-HTT, diminished the growth response to P-EC medium by about 50%, whereas endothelin receptor antagonists had no effect. These data indicate that 5-HT was the main growth factor released by P-EC and acting on PA-SMC. Serotonin was also found to be the main growth factor released by P-EC upon stimulation by angiopoietin-1 (Sullivan et al., 2003). We therefore investigated whether 5-HT in P-EC medium was produced by de novo synthesis of 5-HT by P-EC or was released from an indoleamine pool stored in P-EC. We found that P-EC treatment with PCPA (Koe and Weissman, 1966), a selective Tph inhibitor (Hamon et al., 1979; Lepetit et al., 1991), reduced the levels of 5-HT and 5-HTT to undetectable values in the medium and reduced PA-SMC growth to a similar extent as that obtained by PA-SMC pretreatment with fluoxetine. A strong argument that the effects of PCPA and fluoxetine were mediated specifically through 5-HT-dependent mechanisms is that no further effects were produced by combining the two drugs. Thus, when optimal depletion of intracellular 5-HT was already achieved by 5-HT synthesis inhibition (with PCPA), 5-HTT blockade by fluoxetine did not further inhibit PA-SMC proliferation. Moreover, strong TPH immunoreactivity was seen in the endothelium of normal pulmonary vessels, indicating that Tph expression occurred in vivo in the normal lung. We also found that only the tph1 gene was expressed in whole lung homogenates and in quiescent cultured human P-EC. Thus, serotonin is synthesized by P-EC in the normal lung as a result of Tph1 enzyme activity and appears to be the main growth factor produced by P-EC and acting on PA-SMC in a paracrine fashion. Such cross talk between endothelial cells and smooth muscle cells mediated by 5-HT appears unique to the pulmonary circulation, since 5-HT does not seem to act as a potent mitogenic factor on smooth muscle cells in systemic vessels (Fanburg and Lee, 1997; Nemecek et al., 1986). 3. Chemokines in pulmonary arterial hypertension Inflammatory mechanisms are believed to play a role in PAH (Dorfmuller et al., 2003). Indeed, severe PAH may occur in a subset of patients displaying systemic inflammatory conditions and treatments with corticosteroids and/or immunosuppressants sometimes dramatically improve PAH complicating POEMS (Polyneuropathy, Organomegaly, Endocrinopathy, Monoclonal immunoglobulin, Skin changes) syndrome (Jouve et al., 2007), Castleman's disease (Montani et al., 2005), systemic lupus erythematosus (Jais et al., 2008; Sanchez et al., 2006), mixed connective tissue disease (Jais et al., 2008; Sanchez et al., 2006) and primary Sjögren's syndrome (Launay et al., 2007). Autoimmunity and inflammation have also been demonstrated to contribute to idiopathic PAH pathogenesis (Humbert et al., 1995; Mouthon

116

M. Humbert et al. / Vascular Pharmacology 49 (2008) 113–118

et al., 2005; Nicolls et al., 2005, Tamby et al., 2005; Tamby et al., 2006). Idiopathic PAH patients are characterized by circulating autoantibodies (antinuclear, anti-endothelial and anti-fibroblast antibodies) and elevated serum concentrations of pro-inflammatory cytokines (interleukin (IL)-1 and IL-6) (Humbert et al., 1995; Mouthon et al., 2005; Nicolls et al., 2005; Tamby et al., 2005). Of note, pathogenic auto-antibodies targeting endothelial cells might be capable of inducing vascular endothelial apoptosis which may in turn promote PAH development (Nicolls et al., 2005). Lung pathology of patients displaying severe PAH in the context of connective tissue diseases further indicates that inflammation and remodeling are key contributors to pulmonary vascular disease complicating inflammatory diseases (Dorfmuller et al., 2007). In addition, idiopathic PAH frequently reveals inflammatory infiltrates corresponding to macrophages, lymphocytes and dendritic cells in the range of plexiform lesions with local expression of chemokines CCL2 (MCP-1), CCL5 (RANTES) and CX3CL1 (fractalkine) (Balabanian et al., 2002; Dorfmuller et al., 2002; Dorfmuller et al., 2007; Perros et al., 2007b; Sanchez et al., 2007). Experimental data from monocrotaline-induced pulmonary hypertension in rats further support a direct link between pulmonary vascular inflammation and remodeling (Dorfmuller et al., 2003; Perros et al., 2007b). Involvement of leukocytes, such as macrophages and lymphocytes in complex lesions of idiopathic PAH, has been initially described by Tuder et al. (1994). Recent studies confirmed this observation, stressing the possible role of perivascular lymphocytic infiltrates in PAH. This inflammatory pattern has been demonstrated in plexiform lesions as well as in other vascular lesions of PAH-affected lungs (Dorfmuller et al., 2007; Perros et al., 2007b). The role of T lymphocyte recruitment by chemotactic cytokines in PAH and its mechanism has also been evaluated (Balabanian et al., 2002; Dorfmuller et al., 2002). Leukocyte trafficking involves successive events, including rolling, firm adhesion, and extravasation, in response to a chemoattractant gradient that is thought to involve chemokines. Chemokines are soluble, secreted basic proteins that direct the migration of specific subsets of leukocytes. They play a major role in the different steps of leukocyte recruitment, including rolling, activation, adherence, and extravasation into the inflamed tissue (Balabanian et al., 2002; Dorfmuller et al., 2002). Studies have attempted to analyze those chemokine-dependent mechanisms leading to inflammatory cell recruitment in the lungs of patients displaying PAH. Fractalkine (CX3CL1) is a unique chemokine because it exists both in a soluble form as a chemotactic protein, and in a membrane-anchored form as a cell-adhesion molecule on endothelial cells (Balabanian et al., 2002). Its actions are mediated by CX3CR1, a seven-transmembrane receptor that is expressed by monocytes, dendritic cells, microglial cells, neurons, natural killer cells, mast cells, and subpopulations of T lymphocytes. CX3CL1 promotes CX3CR1-expressing leukocyte recruitment, but, unlike other chemokines, it can mediate the rapid capture, integrin-independent adhesion, and activation of circulating CX3CR1+ leukocytes under high blood flow (Balabanian et al., 2002). Several recent findings have reported a polymorphism in CX3CR1 associated with a reduced risk of acute coronary artery disease, suggesting that fractalkine plays a critical role in monocyte/T cell recruitment to the vessel wall. Balabanian et al. have been able to demonstrate that (i) CX3CR1 was upregulated in circulating CD4+ and CD8+ T lymphocytes from PAH patients as compared with controls; (ii) this deregulation of CX3CR1 expression accounted for the increased sensitivity of these cells to soluble CX3CL1; (iii) the abnormal response of T lymphocytes to CX3CL1 was not the mere consequence of pulmonary hypertension because it was not present in patients with pulmonary hypertension secondary to chronic thromboembolic pulmonary hypertension (CTEPH); (iv) elevated soluble CX3CL1 plasma concentrations were measured in PAH patients, as compared with CTEPH patients and normal controls; (v) lung samples from PAH patients showed an increased CX3CL1 mRNA expression as compared with controls, and pulmonary artery endothelial cells from PAH

patients expressed CX3CL1 (Balabanian et al., 2002). RANTES (Regulated upon Activation, Normal T cell expressed and secreted: CCL5) is an important chemoattractant for monocytes and T-cells (Dorfmuller et al., 2002). CCL5 presumably plays a key role in several arterial inflammatory processes such as glomerulonephritis, Kawasaki syndrome, and Takayasu's arteritis (Dorfmuller et al., 2002). In addition, successful antagonization of CCL5 has been reported in animal models of inflammatory diseases. CCL5 may also play an indirect role in PAH through the induction of endothelin-converting enzyme-1 and endothelin-1, a potent endothelium-derived factor with strong vasoconstrictive and mitogenic action. In experiments on PAH patients and healthy controls it was shown that (i) CCL5 mRNA can be detected by competitive reverse transcription polymerase chain reaction in lung samples from all PAH patients and controls; (ii) numbers of CCL5 mRNA copies are significantly elevated in the lungs of PAH patients as compared with controls; (iii) endothelial cells are the major source of CCL5, identified by in situ hybridization and immunohistochemistry in PAH lung samples (Dorfmuller et al., 2002). Two recent studies have further emphasized that chemokines produced in the small pulmonary arteries of PAH patients may contribute to inflammatory cell recruitment and PA-SMC proliferation. First, Perros et al. studied the expression of CX3CL1 and CX3CR1 in monocrotaline-induced pulmonary hypertension by means of immunohistochemistry and RT-PCR in laser-captured microdissected pulmonary arteries (Perros et al., 2007a). They demonstrated that CX3CL1 was expressed by inflammatory cells surrounding pulmonary arterial lesions and that smooth muscle cells from these vessels had increased CX3CR1 expression (Perros et al., 2007a). In addition, they showed that cultured rat pulmonary artery smooth muscle cells expressed CX3CR1 and that CX3CL1 induced proliferation but not migration of these cells (Perros et al., 2007a). Thus, fractalkine may act as a growth factor for pulmonary artery smooth muscle cells (Perros et al., 2007a). Chemokines may thus play a role in pulmonary artery remodeling (Perros et al., 2007a). This hypothesis was further studied by Sanchez et al. (2007). Compared to controls, idiopathic PAH patients were characterized by elevated CCL2 (also known as MCP-1) protein levels in plasma and lung tissue, whereas monocyte CCL2 levels were similar between patients and controls (Sanchez et al., 2007). In addition elevated CCL2 release by pulmonary endothelial cells or pulmonary artery smooth muscle cells was demonstrated (Sanchez et al., 2007). Of note, pulmonary endothelial cells released twice as much CCL2 as did pulmonary artery smooth muscle cells (Sanchez et al., 2007). Monocyte migration was markedly increased in the presence of pulmonary endothelial cells, and the increase was larger with cells from patients with idiopathic PAH (Sanchez et al., 2007). CCL2blocking antibodies reduced pulmonary endothelial cells chemotactic activity by 60%. Compared to controls, pulmonary artery smooth muscle cells from patients exhibited stronger migratory and proliferative responses to CCL2, in keeping with the finding that CCR2 was markedly increased in pulmonary artery smooth muscle cells from patients (Sanchez et al., 2007). 4. Platelet-derived growth factor in pulmonary arterial hypertension In recent years, platelet-derived growth factor (PDGF) has been identified as a novel possible therapeutic target in PAH. Active PDGF is built up by polypeptides (A and B chain) that form homo- or heterodimers and stimulate α and β cell surface receptors. PDGF is synthesized by many different cell types including smooth muscle cells, endothelial cells, and macrophages (Humbert et al., 1998; Schermuly et al., 2005). PDGF has the ability to induce the proliferation and migration of smooth muscle cells and fibroblasts, and it has been proposed as a key mediator in the progression of several fibroproliferative disorders such as atherosclerosis, lung fibrosis, and pulmonary hypertension (Humbert et al., 1998; Schermuly et al., 2005). It can

M. Humbert et al. / Vascular Pharmacology 49 (2008) 113–118

also induce the contraction of rat aorta strips in vitro. Novel therapeutic agents such as imatinib mesylate (Gleevec®) inhibit several tyrosine kinases associated with disease states, including BCR-ABL in patients with chronic myelogenous leukemia, c-kit in patients with gastrointestinal stromal tumours, and PDGF receptors α and β in patients with certain myeloproliferative disorders and dermatofibrosarcoma protuberans, respectively (Schermuly et al., 2005). Imatinib has been demonstrated to reverse pulmonary vascular remodeling in animal models of pulmonary hypertension and a few cases of clinical and hemodynamic improvements have also been reported in human PAH (Ghofrani et al., 2005; Patterson et al., 2006; Souza et al., 2006). Due to the lack of comprehensive human data, we performed a complete analysis of the pathogenic role of PDGF in human PAH (Perros et al., 2008). We first confirmed increased expression of PDGF and PDGF receptors by means of RT-PCR performed on laser capture microdissected pulmonary arteries from lung transplanted patients displaying severe idiopathic PAH (Perros et al., 2008). PDGF-A, PDGFB, PDGF-R α and PDGF-R β mRNA expression was increased in small pulmonary arteries from patients displaying severe idiopathic PAH, as compared to controls (Perros et al., 2008). In small pulmonary arteries, PDGF-B was mainly expressed in endothelial cells, smooth muscle cells, and in some perivascular inflammatory cells, and PDGF-R β was mainly expressed in smooth muscle cells (Perros et al., 2008). PDGF-Binduced proliferation and migration of pulmonary artery smooth muscle cells were inhibited by imatinib (Perros et al., 2008). These data support the concept that PDGF is overproduced within the pulmonary artery wall of PAH patients and promotes pulmonary arterial remodeling (Perros et al., 2008). In conclusion, the pathogenesis of PAH involves a complex and multifactorial process in which endothelial cell dysfunction appears to play an integral role in mediating the structural changes in the pulmonary vasculature (Budhiraja et al., 2004; Humbert et al., 2004). A greater understanding of the role of the endothelium in PAH will presumably facilitate the evolution of newer, targeted therapies. References Adnot, S., 2005. Lessons learned from cancer may help in the treatment of pulmonary hypertension. J. Clin. Invest. 115, 1461–1463. Armulik, A., Abramsson, A., Betsholtz, C., 2005. Endothelial/pericyte interactions. Circ. Res. 97, 512–523. Balabanian, K., Foussat, A., Dorfmuller, P., Durand-Gasselin, I., Capel, F., Bouchet-Delbos, L., Portier, A., Marfaing-Koka, A., Krzysiek, R., Rimaniol, A.C., Simonneau, G., Emilie, D., Humbert, M., 2002. CX(3)C chemokine fractalkine in pulmonary arterial hypertension. Am. J. Respir. Crit. Care Med. 165, 1419–1425. Budhiraja, R., Tuder, R.M., Hassoun, P.M., 2004. Endothelial dysfunction in pulmonary hypertension. Circulation 109, 159–165. Cote, F., Thevenot, E., Fligny, C., Fromes, Y., Darmon, M., Ripoche, M.A., Bayard, E., Hanoun, N., Saurini, F., Lechat, P., Dandolo, L., Hamon, M., Mallet, J., Vodjdani, G., 2003. Disruption of the nonneuronal tph1 gene demonstrates the importance of peripheral serotonin in cardiac function. Proc. Natl. Acad. Sci. U S A 100, 13525–13530. Darmon, M.C., Guibert, B., Leviel, V., Ehret, M., Maitre, M., Mallet, J., 1988. Sequence of two mRNAs encoding active rat tryptophan hydroxylase. J. Neurochem. 51, 312–316. Dorfmuller, P., Zarka, V., Durand-Gasselin, I., Monti, G., Balabanian, K., Garcia, G., Capron, F., Coulomb-Lhermine, A., Marfaing-Koka, A., Simonneau, G., Emilie, D., Humbert, M., 2002. Chemokine RANTES in severe pulmonary arterial hypertension. Am. J. Respir. Crit. Care Med. 165, 534–539. Dorfmuller, P., Perros, F., Balabanian, K., Humbert, M., 2003. Inflammation in pulmonary arterial hypertension. Eur. Respir. J. 22, 358–363. Dorfmuller, P., Humbert, M., Perros, F., Sanchez, O., Simonneau, G., Muller, K.M., Capron, F., 2007. Fibrous remodeling of the pulmonary venous system in pulmonary arterial hypertension associated with connective tissue diseases. Hum. Pathol. 38, 893–902. Eddahibi, S., Fabre, V., Boni, C., Martres, M.P., Raffestin, B., Hamon, M., Adnot, S., 1999. Induction of serotonin transporter by hypoxia in pulmonary vascular smooth muscle cells. Relationship with the mitogenic action of serotonin. Circ. Res. 84, 329–336. Eddahibi, S., Hanoun, N., Lanfumey, L., Lesch, K.P., Raffestin, B., Hamon, M., Adnot, S., 2000. Attenuated hypoxic pulmonary hypertension in mice lacking the 5hydroxytryptamine transporter gene. J. Clin. Invest. 105, 1555–1562. Eddahibi, S., Humbert, M., Fadel, E., Raffestin, B., Darmon, M., Capron, F., Simonneau, G., Dartevelle, P., Hamon, M., Adnot, S., 2001. Serotonin transporter overexpression is responsible for pulmonary artery smooth muscle hyperplasia in primary pulmonary hypertension. J. Clin. Invest. 108, 1141–1150. Eddahibi, S., Adnot, S., 2002. Anorexigen-induced pulmonary hypertension and the serotonin (5-HT) hypothesis: lessons for the future in pathogenesis. Respir. Res. 3, 9.

117

Eddahibi, S., Guignabert, C., Barlier-Mur, A.M., Dewachter, L., Fadel, E., Dartevelle, P., Humbert, M., Simonneau, G., Hanoun, N., Saurini, F., Hamon, M., Adnot, S., 2006. Cross talk between endothelial and smooth muscle cells in pulmonary hypertension: critical role for serotonin-induced smooth muscle hyperplasia. Circulation 113, 1857–1864. Fanburg, B.L., Lee, S.L., 1997. A new role for an old molecule: serotonin as a mitogen. Am. J. Physiol. 272, L795–806. Folkman, J., D'Amore, P.A., 1996. Blood vessel formation: what is its molecular basis? Cell 87, 1153–1155. Gershon, M.D., 1999. Review article: roles played by 5-hydroxytryptamine in the physiology of the bowel. Aliment Pharmacol. Ther. 13 (Suppl 2), 15–30. Ghofrani, H.A., Seeger, W., Grimminger, F., 2005. Imatinib for the treatment of pulmonary arterial hypertension. N. Engl. J. Med. 353, 1412–1413. Giaid, A., Yanagisawa, M., Langleben, D., Michel, R.P., Levy, R., Shennib, H., Kimura, S., Masaki, T., Duguid, W.P., Stewart, D.J., 1993. Expression of endothelin-1 in the lungs of patients with pulmonary hypertension. N. Engl. J. Med. 328, 1732–1739. Guignabert, C., Raffestin, B., Benferhat, R., Raoul, W., Zadigue, P., Rideau, D., Hamon, M., Adnot, S., Eddahibi, S., 2005. Serotonin transporter inhibition prevents and reverses monocrotaline-induced pulmonary hypertension in rats. Circulation 111, 2812–2819. Hamon, M., Bourgoin, S., Youdim, M., 1979. Tryptophan hydroxylation in the central nervous system and other tissues. In: Youdim, M.B.H. (Ed.), Amino Acid Hydrolases and Mental Disease. John Wiley Ltd, pp. 234–297. Hanahan, D., 1997. Signaling vascular morphogenesis and maintenance. Science 277, 48–50. Humbert, M., Monti, G., Brenot, F., Sitbon, O., Portier, A., Grangeot-Keros, L., Duroux, P., Galanaud, P., Simonneau, G., Emilie, D., 1995. Increased interleukin-1 and interleukin-6 serum concentrations in severe primary pulmonary hypertension. Am. J. Respir. Crit. Care Med. 151, 1628–1631. Humbert, M., Monti, G., Fartoukh, M., Magnan, A., Brenot, F., Rain, B., Capron, F., Galanaud, P., Duroux, P., Simonneau, G., Emilie, D., 1998. Platelet-derived growth factor expression in primary pulmonary hypertension: comparison of HIV seropositive and HIV seronegative patients. Eur. Respir. J. 11, 554–559. Humbert, M., Morrell, N.W., Archer, S.L., Stenmark, K.R., MacLean, M.R., Lang, I.M., Christman, B.W., Weir, E.K., Eickelberg, O., Voelkel, N.F., Rabinovitch, M., 2004. Cellular and molecular pathobiology of pulmonary arterial hypertension. J. Am. Coll. Cardiol. 43, 13S–24S. Jain, R.K., 2003. Molecular regulation of vessel maturation. Nat. Med. 9, 685–693. Jais, X., Launay, D., Yaic, i A., Le Pavec, J., Tcherakian, C., Sitbon, O., Simonneau, G., Humbert, M., 2008. Immunosuppressive therapy in lupus- and mixed connective tissue disease-associated pulmonary arterial hypertension: a retrospective analysis of twenty-three cases. Arthritis Rheum. 58, 521–531. Jouve, P., Humbert, M., Chauveheid, M.P., Jais, X., Papo, T., 2007. POEMS syndromerelated pulmonary hypertension is steroid-responsive. Respir. Med. 101, 353–355. Koe, B.K., Weissman, A., 1966. p-Chlorophenylalanine: a specific depletor of brain serotonin. J. Pharmacol. Exp. Ther. 154, 499–516. Launay, D., Hachulla, E., Hatron, P.Y., Jais, X., Simonneau, G., Humbert, M., 2007. Pulmonary arterial hypertension: a rare complication of primary Sjogren syndrome: report of 9 new cases and review of the literature. Medicine (Baltimore) 86, 299–315. Lee, S.L., Wang, W.W., Moore, B.J., Fanburg, B.L., 1991. Dual effect of serotonin on growth of bovine pulmonary artery smooth muscle cells in culture. Circ. Res. 68, 1362–1368. Lepetit, P., Touret, M., Grange, E., Gay, N., Bobillier, P., 1991. Inhibition of methionine incorporation into brain proteins after the systemic administration of pchlorophenylalanine and L-5-hydroxytryptophan. Eur. J. Pharmacol. 209, 207–212. MacLean, M.R., Herve, P., Eddahibi, S., Adnot, S., 2000. 5-hydroxytryptamine and the pulmonary circulation: receptors, transporters and relevance to pulmonary arterial hypertension. Br. J. Pharmacol. 131, 161–168. MacLean, M.R., Deuchar, G.A., Hicks, M.N., Morecroft, I., Shen, S., Sheward, J., Colston, J., Loughlin, L., Nilsen, M., Dempsie, Y., Harmar, A., 2004. Overexpression of the 5hydroxytryptamine transporter gene: effect on pulmonary hemodynamics and hypoxia-induced pulmonary hypertension. Circulation 109, 2150–2155. Montani, D., Achouh, L., Marcelin, A.G., Viard, J.P., Hermine, O., Canioni, D., Sitbon, O., Simonneau, G., Humbert, M., 2005. Reversibility of pulmonary arterial hypertension in HIV/HHV8-associated Castleman's disease. Eur. Respir. J. 26, 969–972. Mouthon, L., Guillevin, L., Humbert, M., 2005. Pulmonary arterial hypertension: an autoimmune disease? Eur. Respir. J. 26, 986–988. Nemecek, G.M., Coughlin, S.R., Handley, D.A., Moskowitz, M.A., 1986. Stimulation of aortic smooth muscle cell mitogenesis by serotonin. Proc. Natl. Acad. Sci. U S A 83, 674–678. Nicolls, M.R., Taraseviciene-Stewart, L., Rai, P.R., Badesch, D.B., Voelkel, N.F., 2005. Autoimmunity and pulmonary hypertension: a perspective. Eur. Respir. J. 26, 1110–1118. Patterson, K.C., Weissmann, A., Ahmadi, T., Farber, H.W., 2006. Imatinib mesylate in the treatment of refractory idiopathic pulmonary arterial hypertension. Ann. Intern. Med. 145, 152–153. Perros, F., Dorfmuller, P., Souza, R., Durand-Gasselin, I., Godot, V., Capel, F., Adnot, S., Eddahibi, S., Mazmanian, M., Fadel, E., Herve, P., Simonneau, G., Emilie, D., Humbert, M., 2007a. Fractalkine-induced smooth muscle cell proliferation in pulmonary hypertension. Eur. Respir. J. 29, 937–943. Perros, F., Dorfmuller, P., Souza, R., Durand-Gasselin, I., Mussot, S., Mazmanian, M., Herve, P., Emilie, D., Simonneau, G., Humbert, M., 2007b. Dendritic cell recruitment in lesions of human and experimental pulmonary hypertension. Eur. Respir. J. 29, 462–468. Perros, F., Montani, D., Dorfmüller, P., Durand-Gasselin, I., Tcherakian, C., Le Pavec, J., Mazmanian, M., Fadel, E., Mussot, S., Mercier, O., Hervé, P., Emilie, D., Eddahibi, S., Simonneau, G., Souza, R., Humbert, M., 2008. Platelet-derived growth factor

118

M. Humbert et al. / Vascular Pharmacology 49 (2008) 113–118

expression and function in idiopathic pulmonary arterial hypertension. Am. J. Respir. Crit. Care Med. 178, 81–88. Sakowski, S.A., Geddes, T.J., Thomas, D.M., Levi, E., Hatfield, J.S., Kuhn, D.M., 2006. Differential tissue distribution of tryptophan hydroxylase isoforms 1 and 2 as revealed with monospecific antibodies. Brain Res. 1085, 11–18. Sanchez, O., Sitbon, O., Jais, X., Simonneau, G., Humbert, M., 2006. Immunosuppressive therapy in connective tissue diseases-associated pulmonary arterial hypertension. Chest 130, 182–189. Sanchez, O., Marcos, E., Perros, F., Fadel, E., Tu, L., Humbert, M., Dartevelle, P., Simonneau, G., Adnot, S., Eddahibi, S., 2007. Role of endothelium-derived CC chemokine ligand 2 in idiopathic pulmonary arterial hypertension. Am. J. Respir. Crit. Care Med. 176, 1041–1047. Schermuly, R.T., Dony, E., Ghofrani, H.A., Pullamsetti, S., Savai, R., Roth, M., Sydykov, A., Lai, Y.J., Weissmann, N., Seeger, W., Grimminger, F., 2005. Reversal of experimental pulmonary hypertension by PDGF inhibition. J. Clin. Invest. 115, 2811–2821. Souza, R., Sitbon, O., Parent, F., Simonneau, G., Humbert, M., 2006. Long term imatinib treatment in pulmonary arterial hypertension. Thorax 61, 736.

Sullivan, C.C., Du, L., Chu, D., Cho, A.J., Kido, M., Wolf, P.L., Jamieson, S.W., Thistlethwaite, P.A., 2003. Induction of pulmonary hypertension by an angiopoietin 1/TIE2/ serotonin pathway. Proc. Natl. Acad. Sci. U S A 100, 12331–12336. Tamby, M.C., Chanseaud, Y., Humbert, M., Fermanian, J., Guilpain, P., Garcia-de-la-PenaLefebvre, P., Brunet, S., Servettaz, A., Weill, B., Simonneau, G., Guillevin, L., Boissier, M.C., Mouthon, L., 2005. Anti-endothelial cell antibodies in idiopathic and systemic sclerosis associated pulmonary arterial hypertension. Thorax 60, 765–772. Tamby, M.C., Humbert, M., Guilpain, P., Servettaz, A., Dupin, N., Christner, J.J., Simonneau, G., Fermanian, J., Weill, B., Guillevin, L., Mouthon, L., 2006. Antibodies to fibroblasts in idiopathic and scleroderma-associated pulmonary hypertension. Eur. Respir. J. 28, 799–807. Tuder, R.M., Groves, B., Badesch, D.B., Voelkel, N.F., 1994. Exuberant endothelial cell growth and elements of inflammation are present in plexiform lesions of pulmonary hypertension. Am. J. Pathol. 144, 275–285. Walther, D.J., Peter, J.U., Bashammakh, S., Hortnagl, H., Voits, M., Fink, H., Bader, M., 2003. Synthesis of serotonin by a second tryptophan hydroxylase isoform. Science 299, 76.