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` new england journal
`The
`
`of
`
` medicine
`
`review article
`
`mechanisms of disease
`Pulmonary Arterial Hypertension
`
`Harrison W. Farber, M.D., and Joseph Loscalzo, M.D., Ph.D.
`
`p
`
`ulmonary hypertension is usually classified as primary (idio
`-
`1
` It is now clear, however, that there are conditions within
`pathic) or secondary.
`the category of secondary pulmonary hypertension that resemble primary pul-
`monary hypertension in their histopathological features and their response to treatment.
`For this reason, the World Health Organization (WHO) classified pulmonary hyperten-
`sion into five groups on the basis of mechanisms, rather than associated conditions
`(Table 1). The most recent revision of the WHO classification uses consistent termi-
`2
`nology and defines pulmonary hypertension more precisely than previous versions.
`Group I of the WHO classification, designated pulmonary arterial hypertension, is the
`principal focus of this review.
`Pulmonary arterial hypertension is defined as a sustained elevation of pulmonary
`arterial pressure to more than 25 mm Hg at rest or to more than 30 mm Hg with ex-
`ercise, with a mean pulmonary-capillary wedge pressure and left ventricular end-dia-
`3
`stolic pressure of less than 15 mm Hg.
` Pulmonary arterial hypertension comprises
`idiopathic pulmonary arterial hypertension (formerly, primary pulmonary hyperten-
`sion); pulmonary arterial hypertension in the setting of collagen vascular disease (e.g.,
`in localized cutaneous systemic sclerosis, also known as the CREST syndrome [cal-
`cinosis cutis, Raynaud’s phenomenon, esophageal dysfunction, sclerodactyly, and tel-
`angiectasia]), portal hypertension, congenital left-to-right intracardiac shunts, and
`infection with the human immunodeficiency virus (HIV); and persistent pulmonary
`hypertension of the newborn. The histologic appearance of lung tissue in each of these
`conditions is similar: intimal fibrosis, increased medial thickness, pulmonary arteriolar
`4
`occlusion, and plexiform lesions predominate.
`Although the pathogenesis of most forms of pulmonary arterial hypertension is un-
`known, there have been many recent developments, especially pertaining to the mo-
`lecular genetics and cell biology of idiopathic pulmonary arterial hypertension. In this
`review, we discuss these developments and relate them to other forms of pulmonary ar-
`terial hypertension, when appropriate. Treatment is discussed briefly as it relates to the
`disease mechanism; more information on treatment can be found in recent reviews of
`5,6
`this topic.
`
`From the Evans Department of Medicine
`(H.W.F., J.L.), the Pulmonary Center (H.W.F.),
`and the Whitaker Cardiovascular Institute
`(J.L.), Boston University School of Medi-
`cine, Boston. Address reprint requests to
`Dr. Loscalzo at the Department of Medicine,
`Boston University School of Medicine, 88
`E. Newton St., Boston, MA 02118, or at
`jloscalz@bu.edu.
`
`N Engl J Med 2004;351:1655-65.
`Copyright © 2004 Massachusetts Medical Society.
`
`imbalance of vascular effectors
`
`The main vascular changes in pulmonary arterial hypertension are vasoconstriction,
`smooth-muscle cell and endothelial-cell proliferation, and thrombosis. These findings
`suggest the presence of perturbations in the normal relationships between vasodila-
`tors and vasoconstrictors, growth inhibitors and mitogenic factors, and antithrombotic
`and prothrombotic determinants. These homeostatic imbalances are probably conse-
`quences of pulmonary endothelial-cell dysfunction or injury.
`
`n engl j med
`
`351;16
`
`www.nejm.org october
`
`14, 2004
`
`1655
`
`Downloaded from www.nejm.org at TEXAS TECH UNIVERSITY HEALTH SCIENCES CENTER on April 20, 2008 .
`Copyright © 2004 Massachusetts Medical Society. All rights reserved.
`
`Liquidia's Exhibit 1051
`Page 1
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`
` new england journal
`The
`
` medicine
`of
`
`prostacyclin and thromboxane a
`2
`Prostacyclin and thromboxane A
` are major arachi-
`2
`donic acid metabolites of vascular cells. Prostacy-
`clin, a potent vasodilator, inhibits platelet activation
`and has antiproliferative properties; in contrast,
`thromboxane A
` is a potent vasoconstrictor and
`2
`7
`platelet agonist.
` In pulmonary arterial hyperten-
`sion, the imbalance between these two molecules
`8
`is shifted toward thromboxane A
`: in the urine of
`2
`patients with pulmonary hypertension, the levels
`of a metabolite of prostacyclin (6-keto-prostacy-
`) are decreased, whereas the levels of a me-
`clin F
`2
`a
`tabolite of thromboxane A
` (thromboxane B
`) are
`2
`2
`increased. Furthermore, the production of pros-
`tacyclin synthase is decreased in the small and me-
`dium-sized pulmonary arteries of patients with
`pulmonary hypertension, particularly those with id-
`9
`iopathic pulmonary arterial hypertension.
`
`Table 1. The Revised World Health Organization Classification of Pulmonary
`Hypertension.*
`
`Group I. Pulmonary arterial hypertension
`Idiopathic (primary)
`Familial
`Related conditions: collagen vascular disease, congenital systemic-
`to-pulmonary shunts, portal hypertension, HIV infection, drugs
`l
`and toxins (e.g., anorexigens, rapeseed oil,
`-tryptophan, metham-
`phetamine, and cocaine); other conditions: thyroid disorders, gly-
`cogen storage disease, Gaucher’s disease, hereditary hemorrhagic
`telangiectasia, hemoglobinopathies, myeloproliferative disorders,
`splenectomy
`Associated with significant venous or capillary involvement
`Pulmonary veno-occlusive disease
`Pulmonary-capillary hemangiomatosis
`Persistent pulmonary hypertension of the newborn
`Group II. Pulmonary venous hypertension
`Left-sided atrial or ventricular heart disease
`Left-sided valvular heart disease
`Group III. Pulmonary hypertension associated with hypoxemia
`Chronic obstructive pulmonary disease
`Interstitial lung disease
`Sleep-disordered breathing
`Alveolar hypoventilation disorders
`Chronic exposure to high altitude
`Developmental abnormalities
`Group IV. Pulmonary hypertension due to chronic thrombotic disease, embol-
`ic disease, or both
`Thromboembolic obstruction of proximal pulmonary arteries
`Thromboembolic obstruction of distal pulmonary arteries
`Pulmonary embolism (tumor, parasites, foreign material)
`Group V. Miscellaneous
`Sarcoidosis, pulmonary Langerhans’-cell histiocytosis, lymphangio-
`matosis, compression of pulmonary vessels (adenopathy, tumor,
`fibrosing mediastinitis)
`
`* The table has been adapted from Simonneau et al.
`
`2
`
`endothelin-1
`Endothelin-1, a potent vasoconstrictor, stimulates
`the proliferation of pulmonary-artery smooth-mus-
`10,11
`cle cells.
` The plasma levels of endothelin-1 are
`12-14
`increased in pulmonary arterial hypertension,
`and the level of endothelin-1 is inversely propor-
`tional to the magnitude of the pulmonary blood
`flow and cardiac output, suggesting that these he-
`modynamic changes are influenced directly by this
`vascular effector.
`
`nitric oxide
`The synthesis of nitric oxide, a potent vasodila-
`tor and inhibitor of platelet activation and vascular
`smooth-muscle cell proliferation, is catalyzed by
`the family of nitric oxide synthase enzymes. De-
`creased levels of the endothelial isoform of nitric
`oxide synthase have been observed in the pulmo-
`nary vascular tissue of patients with pulmonary hy-
`pertension, particularly those with idiopathic pul-
`15,16
` Endothelial nitric
`monary arterial hypertension.
`oxide synthase is, however, increased in the plexi-
`form lesions of idiopathic pulmonary arterial hy-
`pertension, where it probably promotes pulmonary
`17
`endothelial-cell proliferation.
`
`serotonin
`Serotonin (5-hydroxytryptamine) is a vasocon-
`strictor that promotes smooth-muscle cell hyper-
`18
`trophy and hyperplasia.
` Elevated levels of plas-
`ma serotonin and reduced content of serotonin in
`platelets have been found in idiopathic pulmonary
`19
`arterial hypertension
` and persist even after the
`normalization of pulmonary-artery pressures fol-
`lowing lung transplantation. A platelet defect that
`results in a reduced uptake of serotonin (i.e.,
` stor-
`d
`age pool disease) has been associated with pulmo-
`20
`nary hypertension.
` Among patients who took the
`appetite suppressant dexfenfluramine, which in-
`creases the release of serotonin from platelets and
`inhibits its reuptake, for more than three months,
`the incidence of pulmonary arterial hypertension
`21
`increased.
` Recently, mutations in the serotonin
`transporter (5-HTT), the 5-hydroxytryptamine 2b
`receptor (5-HT2B), or both have been described in
`platelets and lung tissue from patients with pulmo-
`22
` Nevertheless, the lev-
`nary arterial hypertension.
`el of serotonin itself is probably not a determinant
`of pulmonary hypertension, because selective sero-
`tonin-reuptake inhibitors (SSRIs), which increase
`serotonin levels but inhibit serotonin transport, are
`
`1656
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`n engl j med
`
`351;16
`
`www.nejm.org october
`
`,
`
`14
`
`2004
`
`Downloaded from www.nejm.org at TEXAS TECH UNIVERSITY HEALTH SCIENCES CENTER on April 20, 2008 .
`Copyright © 2004 Massachusetts Medical Society. All rights reserved.
`
`Liquidia's Exhibit 1051
`Page 2
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`mechanisms of disease
`
`not associated with an increased incidence of pul-
`monary hypertension and may, in fact, be protec-
`23
`tive in the setting of hypoxia.
`
`adrenomedullin
`Adrenomedullin dilates pulmonary vessels, increas-
`es the pulmonary blood flow, and is synthesized by
`several cell populations in the normal lung. High
`levels of messenger RNA (mRNA) for adrenomed-
`ullin and its receptor in the lung suggest a homeo-
`static role for this peptide in the pulmonary circula-
`24
`tion.
` The plasma levels of adrenomedullin are
`elevated in both pulmonary arterial hypertension
`and pulmonary hypertension associated with hy-
`25,26
`poxemia,
` and the elevation correlates with in-
`creases in the mean right atrial pressure, pulmo-
`nary vascular resistance, and the mean pulmonary
`27
` Current data suggest, however,
`arterial pressure.
`that increased adrenomedullin is a marker of pul-
`monary hypertension, rather than a cause.
`
`vasoactive intestinal peptide
`Vasoactive intestinal peptide, a potent systemic
`vasodilator, decreases pulmonary-artery pressure
`and pulmonary vascular resistance in rabbits with
`28
`monocrotaline-induced pulmonary hypertension
`29
`and in healthy human subjects
`; it also inhibits
`30
`platelet activation
` and vascular smooth-mus-
`31
` A recent study reported de-
`cle cell proliferation.
`creased levels of vasoactive intestinal peptide in the
`serum and the lungs in patients with pulmonary
`arterial hypertension; treatment with inhaled
`vasoactive intestinal peptide improved the clinical
`32
`course and the hemodynamics in these patients.
`
`vascular endothelial growth factor
`In acute and chronic hypoxia, the production of
`vascular endothelial growth factor (VEGF) is in-
`creased and that of its receptors, VEGF receptor-1
`(kinase-domain related [KDR], or Flk) and VEGF
`33
`receptor-2 (Flt), in the lung.
` In pulmonary arte-
`rial hypertension, disordered angiogenic respons-
`es appear to underlie the formation of plexiform
`lesions and the clonal expansion of endothelial
`cells within the lesions. VEGF mRNA and protein
`have been detected in such lesions along with in-
`creased amounts of VEGF receptor-2, hypoxia-
`inducible factor
`, and hypoxia-inducible factor
`a
`b
`and decreased amounts of three signaling mole-
`cules essential for the angiogenic response to VEGF,
`34
` These
`phosphoinositide-3-kinase, Akt, and src.
`
`observations suggest an abnormal angiogenic re-
`sponse to hypoxia owing to abnormal signaling
`responses in pulmonary arterial hypertension.
`In summary, there is an imbalance of the vas-
`cular effectors in pulmonary arterial hypertension
`that favors vasoconstriction, vascular-cell prolif-
`eration, and thrombosis (Fig. 1). The treatments
`developed on the basis of these observations (i.e.,
`epoprostenol, nitric oxide, and endothelin-recep-
`tor antagonists) have been effective in improving
`the pulmonary vascular hemodynamics, clinical sta-
`tus, and, in some cases, survival in idiopathic and
`other forms of pulmonary arterial hypertension.
`None of these vasoactive molecules, however, have
`yet been conclusively linked to the primary patho-
`genesis of the disease.
`
`
`associated environmental associated environmental
`factors
`factors
`
`Among the environmental factors associated with
`an increased risk of the development of pulmo-
`nary arterial hypertension, three — hypoxia, ano-
`rexigens, and central nervous system stimulants —
`have plausible mechanistic underpinnings.
`
`hypoxia
`Hypoxia induces vasodilation in systemic vessels,
`but it induces vasoconstriction in the pulmonary
`vasculature. The acute effect of hypoxia is regulat-
`ed, in part, by two endothelial cell–derived vaso-
`constrictors, endothelin and serotonin, and, in part,
`by hypoxia-mediated changes in ion-channel ac-
`
`Vasoconstriction Cell Proliferation
`
`Thrombosis
`
`Increased TxA2
`Decreased PGI2
`
`Increased VEGF
`
`Decreased PGI2
`
`Increased TxA2
`Decreased PGI2
`
`Decreased NO
`
`Decreased NO
`
`Decreased NO
`
`Increased ET-1
`
`Increased ET-1
`
`Increased 5-HT
`
`Increased 5-HT
`
`Increased 5-HT
`
`Decreased VIP
`
`Decreased VIP
`
`Decreased VIP
`
`Figure 1. Mediators of Pulmonary Vascular Responses
`in Pulmonary Arterial Hypertension.
`
`TxA
` denotes thromboxane A
`, PGI
` prostaglandin I
`2
`2
`2
`2
`(prostacyclin), NO nitric oxide, ET-1 endothelin-1, 5-HT
`5-hydroxytryptamine (serotonin), VEGF vascular endothe-
`lial growth factor, and VIP vasoactive intestinal peptide.
`
`n engl j med
`
`351;16
`
`www.nejm.org october
`
`14, 2004
`
`1657
`
`Downloaded from www.nejm.org at TEXAS TECH UNIVERSITY HEALTH SCIENCES CENTER on April 20, 2008 .
`Copyright © 2004 Massachusetts Medical Society. All rights reserved.
`
`Liquidia's Exhibit 1051
`Page 3
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`
` new england journal
`The
`
` medicine
`of
`
`tivity in smooth-muscle cells in the pulmonary
`35
`arteries.
` Acute hypoxia inhibits the function of
`voltage-gated potassium channels in these smooth-
`muscle cells, resulting in membrane depolariza-
`tion, an increase in the concentration of cytoplas-
`36
`mic calcium, and vasoconstriction.
` Acute hypoxia
`causes reversible changes in vascular tone, where-
`as chronic hypoxia induces structural remodeling,
`the proliferation and migration of vascular smooth
`muscle, and an increase in the deposition of vascu-
`lar matrix. Although hypoxia is not of central im-
`portance in the initial development of pulmonary
`arterial hypertension, it may contribute to the re-
`modeling of the pulmonary vasculature as the dis-
`ease progresses.
`
`anorexigens
`An association between the use of anorexigenic
`agents and the development of pulmonary arterial
`hypertension was initially observed in the 1960s,
`when an epidemic of idiopathic pulmonary arterial
`hypertension was noted in Europe after the intro-
`37
`duction of the anorexigen aminorex fumarate.
`Although this medication was withdrawn from the
`market, structurally related compounds, such as
`fenfluramine and dexfenfluramine, were developed
`subsequently, in the 1980s. The use of these agents
`has also been associated with an increased risk of
`21
`pulmonary arterial hypertension.
` Although the
`incidence of pulmonary arterial hypertension in-
`creases with the duration of use, an elevation in pul-
`monary pressure can occur after as little as three
`38,39
`to four weeks of exposure to these agents.
`
`central nervous system stimulants
`The use of the central nervous system stimulants
`methamphetamine and cocaine has been associ-
`ated with an increased risk of pulmonary arterial
`40
` Although it has been suggested
`hypertension.
`that contaminants in synthesized methamphet-
`41
`amine play a causative role,
` pulmonary hyper-
`tension occurs after the use of contaminant-free
`fenfluramines and aminorex fumarate, both am-
`phetamine-like anorexigens. In an autopsy study
`of 20 heavy users of cocaine, the lungs of four
`showed medial hypertrophy of the pulmonary ar-
`teries without evidence of foreign-body microem-
`bolization — findings consistent with pulmonary
`42
`arterial hypertension.
` The cause of these changes
`is unknown, and whether the stimulants alone can
`cause pulmonary arterial hypertension is unclear.
`
`other associated conditions
`
`Several coexisting conditions have been associated
`with pulmonary arterial hypertension. Those with
`plausible mechanistic links include scleroderma,
`infection with the human immunodeficiency virus
`(HIV), human herpesvirus (HHV), portal hyperten-
`sion, thrombocytosis, hemoglobinopathies, and he-
`reditary hemorrhagic telangiectasia.
`
`scleroderma
`A pulmonary arteriopathy occurs in limited sys-
`temic sclerosis (i.e., tight skin limited to the fingers,
`with digital ulcers and often pulmonary fibrosis),
`43-45
`especially in the CREST variant.
` At autopsy,
`up to 80 percent of patients with the CREST syn-
`drome have histopathological changes consistent
`with pulmonary arterial hypertension; however, in
`life, only 10 to 15 percent have clinically signifi-
`cant pulmonary hypertension. Histologic features
`consistent with pulmonary arterial hypertension
`and clinically evident pulmonary hypertension have
`occasionally been observed in systemic lupus ery-
`thematosus, mixed connective-tissue disease, and
`rheumatoid arthritis. In each case, there was an as-
`sociation between the occurrence of pulmonary ar-
`terial hypertension and Raynaud’s phenomenon,
`suggesting similarities in the pathogenesis of these
`46
`vasculopathies.
`
`infection with hiv
`An association between HIV infection and pul-
`monary arterial hypertension was first reported in
`1991; the initial cases occurred primarily in patients
`with hemophilia, who acquired HIV infection af-
`47,48
`ter receiving factor VIII–enriched plasma.
` Since
`then, the number of cases has increased and now
`includes people who acquired HIV infection by any
`route. In population studies in which echocardi-
`ography was used to estimate pulmonary-artery
`pressure, the incidence of pulmonary hypertension
`was approximately 0.5 percent among patients with
`HIV infection, a rate 6 to12 times as high as in the
`general population. The occurrence of pulmonary
`arterial hypertension is independent of the CD4 cell
`count, but it appears to be related to the duration of
`HIV infection. Many of these patients also have for-
`eign-body emboli as a result of the use of intrave-
`nous drugs or portal hypertension due to a con-
`comitant infection with hepatitis B or C. Both these
`disease entities have been associated with pulmo-
`
`1658
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`n engl j med
`
`351;16
`
`www.nejm.org october
`
`,
`
`14
`
`2004
`
`Downloaded from www.nejm.org at TEXAS TECH UNIVERSITY HEALTH SCIENCES CENTER on April 20, 2008 .
`Copyright © 2004 Massachusetts Medical Society. All rights reserved.
`
`Liquidia's Exhibit 1051
`Page 4
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`
`mechanisms of disease
`
`nary hypertension. Because HIV does not directly
`infect endothelial cells, the mechanism of pulmo-
`nary hypertension in HIV infection is unclear.
`
`human herpesvirus
`Human herpesvirus 8 (HHV-8) is the causative
`49
`agent of Kaposi’s sarcoma.
` On the basis of the
`histologic similarities between the plexiform le-
`sions in pulmonary arterial hypertension and en-
`dothelial abnormalities in Kaposi’s sarcoma, a re-
`cent study examined markers of HHV-8 infection
`50
` and found ev-
`in pulmonary arterial hypertension
`idence of HHV-8 infection in specimens of lung tis-
`sue obtained from 10 of 16 patients with pulmo-
`nary arterial hypertension.
`
`portal hypertension
`There is an uncommon association between por-
`tal hypertension and pulmonary arterial hyperten-
`sion. In a large autopsy series, histologic changes
`consistent with pulmonary arterial hypertension
`were found in 0.73 percent of patients with cir-
`rhosis, which is six times the prevalence found
`on autopsy in persons without portal hyperten-
`51
`sion.
` Hemodynamic studies have found pulmo-
`nary hypertension in 2 to 5 percent of patients with
`52
`; the prevalence may be higher (3.5 to
`cirrhosis
`8.5 percent) in patients referred for liver transplan-
`53
`tation.
` Right heart catheterization disclosed the
`presence of pulmonary hypertension in approxi-
`mately 6 percent of patients undergoing evalua-
`54
`tion for liver transplantation.
` The diagnosis of
`pulmonary hypertension is usually made within
`four to seven years after the diagnosis of portal
`55
`56
`hypertension
` but, rarely, may precede it.
` In ad-
`dition, the risk of pulmonary arterial hyperten-
`sion increases with the duration of portal hyper-
`56
`tension.
` The mechanism of this association is
`unclear, but cirrhosis without portal hypertension
`appears to be insufficient for the development of
`pulmonary arterial hypertension.
`
`thrombocytosis
`Pulmonary arterial hypertension has been report-
`ed in patients with chronic myelodysplastic syn-
`57,58
`dromes with thrombocytosis.
` Of 26 patients
`with a chronic myelodysplastic syndrome and un-
`explained pulmonary hypertension, 14 had elevat-
`ed platelet counts (median, approximately 600,000
`58
` The association between
`per cubic millimeter).
`pulmonary arterial hypertension and the myelo-
`
`dysplastic syndromes is probably caused by several
`different features of this syndrome, including sple-
`nectomy, portal hypertension, pulmonary vascu-
`lar obstructive disease as a result of chemothera-
`py, and the infiltration of hematopoietic cells into
`58
`the pulmonary parenchyma.
` However, a corre-
`lation between the platelet count and the level of
`pulmonary hypertension has been found, and in
`two cases, there was evidence of pulmonary-artery
`obstruction by megakaryocytes, suggesting that
`platelets and their precursors play a direct role in
`59
`pathogenesis.
` In addition, rare cases of pulmo-
`nary hypertension have been reported in patients
`60,61
`with idiopathic thrombocythemia.
` It is possi-
`ble that platelet-derived serotonin, platelet-derived
`growth factor, or transforming growth factor
`b
`(TGF-
` ) are important in the development of pul-
`b
`monary arterial hypertension in such patients.
`These agents derive from platelets and are potent
`stimuli of smooth-muscle cell proliferation; in an
`animal model of pulmonary vascular injury, nor-
`malization of the platelet count retarded the devel-
`62
`opment of pulmonary arterial hypertension.
`
`hemoglobinopathies
`Several studies have documented pulmonary hy-
`pertension and right ventricular dysfunction in pa-
`tients with thalassemia, particularly homozygous
`63,64
`-thalassemia.
` Although one study reported
`b
`evidence of pulmonary hypertension in 75 percent
`of patients with
`-thalassemia, this study and oth-
`b
`ers relied on echocardiography for the diagnosis
`of pulmonary hypertension and therefore probably
`overestimated its incidence.
`In sickle cell anemia, the estimated incidence
`of pulmonary hypertension, as determined on echo-
`65
`cardiography, varies from 8 percent to 30 percent.
`In a recent study of 34 adults with sickle cell dis-
`66
` 20 received
`ease who underwent catheterization,
`a diagnosis of pulmonary hypertension, and several
`of these adults had elevated pulmonary-capillary
`wedge pressures consistent with left ventricular
`diastolic dysfunction. The mean pulmonary-artery
`pressure was inversely related to survival: each in-
`crease of 10 mm Hg in the mean pulmonary-artery
`pressure was associated with an increase by a fac-
`tor of 1.7 in the risk of death. Very recent data con-
`firm that pulmonary hypertension increases the
`67
`risk of death in patients with sickle cell disease.
`Historically, recurrent episodes of acute chest syn-
`drome were considered to be the most important
`
`n engl j med
`
`351;16
`
`www.nejm.org october
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`14, 2004
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`1659
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`Downloaded from www.nejm.org at TEXAS TECH UNIVERSITY HEALTH SCIENCES CENTER on April 20, 2008 .
`Copyright © 2004 Massachusetts Medical Society. All rights reserved.
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`Liquidia's Exhibit 1051
`Page 5
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` new england journal
`The
`
`of
`
` medicine
`
`risk factor for the development of pulmonary arte-
`68
`rial hypertension
`; however, recent data suggest
`this may not be the case.
`The destruction of bioactive nitric oxide by free
`69
`hemoglobin
` and an increase in the production of
`70,71
`reactive oxygen species
` may be more impor-
`tant in the development of pulmonary hyperten-
`sion in patients with a hemolytic anemia than in
`those without a hemolytic anemia. For example, in
`sickle cell anemia, the plasma levels of oxyhemo-
`globin are high owing to intravascular hemolysis;
`this cell-free hemoglobin can impair responses to
`intrinsic and exogenously delivered nitric oxide.
`In patients with sickle cell anemia, there are also
`increased circulating and intracellular levels of re-
`active oxygen species, which can inactivate nitric
`oxide.
`
`hereditary hemorrhagic telangiectasia
`Pulmonary hypertension that is clinically and histo-
`logically indistinguishable from idiopathic pulmo-
`nary arterial hypertension occurs in approximately
`15 percent of cases of hereditary hemorrhagic tel-
`angiectasia (the Osler–Rendu–Weber syndrome), an
`72,73
`autosomal dominant vascular dysplasia.
` Mu-
`tations in two genes encoding the TGF-
` receptors,
`b
`endoglin and activin-receptor–like kinase 1 (ALK1),
`have been associated with the pulmonary hyper-
`tension of hereditary hemorrhagic telangiectasia.
`
`associated genetic
`abnormalities
`
`Approximately 100 families worldwide have been
`identified as having idiopathic pulmonary arterial
`74-76
`hypertension.
` The familial form accounts for
`at least 6 percent of all cases of pulmonary arterial
`hypertension, and its distribution between female
`and male patients, the age at onset, and its natural
`history are similar to those in the sporadic form.
`Segregation analysis of affected pedigrees shows
`an autosomal dominant inheritance, but only 10 to
`20 percent of the carriers of the relevant mutation
`have evidence of pulmonary arterial hypertension.
`Inheritance of the appropriate genetic mutation
`shows genetic anticipation: in each successive gen-
`eration in which the disease develops, it occurs at
`a younger age and with greater severity than in the
`preceding generation.
`
` receptor pathway
`tgf-
`b
`Two genes in the ubiquitous TGF-
`receptor fam-
`b
`ily have been strongly linked to familial pulmo-
`
`74
`nary arterial hypertension.
` The first gene, bone
`BMPR2
`morphogenetic protein receptor type 2 (
`),
`modulates the growth of vascular cells by activat-
`ing the intracellular pathways of Smad and LIM
`77
`(Lin-11, Isl-1, and Mec-3 protein) kinase.
` Under
`normal conditions, bone morphogenetic proteins
`2, 4, and 7 signal through heterodimeric complex-
`es of BMPR2 and type 1 receptors to suppress the
`growth of vascular smooth-muscle cells. More than
`BMPR2
`45 different mutations in
` have been iden-
`tified in patients with familial pulmonary arterial
`75,76
` Functional studies have shown
`hypertension.
`that point mutations and truncations in the kinase
`domain exert dominant negative effects on recep-
`78
`tor function.
` Because of incomplete penetrance
`and genetic anticipation (i.e., an increased familial
`prevalence of the phenotype in successive genera-
`BMPR2
` mutations are
`tions), it is probable that the
`necessary, but insufficient alone, to account for the
`clinical expression of the disease.
`A rare group of patients with hereditary hem-
`orrhagic telangiectasia and idiopathic pulmonary
`arterial hypertension was found to harbor muta-
` receptor
`tions in another member of the TGF-
`b
`75
`ALK1
`BMPR2 mutations, muta-
`family,
`.
` As with
`tions in this type 1 receptor are believed to result in
`growth-promoting Smad-dependent signaling.
`Perhaps as many as 10 to 26 percent of patients
`with sporadic idiopathic pulmonary arterial hy-
`pertension also bear a mutation of a member of
`the TGF-b receptor family.79 Recently, Du and col-
`leagues80 have argued that all forms of pulmonary
`arterial hypertension also have defects in a com-
`mon vascular signaling pathway that involves an-
`giopoietin-1 and the phosphorylated form of its
`endothelial-specific receptor, TIE2. These investi-
`gators showed that this signaling pathway is up-
`regulated in the lungs of patients with pulmonary
`hypertension, regardless of the cause of the dis-
`ease; the increase in angiopoietin signaling is ac-
`companied by a decrease in another member of
`the TGF-b receptor family, BMPR1A, a complemen-
`tary type 1 receptor required for normal BMPR2 sig-
`naling. Some of these observations, however, run
`counter to previous findings regarding the role of
`angiopoietin in the pulmonary vasculature.81,82
`
`serotoninergic pathway
`The increased serotonin-dependent proliferation
`of cultured pulmonary vascular smooth-muscle
`cells in specimens obtained from patients with id-
`iopathic pulmonary arterial hypertension is, in part,
`a consequence of an increase in the serotonin trans-
`
`1660
`
`n engl j med
`
`351;16
`
`www.nejm.org october
`
`,
`
`14
`
`2004
`
`Downloaded from www.nejm.org at TEXAS TECH UNIVERSITY HEALTH SCIENCES CENTER on April 20, 2008 .
`Copyright © 2004 Massachusetts Medical Society. All rights reserved.
`
`Liquidia's Exhibit 1051
`Page 6
`
`
`

`

`mechanisms of disease
`
`porter 5-HTT.22 The L-allelic variant of the 5HTT
`gene is associated with an increased expression of
`the transporter and an increase in the growth of vas-
`cular smooth-muscle cells, and it is more prevalent
`among patients with idiopathic pulmonary arterial
`hypertension than among controls.
`A complementary study by Launay and col-
`leagues83 showed that hypoxia-induced pulmonary
`arterial hypertension in mice is associated with an
`increase in the expression of 5-HT2B, resulting in
`serotonin-dependent vascular remodeling. In ad-
`dition, they showed that the main metabolite of
`dexfenfluramine, nor-dexfenfluramine, is a potent
`vascular-cell growth–promoting agonist for this
`receptor, thus linking anorexigenic pulmonary ar-
`terial hypertension to signaling pathways in pul-
`monary vascular cells that are up-regulated by hy-
`poxia and activated by serotonin.
`Possible associations among these genetically
`determined pathways are summarized in Figure 2.
`Environmental factors that may influence the func-
`
`tioning of the pathways are also included in the
`figure.
`
`molecular basis
`of treatment strategies
`
`There is no cure for pulmonary arterial hyperten-
`sion. Treatment, however, has improved dramati-
`cally during the past decade, offering both relief
`from symptoms and prolonged survival. The main-
`stays of current medical therapy fall into several
`classes, including vasodilators, anticoagulants, an-
`tiplatelet agents, antiinflammatory therapies, and
`vascular-remodeling therapies. Many of the most
`effective agents have pleiotropic effects. For exam-
`ple, epoprostenol is a vasodilator, a platelet inhib-
`itor, and an antiinflammatory agent, whereas the
`endothelin-receptor antagonist bosentan is a vaso-
`dilator, an antiinflammatory agent, and a remod-
`eling mediator. Many of these therapies can be
`viewed as pharmacologic surrogates for endothe-
`
`Figure 2. Mechanistic Pathways Promoting Pulmonary Arterial Hypertension.
`In the serotoninergic pathway, hypoxia increases the expression of the serotonin receptor 5-HT2B. Increased expression
`of the serotonin transporter 5-HTT is accompanied by an enhanced sensitivity to serotonin as a stimulus of the prolifer-
`ation of vascular smooth-muscle cells and vascular remodeling. Anorexigens, especially nor-dexfenfluramine, boost (+)
`the serotonin-dependent increase in 5-HT2B responses and suppress (¡) the serotonin-dependent increase in 5-HTT
`responses. In the transforming growth factor b (TGF-b) receptor pathway, an unknown stimulus increases the expres-
`sion of angiopoietin-I as well as that of its receptor TIE2, which, in turn, leads to a decrease in the expression of bone
`morphogenetic protein receptor type 1A (BMPR1A). This member of the TGF-receptor family is required for optimal sig-
`naling with its partner receptor BMPR type 2 (BMPR2). Mutant forms of BMPR2 (mBMPR2) and activin-receptor–like ki-
`nase (mALK1) are associated with familial forms of idiopathic pulmonary arterial hypertension, and both mutations
`result in enhanced (unrestrained) signaling through the growth-promoting Smads, which ultimately stimulate vascular
`smooth-muscle cell proliferation and vascular remodeling. PO2 denotes the partial pressure of oxygen.
`
`n engl j med 351;16 www.nejm.org october 14, 2004
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`Copyr