Cellular and Molecular Basisof Pulmonary Arterial Hypertension
Nicholas W. Morrell, MA, MD,* Serge Adnot, MD, PHD,† Stephen L. Archer, MD,‡Jocelyn Dupuis, MD, PHD,§ Peter Lloyd Jones, PHD,� Margaret R. MacLean, PHD,¶Ivan F. McMurtry, PHD,# Kurt R. Stenmark, MD,** Patricia A. Thistlethwaite, MD, PHD,††Norbert Weissmann, PHD,‡‡ Jason X.-J. Yuan, MD, PHD,§§ E. Kenneth Weir, MD��
Cambridge, United Kingdom; Créteil, France; Chicago, Illinois; Montreal, Québec, Canada;Philadelphia, Pennsylvania; Glasgow, Scotland; Mobile, Alabama; Denver, Colorado; La Jolla, California;Giessen, Germany; and Minneapolis, Minnesota
ublished by Elsevier Inc. doi:10.1016/j.jacc.2009.04.018
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espite the recognized success of existing drug interven-ions in the relief of symptoms of pulmonary arterialypertension (PAH), and possibly improvement in sur-ival, most patients eventually become resistant to ther-py and succumb to the disease. The past few years have
rom the *Pulmonary Vascular Diseases Unit, Department of Medicine, University ofambridge School of Clinical Medicine, Cambridge, United Kingdom; †Medicalchool of Créteil, Hôpital Henri Mondor, Créteil, France; ‡University of Chicago,hicago, Illinois; §Research Center of the Montreal Heart Institute, Department ofedicine, University of Montreal, Montreal, Québec, Canada; �University of Penn-
ylvania, Penn/CMREF Center for Pulmonary Arterial Hypertension Research,hiladelphia, Pennsylvania; ¶Institute of Biomedical and Life Sciences, University oflasgow, Glasgow, Scotland; #Departments of Pharmacology and Medicine andenter for Lung Biology, University of South Alabama, Mobile, Alabama; **Devel-pmental Lung Biology Laboratory and Pediatric Critical Care Medicine, Universityf Colorado at Denver and Health Sciences Center, Denver, Colorado; ††Depart-ent of Cardiothoracic Surgery, University of California, San Diego, La Jolla,alifornia; ‡‡University of Giessen Lung Center, Department of Internal Medicine
I/V, Justus-Liebig-University, Giessen, Germany; §§Department of Medicine,niversity of California San Diego, La Jolla, California; and the � �University ofinnesota, Veterans Affairs Medical Center, Minneapolis, Minnesota. Please see the
rnd of this article for each author’s conflict of interest information.
Manuscript received February 6, 2009, accepted April 15, 2009.
een a remarkable increase in our knowledge of theellular and molecular mechanisms responsible for theathobiology of PAH. This summary aims to present theurrent state of our understanding of some of the keyechanisms (Fig. 1). We also indicate further areas and
irections of research and suggest novel approaches toherapy.
ndothelial Dysfunction in PAH
ndothelial cells (ECs) are recognized as major regulators ofascular function, and endothelial dysfunction has come toean a multifaceted imbalance in EC production of vasocon-
trictors versus vasodilators, activators versus inhibitors ofmooth muscle cell (SMC) growth and migration, prothrom-otic versus antithrombotic mediators, and proinflammatoryersus anti-inflammatory signals.ho guanosine triphosphatases (GTPases) in endothelialysfunction. Rho (Ras homologous) GTP-binding proteins
egulate many cellular processes, including gene transcription,
S21JACC Vol. 54, No. 1, Suppl S, 2009 Morrell et al.June 30, 2009:S20–31 Cellular and Molecular Basis of PAH
ifferentiation, proliferation, hypertrophy, apoptosis, phagocy-osis, adhesion, migration, and contraction (1). In therototypical mechanism of RhoA GTPase signaling,nvironmental cues, acting through G-protein– coupledeceptors or receptor-dependent and receptor-independentyrosine kinases, activate guanine nucleotide exchange factors,hich induce exchange of guanosine diphosphate for GTPinding and translocation of GTP-RhoA to the plasma mem-rane. The membrane translocation requires post-translationalrenylation. Upon translocation to the plasma membrane,TP-RhoA activates its effectors, including the 2 isoforms ofho kinase (ROCK), ROCK I (ROK�) and ROCK II
ROK�). Negative regulators of RhoA activation includeuanine nucleotide disassociation inhibitors, which oppose thexchange of GTP for guanosine diphosphate; GTPase activat-ng proteins, which catalyze dephosphorylation and inactiva-ion of membrane-bound GTP-RhoA; statins, which inhibitsoprenylation of RhoA and thereby prevent translocation of
TP-RhoA to the cell membrane (2); and protein kinases And G, which, by phosphorylating RhoA, also prevent mem-rane translocation of the GTP-bound protein (3).ho GTPases and EC permeability. An increase in ECermeability may be an important component of the patho-enesis of PAH. The GTPases RhoA and Rac1 playpposing roles in the regulation of EC barrier function.
hile stimuli such as thrombin activate RhoA/ROCK,hich increases formation of F-actin stress fibers, cell
ontraction, and permeability, barrier-enhancing mediatorsuch as sphingosine-1-phosphate and prostacyclin (PGI2)timulate Rac1/p21-activated kinase (PAK), which coun-eracts the effects of RhoA/ROCK and promotes cortical-actin ring formation and barrier integrity (4). Pulmonaryrtery ECs cultured from chronically hypoxic piglets dem-nstrate low Rac1 and high RhoA activities, which correlateith increased stress fiber formation and permeability (5).ctivation of Rac1/PAK-1 and inhibition of RhoA reverse
hese changes.ho GTPases and EC proliferation, migration, and
poptosis. Rho GTPases participate in EC proliferationnd apoptosis. Interestingly, the hyperproliferative,poptosis-resistant phenotype of PAH ECs may be due toersistent activation of signal transducer and activator ofranscription 3 (6), a downstream target of Rho GTPases.ignal transducer and activator of transcription 3 mediateshoA-induced nuclear factor-�B and cyclin D1 transcrip-
ion and is involved in nuclear factor-�B nuclear transloca-ion (7).
ole of rho GTPases in thrombosis. In situ thrombosisf small peripheral pulmonary arteries contributes toAH. The ECs are directly involved in the fibrinolyticrocess through synthesis and release of the profibrino-
ytic tissue plasminogen activator and the antifibrinolytic/rothrombotic plasminogen activator inhibitor (PAI)-1.he stimulation of systemic artery EC PAI-1 expressiony angiotensin II, C-reactive protein, high glucose, and
onocyte adhesion is dependent on activation of RhoA/ n
OCK signaling. Similarly, ECxpression of tissue factor, anotherrothrombotic mediator, increasedn the pulmonary arteries (PAs) ofAH lungs, is upregulated byhoA/ROCK signaling (8). ThehoA/ROCK and Rac/PAK sig-aling pathways are implicated inhrombin- and thromboxane A2-nduced platelet activation andggregation (9).itric oxide (NO) and PGI2.ndothelial dysfunction in PAH
s reflected by reduced productionf the vasodilators/growth inhibi-ors NO and PGI2 and increasedroduction of the vasoconstrictor/o-mitogens, for example, endo-helin-1 and thromboxane A2.itric oxide signaling is mediatedainly by the guanylate cyclase/
yclic guanosine monophosphatecGMP) pathway. Degradationf the second messenger ofO, cGMP, by phosphodies-
erases is mainly accomplishedy phosphodiesterase-5.Reduced NO bioavailability in
AH can be due to decreasedxpression of endothelial NO syn-hase (eNOS), inhibition of eNOSnzymatic activity, and inactiva-ion of NO by superoxide anion.ctivation of endothelial RhoA/OCK signaling can be involved
n at least the first 2 processes. Forxample, RhoA/ROCK activationediates hypoxia- and thrombin-
nduced inhibition of both eNOSxpression and its activity in cul-ured ECs (10). The activity ofrginase II, which reduces NOynthesis by competing withNOS for the substrate L-rginine, is increased in PAHCs (11), and RhoA/ROCK
ignaling mediates thrombin-nd tumor necrosis factor-�/ipopolysaccharide-induced ac-ivation of eNOS (12). Patientsith idiopathic PAH (IPAH)ave increased plasma levels ofhe endogenous inhibitor ofNOS, asymmetric dimethyl-rginine (13), and the levels of asymmetric dimethylargi-
Abbreviationsand Acronyms
ALK � activin-receptorlikekinase
Ang � angiopoeitin
BMP � bonemorphogenetic protein
BMPR � bonemorphogenetic proteinreceptor
cGMP � cyclic guanosinemonophosphate
EC � endothelial cell
ECM � extracellular matrix
enMT � endothelialmesenchymal transition
eNOS � endothelial nitricoxide synthase
GTPase � guanosinetriphosphatase
5-HT � hydroxytryptamine(serotonin)
5-HTT � hydroxytryptamine(serotonin) transporter
IPAH � idiopathicpulmonary arterialhypertension
MLC � myosin light chain
MLCK � myosin light chainkinase
MLCP � myosin light chainphosphatase
NO � nitric oxide
PA � pulmonary artery
PAEC � pulmonary arteryendothelial cell
PAH � pulmonary arterialhypertension
PAI � plasminogen-activator inhibitor
PAK � p21-activated kinase
PASMC � pulmonary arterysmooth muscle cell
PGI2 � prostacyclin
PH � pulmonaryhypertension
PK � protein kinase
ROCK � Rho kinase
SMC � smooth muscle cell
TGF � transforming growthfactor
TRPC � canonicaltransient receptor potential
ine and the enzyme that degrad
es it, dimethylarginine
dd
sooaon
pPAoagtD
S22 Morrell et al. JACC Vol. 54, No. 1, Suppl S, 2009Cellular and Molecular Basis of PAH June 30, 2009:S20–31
imethylaminohydrolase, are, respectively, increased andecreased in the PA endothelium of IPAH patients (13).Prostacyclin stimulates the formation of cyclic adeno-
ine monophosphate, which also inhibits the proliferationf SMCs and decreases platelet aggregation. A deficiencyf PGI2 and PGI2 synthase and an excess of thromboxanere found in PAH (14). Moreover, PGI2-receptor knock-ut mice develop more severe hypoxia-induced pulmo-
Endothelial cell
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Figure 1 Potential Mechanisms Involved in the Development of
Schematic diagram depicting potential mechanisms involved in the developmenvolume decrease; BMP � bone morphogenetic protein; BMPR � bone morphoging protein; DAG � diacylglycerol; Em � membrane potential; EGF � epidermalGTPase activating protein; GPCR � G protein-coupled receptor; HHV � humannin) transporter; IP3 � inositol 1,4,5-trisphosphate; Kv � voltage-gated K�; MAlight chain kinase; NA(D)PH � nicotinamide adenine dinucleotide phosphate; Nderived growth factor; PGI2 � prostacyclin; PKC � protein kinase C; PLC � phospecies; RTK � receptor tyrosine kinase; SR � sarcoplasmic reticulum; SRF �
kinase; VDCC � voltage-dependent calcium channel.
ary hypertension (PH) (15). Conversely, PGI2 overex- e
ressing mice are protected against hypoxia-inducedH (16).ngiopoietin and TIE2. Angiopoietin (Ang)-1 is anligomeric-secreted glycoprotein, which, along withngiopoietin-2 and angiopoietin-3/4, comprises a family ofrowth factors. The angiopoietin ligands exert their effectshrough the endothelial-specific tyrosine kinase, TIE2 (17).uring lung development, both Ang-1 and TIE2 are
T
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EGFR
ulmonary arterial hypertension (PAH). Ang � angiopoeitin; AVD � apoptoticprotein receptor; CaM � calmodulin; CREB � cAMP-response element bind-
h factor; EGFR � epidermal growth factor receptor; ET � endothelin; GAP �
response factor; TCF � T cell factor; TIE � endothelial-specific tyrosine
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S23JACC Vol. 54, No. 1, Suppl S, 2009 Morrell et al.June 30, 2009:S20–31 Cellular and Molecular Basis of PAH
ecreted by vascular SMCs and pericytes, whereas TIE2 is aransmembrane receptor expressed on endothelial cells (18).n the adult, Ang-1 expression in the lung is minimal,hereas TIE2 expression remains constitutive (19).Several lines of evidence suggest that Ang-1 regulates
athologic SMC hyperplasia in PAH. Ang-1 is overex-ressed in most forms of nonfamilial PAH (18,20). InAH, Ang-1 causes activation of the TIE2 receptor by
yrosine autophosphorylation in the pulmonary vascularndothelium (20,21). Enhanced TIE2 levels and a 4-foldncrease in TIE2 phosphorylation are found in human PAHung tissue, compared with control subjects (20,22).
Virally mediated overexpression of Ang-1 in the rat lungesults in PH (21,23). Ang-1 transgenic animals showncreased pulmonary vascular endothelial TIE2 phosphory-ation and SMC hyperplasia in small pulmonary arterioles.urther, overexpression of a soluble TIE2 ectodomain,hich sequesters Ang-1, suppresses the PH phenotype inonocrotaline- and Ang-1–induced models of this disease (24).There is a reciprocal relationship between bone morpho-
enetic protein receptor (BMPR) 1A and Ang-1 expressionn the lungs of patients with nonfamilial PAH (20). Ang-1ownregulates BMPR1A expression through a TIE2 path-ay in human pulmonary artery endothelial cells (PAECs).timulation of human PAECs with Ang-1 induces releasef 5-hydroxytryptamine (HT [serotonin]), a potent stimu-ator of SMC proliferation (21,22). There is controversy inhis field. In contrast to a causative role, Ang-1 has beeneported to protect against the development of PAH in theat monocrotoline and hypoxia models of disease (25).
he SMC in PAH
erotonin, serotonin transporter, and receptors. Patientsith IPAH have increased circulating 5-HT levels, even
fter heart-lung transplantation (26). In contrast to theonstricting action of 5-HT on SMCs, which is mainlyediated by 5-HT receptors 1B/D, 2A, and 2B (27), theitogenic and co-mitogenic effects of 5-HT require inter-
alization through the serotonin transporter, 5-HTT (28).hat may require co-stimulation of the 5-HT1B receptor
29). Drugs that competitively inhibit 5-HTT block theitogenic effects of 5-HT on SMCs (30). The appetite
uppressants fenfluramine, d-fenfluramine, and aminorexiffer from selective 5-HTT inhibitors in that they not only
nhibit 5-HT reuptake but also trigger indoleamine releasend interact with 5-HTT and -HT receptors in a specificanner (30).
EROTONIN TRANSPORTER. 5-HTT is abundantly expressedn pulmonary artery smooth muscle cells (PASMCs) (31).ice with targeted 5-HTT gene disruption develop less
evere hypoxic PH than do wild-type controls (32,33).onversely, increased 5-HTT expression is associated with
ncreased severity of hypoxic PH (34,35). Indeed, specificverexpression of 5-HTT in PASMCs is sufficient to
roduce spontaneous PH (33). p
-HT receptors in PH. Of the 14 distinct 5-HT recep-ors, the 5-HT2A, 5-HT2B, and 5-HT1B receptors arearticularly relevant to PAH.
-HT2A RECEPTOR. In most nonhuman mammals, the-HT2A receptor mediates vasoconstriction in both theystemic and pulmonary circulations (36). However, the-HT2A receptor antagonist ketanserin is not specific forhe pulmonary circulation, and systemic effects have limitedts use in PAH, where it fails to improve pulmonaryemodynamics significantly (37).
-HT2B RECEPTOR. The development of hypoxia-induced PHn mice is ablated in 5-HT2B receptor knockout mice (38),nd this receptor may control 5-HT plasma levels in mice.owever, the 5-HT2B receptor may also mediate vasodila-
ion of the PA (39), and loss of the 5-HT2B receptor functionay predispose to fenfluramine-associated PH in humans
40).
-HT1B RECEPTOR. The 5-HT1B receptor mediates constric-ion in human PAs (41) and plays a role in the developmentf PAH (36,42), because inhibition, either by geneticnockout or pharmacologic antagonism, reduces hypoxia-nduced pulmonary vascular remodeling (36). There isooperation between the 5-HT1B receptor and the 5-HTTn mediating pulmonary vascular contraction (43). In addi-ion, 5-HT1B receptor expression is increased in miceverexpressing the human 5-HTT and in the fawn-hoodedat, which also demonstrates increased 5-HTT expression43). Both these models are predisposed to hypoxia-inducedulmonary vascular remodeling. Remodeled PAs from pa-ients with PAH overexpress the 5-HT1B receptor. 5-HT1Beceptor-mediated changes are specific to the pulmonaryirculation, making this receptor an attractive therapeuticarget for PH.
-HT SYNTHESIS IN PH. The rate-limiting step in 5-HT bio-ynthesis is catalyzed by the enzyme tryptophan hydroxylase.lthough peripheral 5-HT is synthesized chiefly by the en-
erochromaffin cells in the gut, human PAECs produce 5-HTnd express the tryptophan hydroxylase-1 isoform. Both 5-HTynthesis and tryptophan hydroxylase-1 expression are in-reased in cells from patients with IPAH compared withontrols (44). Mice lacking tryptophan hydroxylase-1 areesistant to hypoxia- and dexfenfluramine-induced PH45,46).
� and Ca� channels in PAH. In PASMCs, the freea2� concentration in the cytosol ([Ca2�]cyt) is an impor-
ant determinant of contraction, migration, and prolifera-ion. The [Ca2�]cyt in PASMCs can be increased by:) Ca2� influx through voltage-dependent Ca2� channels,eceptor-operated Ca2� channels, and store-operated Ca2�
hannels; and 2) Ca2� release from intracellular stores (e.g.,arcoplasmic reticulum) through Ca2� release channelse.g., inositol 1,4,5-trisphosphate receptors and ryanodineeceptors). Inward transport of Ca2� through Ca2� trans-
orters in the plasma membrane, such as the reverse mode
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S24 Morrell et al. JACC Vol. 54, No. 1, Suppl S, 2009Cellular and Molecular Basis of PAH June 30, 2009:S20–31
f Na�/Ca2� exchanger, is also an important pathway forncreasing [Ca2�]cyt. In contrast, [Ca2�]cyt in PASMCs cane decreased by: 1) Ca2� extrusion by the Ca2�-Mg2�
denosine triphosphatase (Ca2� pump) and by the forwardode of Na�/Ca2� exchanger in the plasma membrane;
nd 2) Ca2� sequestration by the Ca2�-Mg2� adenosineriphosphatase in the sarcoplasmic reticulum.
NHIBITION OF K� CHANNEL ACTIVITY. Decreased expressionnd/or function of K� channels leads to membrane depo-arization and contributes to sustained elevation of [Ca2�]cyty: 1) activating voltage-dependent calcium channelVDCC); 2) facilitating the production of inositol 1,4,5-risphosphate, which stimulates the release of sarcoplasmiceticulum Ca2� into the cytoplasm; and 3) promoting Ca2�
ntry through the reverse mode of Na�/Ca2� exchange.
OLE OF RECEPTOR-OPERATED AND STORE-OPERATED CA2�
HANNELS IN REGULATING [CA2�]CYT. The influx of Ca2�
hrough store-operated calcium channels, referred to asapacitative Ca2� entry, is critical for refilling the emptyarcoplasmic reticulum with Ca2�. Store-operated calciumhannels in vascular SMC include the transient receptorotential channels. Some canonical transient receptor po-ential (TRPC) channel genes are expressed in humanASMCs and PAECs.Proliferation of PASMC is associated with a significant
ncrease in messenger ribonucleic acid and protein expres-ion of TRPC channels such as TRPC1, TRPC3, andRPC6 (47,48). Inhibition of TRPC expression with an-
isense oligonucleotides markedly decreases the amplitudef capacitative calcium entry and significantly inhibitsASMC proliferation. Thus, upregulation of TRPC chan-els may be a significant mechanism in the induction ofASMC proliferation.
ATHOGENIC ROLE OF DOWNREGULATED KV CHANNELS AND
PREGULATED TRP CHANNELS. In PASMCs from IPAHatients, the amplitude of whole-cell IK(V) and mRNA/rotein expression levels of Kv channel subunits (e.g., Kv1.2nd Kv1.5) are both significantly decreased in comparisonith cells from controls or patients with secondary PH (49).he downregulated Kv channels and decreased IK(V) are
ssociated with a more depolarized Em in IPAH PASMCs,nd the resting [Ca2�]cyt is much higher than in PASMCsrom controls. The magnitude of capacitative calcium entry,voked by passive store depletion with cyclopiazonic acid, isignificantly greater in PASMCs from IPAH patients thann cells from secondary PH patients. Enhanced capacitativealcium entry, possibly by upregulation of TRPC channels,ay represent a critical mechanism involved in the devel-
pment of severe PAH.
V CHANNELS, MITOCHONDRIAL METABOLISM, AND PAH.
arburg (50) proposed that a metabolic shift from oxida-ive phosphorylation to glycolysis, occurring despite ade-uate oxygen availability, was a characteristic of cancers.
ecent data suggest that PAH and cancer share this k
Warburg phenotype” (51,52). Both are characterized byitochondrial hyperpolarization, depressed pyruvate dehy-
rogenase complex activity, and depressed H2O2 produc-ion (53). In both, there is also an O2-independent perpet-ation of the rapid, reversible metabolic/redox shifts thatormally occur in response to hypoxia and initiate hypoxiculmonary vasoconstriction (54,55). This metabolic shiftreates a “pseudohypoxic environment” with glycolytic pre-ominance and normoxic hypoxia-inducible factor-1� activa-ion. The metabolic shift suppresses Kv1.5 expression, leadingo membrane depolarization and elevation of cytosolic K� anda2�. In both PAH PASMCs and cancer cell lines, this
reates a proliferative, apoptosis-resistant phenotype.As in familial PAH, PAH in the fawn-hooded rat is
eritable. The fawn-hooded rat’s PASMC mitochondrialeticulum is fragmented even before PAH develops. Thebserved hyperpolarization of ��m and reduction in pro-uction of reactive oxygen species also occurs in PASMCsrom IPAH patients (51). In PAH, mitochondrial abnor-alities that shift metabolism away from oxidative phos-
horylation toward glycolysis lead to a decreased electronux and reduced reactive oxygen species production, whichalsely signifies hypoxia, resulting in normoxic hypoxia-nducible factor-1� activation. Both the hypoxia-inducibleactor-1� activation and the related decrease in Kv1.5xpression are reversed by low doses of exogenous
2O2,,consistent with the redox theory for their etiology. Aypoxia-inducible factor-1� dominant-negative constructlso restores Kv1.5 expression in fawn-hooded rat PASMC51). Decreased Kv expression is an emerging hallmark ofhe PAH PASMC, occurring in human PAH (49,51) andll known experimental models (56–58). Interestingly, bothv channels involved in hypoxic pulmonary vasoconstriction
Kv1.5 and Kv2.1) are inhibited by the anorexigens (59) andy 5-HT (60). In addition, endothelin-1 reversibly reduceshe Kv1.5 currents (61). Restoring Kv1.5 expression reduceshronic hypoxic PH and restores hypoxic pulmonary vaso-onstriction (62).
Mitochondrial therapy, for example, inhibition of pyru-ate dehydrogenase kinase by dichloroacetate or Kv1.5 geneherapy partially regresses both PAH and cancer (51,52,62),onsistent with the concept that PAH and cancer share aitochondrial basis. Dichloroacetate restores oxidative me-
abolism in fawn-hooded rat PASMCs, shifting them awayrom the proliferative/apoptosis resistant glycolytic state.ichloroacetate also causes regression of PAH induced by
hronic hypoxia or monocrotaline (51,56,57).hoA/ROCK-mediated vasoconstriction. It is now clear
hat activation of RhoA/ROCK signaling is a major regu-ator of vascular tone (63). Smooth muscle cell tension isetermined primarily by phosphorylation (contraction) andephosphorylation (relaxation) of the regulatory myosin
ight chain (MLC), as described in the preceding text. At aiven level of cytosolic Ca2�, second messenger-mediatedathways can modulate the activity of myosin light chain
inase (MLCK) and myosin light chain phosphatases
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S25JACC Vol. 54, No. 1, Suppl S, 2009 Morrell et al.June 30, 2009:S20–31 Cellular and Molecular Basis of PAH
MLCPs) (e.g., MYPT1) to modify MLC phosphorylationnd force, namely, to modify the Ca2� sensitivity ofontraction. Two major pathways in vascular smooth muscleSM) are inhibition of MLCP action by ROCK-mediatedhosphorylation of MYPT1, and protein kinase C-mediatedhosphorylation and activation of the MLCP-inhibitor pro-ein CPI-17.
Desensitization of Ca2� is also a mechanism of vasodi-ation. Besides inducing SMC relaxation by desensitizingeceptors and decreasing cytosolic [Ca2�] and MLCKctivity, the NO/soluble guanylate cyclase/cGMP/PKGathway also decreases Ca2� sensitivity by phosphorylatingnd inactivating RhoA protein, or by directly phosphory-ating MLCP, which increases MLCP activity (3). Sim-larly, vasodilation by stimuli that activate the adenylateinase/cyclic adenosine monophosphate/PKA pathway islso attributable partly to inhibition of RhoA/ROCKignaling (3).hoA/ROCK in acute pulmonary vasoconstriction.OCK-mediated Ca2� sensitization is necessary for the
ustained phase of acute hypoxic pulmonary vasoconstric-ion (64). Similarly, hypoxia directly activates RhoA inultured PASMCs (65). Many studies have demonstratedhe participation of ROCK in acute pulmonary vasocon-triction due to a variety of stimuli.hoA/ROCK in human PAH. Studies of RhoA/ROCK
ignaling in human PAH are limited. Low intravenousoses of fasudil acutely cause modest decreases in pulmonaryascular resistance in patients with PAH (66). Clinical trialsxamining the inhibition of RhoA/ROCK are under way.
rosstalk Between Vascular Cells
hether SM hyperplasia results from inherent characteris-ics of PASMCs or from dysregulation of molecular eventshat govern PASMC growth, such as signals originatingrom PAECs, remains an open question (67). In addition,here is evidence of crosstalk between adventitial cells andedial SMCs.Endothelial dysfunction in PAH may follow excessive
elease of paracrine factors that act either as growth factorso induce PASMC proliferation or as chemokines to recruitirculating inflammatory cells (44,68). Thus, exposure ofASMCs to culture medium from PAECs inducesASMC proliferation, and this effect is exaggerated whenAECs from patients with PAH are used (44).The role of ECs in angiogenesis and remodeling is now
etter understood (69,70). In maturation, ECs no longerroliferate or migrate but promote vessel stabilization byecruiting periendothelial support cells, which differentiatento SM-like cells (71). Failure of interactions between the
cell types, as seen in numerous genetic mouse models,esults in severe and often lethal cardiovascular defects.eficiencies in this process may lead to abnormal dilation of
esistance pulmonary vessels, such as that seen in hereditary
emorrhagic telangectasia. Several studies suggest that the w
rosstalk between PAECs and PASMCs may be under theontrol of diverse pathways including the angiopoietin-1/IE2, transforming growth factor (TGF)-�/activin-
eceptorlike kinase (ALK)-1, and bone morphogenetic pro-ein (BMP)/BMPR-II pathways (21,22,72). PAECsonstitutively produce and release excessive amounts ofoluble factors that act on PASMCs and inflammatoryirculating cells to initiate or enhance pulmonary vascularemodeling and inflammation.
ellular and Molecularonsequences of BMPR-II Mutation
utations in the BMPR2 gene have been found in �70%f families with PAH (73,74). In addition, up to 25% ofatients with apparently sporadic IPAH harbor muta-ions (75).
ormal BMP/TGF-� signaling. BMPs are the largestroup of cytokines within the TGF-� superfamily (76).MPs are now known to regulate growth, differentiation,nd apoptosis in a diverse number of cell lines (77). TheGF-� superfamily type II receptors are constitutively
ctive serine/threonine kinases. BMPR-II initiates intracel-ular signaling in response to specific ligands (78). Ligandpecificity for different components of the receptor complexay have functional significance to the tissue-specific nature
f BMP signaling (79,80). Recently, BMP9 was identifieds a ligand that signals through a complex comprisingMPR-II and ALK-1 (81). This important finding mightrovide a mechanism for the rare occurrence of severe PAHn some families with hereditary hemorrhagic telangiectasiaue to ALK-1 mutations (82). After ligand binding, theype II receptor phosphorylates a glycine-serine–rich do-ain on the proximal intracellular portion of an associated
ype I receptor (usually BMPR-IA [ALK-3] or BMPR-IBALK-6]). Activated type I receptors in turn phosphorylateytoplasmic signaling proteins known as Smads, which areesponsible for TGF-� superfamily signal transduction (83).MPs signal through a restricted set of receptor-mediatedmads (R-Smads), Smads-1, -5, and -8, which must com-lex with the common partner Smad (Co-Smad), Smad-4,o translocate to the nucleus. Switching off Smad signalingn the cell is achieved by Smad ubiquitination and regulatoryactors (Smurfs) (84) and by recently identified Smadhosphatases (85).he consequences of BMPR2 mutation for BMP/GF-� signaling. The mechanism by which BMPR-IIutants disrupt BMP/Smad signaling is heterogeneous andutation specific (86). Of the missense mutations, substi-
ution of cysteine residues within the ligand binding orinase domain of BMPR-II leads to reduced trafficking ofhe mutant protein to the cell surface. At least for the ligandinding domain mutants, the mistrafficking can be rescuedith chemical chaperones, resulting in improvements inmad signaling (87). In contrast, noncysteine mutations
ithin the kinase domain reach the cell surface but fail to
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S26 Morrell et al. JACC Vol. 54, No. 1, Suppl S, 2009Cellular and Molecular Basis of PAH June 30, 2009:S20–31
ctivate Smad-responsive luciferase reporter genes. Manyutations lead to nonsense-mediated mRNA decay of theutant transcript, leading to a state of haploinsufficiency.ASMCs from mice heterozygous for a null mutation in theMPR2 gene are also deficient in Smad signaling (88,89).hus, haploinsufficiency or missense mutation leads to a lossf signaling by the Smad1/5 pathway in response to BMP2nd BMP4. However, marked siRNA knockdown ofMPR-II leads to increased Smad signaling in response to
ome ligands, for example, BMP7 (80,89). This effect isediated by increased signaling through the ActR-II recep-
or. In PASMCs, BMPR-II appears to mediate growthnhibition and differentiation, whereas ActR-II mediatessteoblastic differentiation (90).tudies of BMP signaling cells and tissues from PAHatients. In the lung, BMPR-II is highly expressed on theascular endothelium of the PAs (91) and at a lower level inASMCs and fibroblasts. The expression of BMPR-II isarkedly reduced in the pulmonary vasculature of patientsith mutations in the BMPR-II gene (91). BMPR-II
xpression is also reduced in the pulmonary vasculature ofatients with IPAH in whom no mutation in the BMPR2ene was identified. A reduction in the expression ofMPR-II may be important to the pathogenesis of PAH,hether or not there is a mutation in the gene. In addition,
ince the level of BMPR-II expression in familial cases wasonsiderably lower than predicted from the state of haplo-nsufficiency, this suggests that some additional environ-
ental or genetic factor may be necessary to further reduceMPR-II expression below a threshold that triggers vascu-
ar remodeling.Phosphorylation of Smad1/5 is also reduced in the
ulmonary arterial wall of patients with underlyingMPR-II mutations and in patients with IPAH with no
dentifiable mutation (92). The response of PASMCs toMP ligands depends to some extent on the anatomicalrigin of cells. The serum-stimulated proliferation of cellsarvested from the main or lobar PAs tends to be inhibitedy TGF-�1 and BMPs 2, 4, and 7 (92). Indeed, BMPs maynduce apoptosis in these cells (93). The growth inhibitoryffects of BMPs have been shown to be Smad1 dependent92). In contrast, in PASMCs isolated from PAs of 1 to 2m diameter, BMPs 2 and 4 stimulate proliferation (92).his pro-proliferative effect of BMPs is dependent on the
ctivation of ERK1/2 and p38MAPK. Both Smad andAPK pathways are activated to a similar extent in cells
rom both locations, but the integration of these signals byhe cell differs. This integration may be at the level of anmportant family of transcription factors, the inhibitors of
NA binding (Id genes) (94).The response of vascular ECs to BMPs is dependent on
he specific BMP ligand. Endothelial cells proliferate, mi-rate, and form tubular structures in response to BMP4 andMP6 (95). In addition, BMPs in general protect endothe-
ial cells from apoptosis (96). Interestingly, BMP9, which
cts through BMPR-II and ALK-1, seems to inhibit (
AEC proliferation. Knockdown of BMPR-II with siRNAncreases the susceptibility of PAECs to apoptosis (96).
The contrasting effects of BMPs in pulmonary vascularCs and the underlying PASMCs provide a hypothesis forulmonary vascular damage and remodeling in familialAH. A critical reduction in BMPR-II function in thendothelium may promote increased endothelial apoptosis,hich compromises the endothelial barrier. This would
llow ingress of serum factors and stimulate activation ofascular elastases. High rates of apoptosis in the endothe-ium could favor the development of apoptosis-resistantlones of ECs and lead to plexiform lesion formation. In thenderlying media, PASMCs already compromised in theirbility to respond to the growth-suppressive effects of BMPsre exposed to growth factors stimulating proliferation.MP signaling in rodent models of PAH. ReducedRNA and protein expression of BMPR-II have been
eported in the lungs of animals with experimental PH97,98). In the monocrotaline rat model, adenoviral deliveryf BMPR-II through the airways failed to prevent PH (99).owever, targeted gene delivery of BMPR-II to the pul-onary endothelium did significantly reduce PH in chron-
cally hypoxic rats (100).Studies in knockout mice reveal the critical role of the
MP pathway in early embryogenesis and vascular devel-pment (101). However, heterozygous BMPR-II �/�ice survive to adulthood with no discernable phenotype
88). When heterozygotes are exposed to lung overexpres-ion of interleukin-1� (102) or chronically infused with-HT (88), they develop more PH compared with wild-ype littermates. Thus, BMPR-II dysfunction increases theusceptibility to PH when exposed to other environmentaltimuli. The relatively low penetrance of PAH withinamilies supports a “two-hit” hypothesis, in which theascular abnormalities are triggered by accumulation ofenetic and/or environmental insults in a susceptible person.
Transgenic mice overexpressing siRNA targetingMPR-II exhibit �10% of the normal levels of BMPR-IIuring development. These mice survive but do not developpontaneous PAH. Intriguingly, they display a phenotypeuggestive of hereditary hemorrhagic telangiectasia, withascular ectasia and anemia (103). Conditional overexpres-ion of a dominant negative kinase domain mutantMPR-II in vascular SMCs of adult mice causes increasedulmonary vascular remodeling and PH (104). Conditionalnockout of endothelial BMPR-II in adult mice has alsoeen shown to predispose to PH (105).
he Extracellular Matrix
he extracellular matrix (ECM) not only represents aubstrate for tissue morphogenesis, but also instructs almostll forms of cell behavior at the biophysical and biochemicalevels through interactions with multiple receptors, includ-ng heterodimeric integrins composed of � and � subunits
106). Importantly, major qualitative and quantitative
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S27JACC Vol. 54, No. 1, Suppl S, 2009 Morrell et al.June 30, 2009:S20–31 Cellular and Molecular Basis of PAH
hanges in the ECM underscore a number of humanathologies, including cancer and PAH. Functional differ-ntiation of the breast epithelium relies upon contact withn appropriate basement membrane by �1 integrins thatromote both proper cell polarity and patterns of genexpression (107). Similarly, the underlying ECM dictateshether human stem cells will differentiate into adipocytesr osteoblasts (108). In this instance, differentiation reliespon cytoskeletal tension generated by RhoA and ROCK.any studies highlight the critical importance of under-
tanding the reciprocal relationships between the ECM andignaling pathways, such as Rho GTPases. The connectionsetween integrins, ECM ligands, and actin-based micro-laments inside the cell are indirect and are linked throughcaffolding proteins, such as talin, paxillin, and �-actinin106). These scaffolds activate or recruit numerous signalingolecules, including focal adhesion kinase and Src kinase
amily members, which then phosphorylate their substrates109).
enascin-C in PAH. Tenascin-C, a large ECM glyco-rotein, is expressed within the medial SMC layer of injurednd remodeling PAs from hypertensive animals (110) andumans (111,112). It surrounds proliferating PASMCsithin arteries from hypertensive individuals (110,111).urthermore, tenascin-C promotes PASMC proliferationnd survival. For example, exogenous tenascin-C proteinmplifies the SMC proliferative response to soluble growthactors, including epidermal growth factor and basic fibro-last growth factor (110), by promoting clustering andctivation of receptor tyrosine kinases, such as epidermalrowth factor receptors (113). Moreover, studies usingsolated PASMCs and PAs from monocrotaline-exposedypertensive rats revealed that suppression of tenascin-Csing an antisense approach induces SMC apoptosis andegression of pulmonary vascular lesions (114).
rigins of the Myofibroblast in PAH
ulmonary hypertension is characterized by cellular changesn the walls of PAs. Virtually all of these changes areharacterized by increased numbers of cells expressing �-M actin (115). It has been thought that the SM-like cellshat express �-SM actin and accumulate in vascular lesionsere derived from the expansion of resident vascular SMCsr adventitial fibroblasts. However, new data suggest otherossible sources of �-SM actin-expressing cells (SM-likeells and/or myofibroblasts) in various vascular diseases.irculating progenitor cells can assume an SM-like pheno-
ype (116). Resident vascular progenitor cells have also beenemonstrated to express SM-like characteristics in severalascular injury states (117). Finally, the possibility that bothpithelial and endothelial cells have the capability of tran-itioning into a mesenchymal or SM-like phenotype haseen raised.ndothelial-mesenchymal transition. The term endothelial-
esenchymal transition (EnMT), rather than transformation e
r transdifferentiation, relates to epithelial biology, where therocess of epithelial-mesenchymal transition has been morehoroughly investigated. Epithelial-mesenchymal transition isprocess in which epithelial cells lose cell-to-cell contacts andolarity and undergo dramatic remodeling of the cytoskeleton118), with repression of epithelial markers. Concurrently, cellsegin to express mesenchymal antigens, including FSP-1,-SM actin, fibronectin, and types I and III collagens, andanifest a proliferative and migratory phenotype. The transi-
ion of epithelial cells toward a mesenchymal phenotype occursuring embryonic development, and recent data suggest thatpithelial-mesenchymal transition is important in cancer biol-gy. A role for epithelial-mesenchymal transition during tissuenjury leading to organ fibrosis is also becoming clear.
Less is known regarding EnMT than epithelial-esenchymal transition. However, several groups have pro-
ided evidence that EnMT is critical in aortic and PAevelopment (119). Endothelial cells labeled at an earlytage of development appear later (at the onset of SMCifferentiation) in the subendothelial space of the develop-ng aorta and express �-SM actin (120). Morphologictudies in human embryos suggest that endothelial-like cellsay give rise to SMC during the maturation of both PAs
nd veins (121). Findings in experimental wound repairave suggested that EnMT may also take place in the adult.imilarly, microvascular ECs transition into mesenchymalells in response to chronic inflammatory stimuli (122). Aole for EnMT in the neointimal thickening observed inransplant atherosclerosis and restenosis has also been sug-ested (120).
Endothelial cells from a variety of vascular beds retain theapability of transitioning into mesenchymal or even SM-ike cells under several culture conditions (119). Endothelialells derived from the adult bovine aorta convert to spindle-haped �-SM actin-expressing cells when treated withGF�-1(123). Human dermal microvascular ECs can be
nduced to transform into myofibroblasts in vitro, afterong-term exposure to inflammatory cytokines (124). Re-ent studies have demonstrated that hypoxia is also capablef inducing transdifferentiation of PAECs into myofibro-last or SM-like cells in a process regulated by myocardin125).
irculating Mesenchymal Progenitorells in Pulmonary Vascular Remodeling
one marrow-derived circulating cells, known as fibrocytes,ay be a source for myofibroblast accumulation during
eparative processes in the lung (126). Fibrocytes are mes-nchymal progenitors that coexpress hematopoietic stemell antigens, markers of the monocyte lineage, and fibro-last products. They constitutively produce ECM compo-ents as well as ECM-modifying enzymes and can furtherifferentiate into myofibroblasts. These cells can contributeo the new population of fibroblasts and myofibroblasts that
merge at tissue sites during normal or aberrant wound
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ealing, in ischemic or inflammatory fibrotic processes, ands part of the stromal reaction to tumor development (127).
The fibrocyte may differentiate into mature mesenchymalells in vivo. Differentiation of fibrocytes into myofibro-lastlike cells occurs where there is increased production ofGF�-1 and/or endothelin. In these settings, fibrocytes orbrocyte precursor cells demonstrate downregulation of
eukocytic markers (e.g., CD34 and CD45) with a concom-tant upregulation of mesenchymal markers. A causal linketween accumulation of fibrocytes at injured sites andngoing tissue fibrogenesis or vascular remodeling has beenrovided in animal models of pulmonary disease (116).nhibition of fibrocyte accumulation results in reducedollagen deposition and reduced accumulation of myofibro-lasts. In the chronically hypoxic rat, monocyte/fibrocyteepletion markedly attenuated pulmonary vascular remod-ling (116).
The transition of any cell type including ECs, progenitorells, fibroblasts, or even SMC into a myofibroblast becomeselevant to a better understanding of PH, as myofibroblastsan generate long-lasting constriction regulated at the levelf Rho/Rho-kinase–mediated inhibition of MLC phospha-ase (128). Thus, cells that have transitioned into fibroblast-ike and myofibroblastlike cells may play a role in thenability of the vessel wall to dilate in response to traditionalasodilating stimuli.
uthor Disclosures
r. Morrell has received research grants from the Britisheart Foundation, Cambridge NIHR Biomedical Researchenter, and Novartis and has received honoraria for educa-
ional lectures from Actelion, GlaxoSmithKline, and Pfizer.r. Archer has received grant support from the National
nstitutes of Health, and has received an honorarium fromilead. Dr. Dupuis has served as a consultant for Actelion,fizer, and Encysive, and is president and a shareholder ofulmoScience Inc. Dr. Jones has received an honorarium fromovartis. Dr. MacLean has received funding from the Bio-
echnology and Biological Sciences Research Council and theritish Heart Foundation. Dr. McMurtry has received a
esearch grant from the National Heart, Lung and Bloodnstitute. Dr. Thistlethwaite has received grant support fromhe National Institutes of Health and the Center for Medicalesearch and Education Fund. Dr. Weissmann has received
esearch grants from the Deutsche ForschungsgemeinschaftExcellence Cluster Cardio-Pulmonary System,” BayerealthCare, Sanofi-Aventis, and Solvay Pharmaceuticals, and
as received lecture fees from Nycomed, Sanofi-Aventis, andolvay Pharmaceuticals. Dr. Yuan has received grant supportrom the National Institutes of Health. Dr. Weir has receivedrant support from NIH RO1 HL 65322. Drs. Adnot and
tenmark report no conflicts of interest.
eprint requests and correspondence: Dr. Nicholas W. Morrell,ox 157, Addenbrooke’s Hospital, Hills Road, Cambridge CB2QQ, United Kingdom. E-mail: [email protected].
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ey Words: pulmonary arterial hypertension y cellular y molecular
