CHEST Translating Basic Research Into Clinical Practice

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1 CHEST Translating Basic Research Into Clinical Practice Molecular Mechanisms of Pulmonary Arterial Hypertension* Role of Mutations in the Bone Morphogenetic Protein Type II Receptor Rachel J. Davies, MD; and Nicholas W. Morrell, MD Pulmonary arterial hypertension (PAH) is characterized by abnormal remodeling of small, peripheral resistance vessels in the lung involving proliferation and migration ofvascular smooth muscle, endothelial cell and fibroblasts. The increase in pulmonary vascular resistance leads to right heart failure, and, without treatment, death occurs within 3 years ofdiagnosis. The etiology of PAH is multifactorial. In some patients, there is a major genetic predisposition in the form of heterozygous germline mutations in a transforming growth factor-p superfamily receptor, the bone morphogenetic type II receptor (BMPR-II). In addition, it is likely that additional factors, such as inflammation, are important to manifest disease. The currently available treatments for PAH were developed mainly as vasodilators, and although they improve symptoms they have limited impact on survival. This review examines the role ofthe BMPR-II signaling pathway in the process of pulmonary vascular remodeling. We discuss the ways in which manipulation of BMPR-II signaling might be targeted with the aim of preventing or reversing vascular remodeling and improving survival in patients with PAH. (CHEST 2008; 134: ) Key words: bone morphogenetic type II receptor; familial pulmonary hypertension; pulmonary vascular remodeling Abbreviations: ALK = activin receptor-like kinase; BMP = bone morphogenetic protein; BMPR = bone morphogenetic protein receptor; IPAH = idiopathic pulmonary arterial hypertension; PAH = pulmonary arterial hypertension; PASMC = pulmonary artery smooth muscle cell; PDGF = platelet-derivedgrowth factor; TGF = transforming growth factor I diopathic pulmonary arterial hypertension (IPAH) is a rare, but devastating condition. It is estimated to affect 2 to 3 individuals per million per year' and typically affects young women (female/male ratio, 2.3:1).2 The occlusion of small, peripheral pulmonary arteries leads to a persistently elevated pulmonary 'From the Department of Medicine, University of Cambridge, Addenbrooke's Hospital, Cambridge, UK. The authors have reported to the ACCP that no significant conflicts of interest exist with any companies/organizations whose products or services may be discussed in this article. Manuscript received May 27, 2008; revision accepted July 9, Reproduction of this article is prohibited without written permission from the American College of Chest Physicians ( orglmisc/reprints.shtml). Correspondence to: Nicholas W. Morrell, MD, Department of Medicine, University ofcambridge, Box 157, LevelS, Addenbrooke's Hospital, Hills Rd, Cambridge CB2 2QQ, UK; T1wm23@cam.ac.uk 001: IO.1378/chest I vascular resistance, a raised pulmonary arterial pressure, and ultimately death from right heart failure. Before the modern treatment era, therapy was usually symptomatic and life expectancy was short." The term pulmonary arterial hypertension (PAH)4 refers to a variety of forms of precapillary pulmonary hypertension. PAH is known to be associated with systemic diseases such as collagen vascular disease, HIV infection, or the ingestion of drugs or toxins. It may also be found in association with congenital heart disease. IPAH (formerly known as primary pulmonary hypertension) remains a diagnosis of exclusion. In 6 to 10% of cases of IPAH, there is more than one affected family member, and the condition is referred to as familial PAH or heritable PAH. Despite the diverse etiology of PAH in these subdivisions, the group may share a common CHEST/134/6/ DECEMBER,

2 pathogenesis and as such may be amenable to new advances in therapeutic approaches to the disease. This review aims to present a major advance in our understanding of the pathogenesis of PAR, namely, the discovery that the majority of familial cases of the disease are due to mutations in a transforming growth factor (TGF)-13 superfamily receptor, bone morphogenetic protein receptor (BMPR) type II. We shall discuss how this discovery has furthered our understanding of PAR as well as the potential therapeutic approaches aimed at rescuing this signaling pathway. THE PATHOLOGY OF PAR The pathogenesis of PAR is characterized by vascular remodeling, in situ thrombus formation, and varying degrees of inflammation," Vascular remodeling occurs as a result of abnormal proliferation, and the survival of vascular smooth muscle cells and myoflbroblasts.s leading to medial hypertrophy, concentric intimal lesions, and a thickened adventitia? (Fig 1). Altered proliferation and survival of endothelial cells contributes to the formation of so-called plexifonn lesions. 8 Endothelial dysfunction leads to changes in the release of mediators of vascular tone. In the pulmonary circulation of healthy persons, there is a finely regulated balance between locally released and circulating vasoconstrictive agents and vasodilators. In contrast, pulmonary hypertension is associated with increased levels ofthe vasoconstrictors endothelin-i," angiotensin 11,3 and serotonin.!" concomitant with a reduction in vasodilators or their Signaling pathways, such as nitric oxide signaling ll and prostaglandins.v The resulting imbalance favors the progression of pulmonary hypertension and has become a useful target for therapy. A further characteristic abnormality of PAR is the development of an in situ thrombus. It has been widely reported'" that patients with PAR have a procoagulant state with evidence for abnormal platelet function, which provides the basis for the frequent use of anticoagulants in patients with PAR. Despite the development of several new therapies for PAR in recent years, the majority of these drugs primarily serve to redress the imbalance between vasoconstrictor and vasodilator pathways. They may also, but to a lesser extent, impact on the process of vascular remodeling. The clinical course of the majority of patients remains one of eventual deterioration, suggesting that vascular remodeling eventually progresses. GENETICS OF FAMILIAL PAR Up to 10% of patients with idiopathic PAR have a family history of the disease.l- Segregation studies FIGURE 1. The pathogenesis of PAH: an illustration representing the progressive muscularization of small, peripheral pulmonary arteries. With time, abnormal proliferation of vascular smooth muscle cells and altered apoptosislgrowth of endothelial cells reduces the lumen diameter of the vessel, leading to a rise in pulmonary vascular resistance and hence to an elevated pulmonary artery pressure. The immunohistochemical image shows a typical "onion skin-like" concentric lesion. The artery now has a thick muscularized intima, as demonstrated by the heavy staining for the smooth muscle marker a-smooth muscle actin Translating Basic Research Into Clinical Practice

3 have demonstrated an autosomal-dominant pattern of inheritance with reduced penetrance. In 2000, two independent groups15,16 identified heterozygous germline mutations in the BMPR-II in families with PAH. Since this discovery, mutations in the BMPR2 gene have been found in approximately 80% of cases offamilial PAHP Patients with apparently sporadic PAH may also harbor BMPR-II mutations in 11 to 40% of cases. The often markedly reduced penetrance in families with PAH provides evidence that some form of "second hit" is required in addition to the mutation to lead to the manifestation of clinical disease. Studies in heterozygous BMPR2 knockout mice have suggested that additional hits could include inflammatory insults-" or the increased activity of, for example, serotonin.l? Such second hits may further compound the deficiency in BMPR function by impacting on receptor expression, downstream Signaling, or gene transcription. Intriguingly, mutations in the TGF-13 type I receptor, activin receptorlike kinase (ALK)-I, which is usually found in patients with type 2 hereditary hemorrhagic telangiectasia, have also been identified in patients with severe PAH.20 It is becoming increasingly apparent that dysfunctional BMPR Signaling is a feature of diverse forms of PAH. For example, the expression of BMPR-II protein is markedly reduced in the lungs of patients with idiopathic PAH with no detectable mutation in BMPR-IJ.7 Reduced expression ofbmpr-ii has also been found in the lungs of rats with monocrotalineinduced pulmonary hypertension. This is associated with functional effects on downstream bone morphogenetic protein (BMP) signaling. 21 These studies have suggested that a critical reduction in the expression of BMPR-II or a reduction in BMPR-II Signaling may be important in the pathogenesis of PAH, whether or not there is a mutation in the BMPR2 gene. NORMAL BMPR-II SIGNALING In common with other type II receptor members of the TGF-13 superfamily, BMPR-II is a constitutively active serine-threonine receptor kinase. The type II receptor activates an associated type I receptor (BMPR-IA [or ALK-3] or BMPR-IB [or ALK-6]) in the presence of ligand (BMPs 2, 4,6, 7, and 9, and growth differentiation factors 5 and 6). The type I receptor phosphorylates the downstream Signaling molecules, termed Smads 1, 5, and 8. BMP9 has been identified 22 as a ligand for a receptor complex comprising BMPR-II and ALK-l, providing new insight into the link between PAH and hereditary hemorrhagic telangiectasia. The phosphorylated Smads are translocated to the nucleus in a complex with a common partner Smad (Smad4) where they regulate the transcription of multiple genes (Fig 2). Smad-regulated genes have profound effects on developmental processes ranging from soft tissue and skeletal development to vasculogenesis Homozygosity for a null allele for the BMPR2 gene is lethal to the embryo in early gestation, whereas heterozygotes appear normal.w There is a strong gene dosage effect since mice expressing approximately 10% of the normal levels of BMPR-II survive to adulthood but show features of abnormal blood vessel development. Smad-regulated genes are known to play significant roles in the maintenance and repair of adult tissues by controlling the growth, differentiation, and apoptosis of a wide range of cells It is thought that a critical alteration in the control of BMP-regulated genes in pulmonary vascular smooth muscle and endothelial cells predisposes patients to the development of pulmonary vascular remodeling. THE FUNCTIONAL CONSEQUENCES OF MUTATIONS IN BMPR-II The majority of mutations in BMPR-II (approximately 70%) are nonsense or frameshift mutations, which are likely to result in the transcript being degraded by nonsense-mediated messenger RNA decay. Thus, only the nonmutant allele results in the production of protein, leading to a state of haploinsufficiency.22 About 30% of mutations lead to missense mutations in highly conserved amino acids in functional domains of the receptor. Some of these interrupt trafficking of the receptor to the cell surface.s? Kinase domain mutations lead to a failure to activate the type I receptor. Missense mutations also occur in the long cytoplasmic tail of BMPR-II. Studies 28 in cells from patients harboring mutations in BMPR-II demonstrate that all mutations lead to a reduced capacity for Smad phosphorylation and the reduced transcription of BMP target genes. Mutations in BMPR-II lead to the disruption of BMP-mediated function, including growth, migration, and differentiation. Normal pulmonary artery smooth muscle cells (PASMCs) isolated from lobar pulmonary arteries have growth inhibited by BMPs 2,4, and 7, and are stimulated to undergo apoptosis, whereas, lobar PASMCs from patients with mutations in BMPR-II are resistant to these growth inhibitory effects. 28 Remarkably, PASMCs harboring BMPR-II mutations are also resistant to the growth inhibitory effects oftgf-i3, which may be a further mechanism by which these cells evade homeostatic controls on cell turnover.w Increased expression and activity oftgf-13 is known to occurin the hypertensive pulmonary circulation.p? CHEST /134 / 6 / DECEMBER,

4 FIGURE 2. Normal TGF-[3 superfamily signaling. A diagrammatic representation of the expression of type I and type II TGF-[3 superfamily receptors on pulmonary artery smooth muscle and endothelial cells. Activation of the receptors by specific ligands leads to the phosphorylation of Smad second messengers. Phosphorylated receptor Smads combine with the common Smad, Smad 4, before the complex is translocated to the nucleus, where it leads to the transcription of multiple genes. NO = nitric oxide; enos = endothelial nitric oxide synthase; GTP = guanosine triphosphate; GMP = guanosine monophosphate; cgmp = cyclic guanosin monophosphate; ATP = adenosine triphosphate; AMP = adenosine monophosphate; camp = cyclic adenosine monophosphate, ET = endothelin; PDE = phosphodiesterase. PAH is thought to be associated with early endothelial apoptosis but also with the subsequent survival of apoptosis-resistant clones of endothelial cells, possibly giving rise to plexiform lesions. In contrast to the effects in PASMCs, endothelial cells proliferate, migrate, and form tubular structures in response to BMP4 via a Smad li5-dependent mechanism.v! In addition, BMPs protect endothelial cells from apoptosis. 32 These effects occur directly via BMPR-II as knocking down BMPR-II by small interfering RNA increases the susceptibility of endothelial cells to apoptosis. Hence, the loss of BMPR-II signaling is likely to playa significant role in the early endothelial dysfunction observed in patients with PAH. POTENTIAL TREATMENTS TARGETING THE BMPR-II SIGNALING PATHWAY Studies have demonstrated that at least some mutations in the BMPR-II lead to retention of the mutant protein in the endoplasmic reticulum. This occurs especially if cysteine residues are substituted. Preliminary results suggest the potential for rescue of the mutant protein and restoration of signaling by chemical chaperones. Studies in animal models-! have demonstrated that BMPR-II levels may also be reduced at the transcriptional level. Thus, restoration of BMPR-II gene expression by gene therapy is an attractive proposition. In 2006, Reynolds et al 3 :3 developed an adenoviral vector containing a BMPR2 gene that was targeted to the pulmonary endothelium. IV administration of the adenovirus linked to an antibody targeting angiotensin-converting enzyme was used to deliver the BMPR2 gene to the lungs. Rats injected with the BMPR2-Ad virus prior to exposure to 3 weeks of hypoxia had significantly lower pulmonary artery pressures at the end of the study in comparison with mock-infected controls. A further study,34 attempted to ameliorate monocrotalineinduced pulmonary hypertension in rats by nebulized delivery of an adenoviral vector containing BMPR2. Animals were given intratracheal adenovirus encoding BMPR2 14 days after intraperitoneal injection of monocrotaline. There was no reduction in pulmonary artery pressure in the treated animals comparedwith controls, though one limitation ofthis study would be the lack of endothelial targeting. Further studies are needed in this important area to determine whether targeted delivery of BMPR-II prevents the reversal of PAR in animal models Translating Basic Research Into Clinical Practice

5 Statins have long been known to suppress vascular inflammation and vascular smooth muscle cell proliferation through a variety of mechanisms." Simvastatin has been shown to both ameliorate the development of monocrotaline-induced pulmonary hypertension and reduce existing disease in the rat model. A series of case reports of patients with idiopathic and secondary pulmonary hypertension treated with simvastatin in conjunction with standard treatment has also shown a potential limited ability for statins to halt the progression of disease. Hu et a1 36 have shown that simvastatin increases the transcription of BMPR2and increases posttranscriptional messenger RNA stabilization leading to increased BMPR-II protein expression. Thus, the stabilization of BMPR2 transcription may offer a further therapeutic approach in PAH patients. A further approach that is yet to be tested is the use of recombinant BMP ligand. At least in cell cultures, some of the defects in BMP signaling in BMPR-II mutant cells can be overcome by increasing the concentration of the ligand. In vivo studies in animal models are required to provide proof of concept for this approach. INHIBITING TGF-13 SIGNALING There is evidence for enhanced TGF-13 Signaling in the setting of pulmonary hypertension. In addition, PASMCs from patients with idiopathic PAH are resistant to the antiproliferative effects of TGF-I3. This is also apparent in PASMCs harboring mutations in BMPR-II or cells in which BMPR-II expression has been knocked down by RNA interference. The precise mechanisms behind these observations are under investigation, but they raise the possibility that blocking the TGF-13 type I receptor, ALK 5, may be of potential therapeutic benefit. Indeed, Zaiman et a1 30 used a small molecule inhibitor of ALK5 in the rat monocrotaline model. Rats that were given the inhibitor 14 days after monocrotaline administration did not develop pulmonary hypertension; whereas, the control monocrotaline-treated animals had Significantly raised pulmonary artery pressures. The treated group also had decreased early vascular cell apoptosis, reduced adventitial cell proliferation, and reduced metalloprotease expressions, which are all markers of vascular remodeling. THE PLATELET-DERIVED GROWTH FACTOR PATHWAY Abnormal TGF-13 Signaling has been linked to alterations in the expression and Signaling of plateletderived growth factors (PDGFs). PDGF, independently of TGF-13 Signaling, has emerged as an important pathway in the pathogenesis of pulmonary hypertension. Hypoxia is known-'? to induce PDGF FIGURE 3. The future of treatment for PAH. A diagram depicting the role of the BMPR-II and POCF pathways involved in the pathogenesis of PAH (black) and how novel therapies may reduce vascular remodeling (blue). PASMC = pulmonary arterial smooth muscle cell; COF = growth differentiation factor; TCFR = TCF receptor; POCFR = POCF receptor; MMP = matrix metalloprotease. CHEST/134/6/ DECEMBER,

6 gene expression in cultured vascular endothelial cells derived from bovine pulmonary arteries and human umbilical vein. In addition, rats that developed pulmonary hypertension following long-term exposure to hypoxia demonstrate increased expression of PDGF-A and PDGF-B isoforms.v' and lung biopsy specimens from patients with severe pulmonary hypertension show Significantly increased PDGF-A expression.39 PDGF may contribute to pulmonary vascular remodeling via several mechanisms. First, PDGF is a potent mitogen, causing increased PASMC proliferation, mainly by the activation of extracellular signalrelated kinase 1/2 pathway.s? Interestingly extracellular signal-related kinase 1/2 activation can exert an inhibitory effect on Smad 1/5 Signaling, thus compounding the defects due to the loss of BMPR-II. In addition, PDGF increases cell migration and extracellular matrix deposition by inducing the expression of metallomatrix proteases, particularly matrix metalloproteinase 1, 3, and 9. PDGF also potently inhibits apoptosis in vascular smooth muscle cells through the phosphatidylinositol 3 kinase (or PI3K)1 Akt pathway." Furthermore, TGF-~ is recognized to increase the expression of PDGF isoforms, particularly in scleroderma. Imatinib, a tyrosine kinase inhibitor licensed for the treatment of chronic myeloid leukemia, has been investigated as a possible therapy for PAH. Imatinib is a small molecular inhibitor that targets the adenosine triphosphate binding site of tyrosine kinases, thus blocking the actions of PDGF receptors as well as other kinases. Schermuly et al 42 used the monocrotaline rat model and a mouse hypoxic model to demonstrate that animals treated with imatinib had significantly reduced pulmonary artery pressures, higher cardiac index, increased arterial oxygenation, and improved survival when compared to sham-treated animals. The inhibition ofpasmc proliferation was observed in the imatinib-treated groups. A Single clinical case study43 in a patient with familial PAH showed dramatic improvements after imatinib therapy in 6-min walk distance as well as in a reduction in pulmonary vascular resistance. Two further successful casesv' of longer term treatment have since been reported. A multicenter randomized trial of imatinib therapy is due to report later this year. CONCLUSIONS The pathogenesis of PAH is clearly complex, involving abnormal growth and the survival of pulmonary vascular smooth muscle, fibroblasts, myofibroblasts, and endothelial cells. This review has highlighted the more recent advances in our under- standing of the role of BMPR-II signaling and has suggested potentially new therapeutic approaches based on our improved understanding of these mechanisms. Figure 3 summarizes the interaction among the BMPtfGF-B signaling pathways and receptor tyrosine kinases, and indicates potential points for intervention with new therapies. 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