Pathobiology of Pulmonary Hypertension: Marlene Rabinovitch

ANRV295-PM02-14 ARI 13 December 2006 18:31
Pathobiology of Pulmonary
Hypertension
Marlene Rabinovitch
Annu. Rev. Pathol. Mech. Dis. 2007.2:369-399. Downloaded from www.annualreviews.org
by Universita degli Studi di Napoli Frederico II on 08/11/13. For personal use only.
Department of Pediatrics, Stanford University School of Medicine, Stanford,

California 94305; email: marlener@stanford.edu
Annu. Rev. Pathol. Mech. Dis. 2007. Key words

2:369–99
pulmonary vascular disease, pulmonary vascular development,
The Annual Review of
Pathology: Mechanisms of Disease is online at bone morphogenetic protein receptor, elastase
pathmechdis.annualreviews.org
Abstract
This article’s doi:
10.1146/annurev.pathol.2.010506.092033 A variety of conditions can lead to the development of pulmonary
arterial hypertension (PAH). Current treatments can improve symp-
Copyright c 2007 by Annual Reviews.
All rights reserved toms and reduce the severity of the hemodynamic abnormality, but
most patients remain quite limited, and deterioration in their con-
First published online as a Review in
Advance on October 19, 2006 dition necessitates a lung transplant. This review discusses current
experimental and clinical studies that investigate the pathobiology
1553-4006/07/0228-0369$20.00
of PAH. An emerging theme is the consideration of ways in which
one might reverse the advanced occlusive structural changes in the
pulmonary circulation causing PAH. The current debate concerning
the role of regeneration through stem cells is presented. This review
also highlights investigations in a number of laboratories relating the
pathobiology of PAH to mutations causing loss of function of bone
morphogenetic protein receptor II in patients with familial PAH, as
well as sporadic cases.
369
DEVELOPMENT OF THE actin cytoskeleton (5), which likely involves

PULMONARY VASCULATURE IN the Rho kinase pathway (6, 7), and by changes
EMBRYONIC AND FETAL LIFE in the production of connective tissue, es-
pecially elastin and collagen (8–10). Experi-
Normal Lung Vascular Development ments have also shown that there are SMCs
In the fetus, the pre-acinar arteries and with differing proliferative potential (11, 12),
those at the level of the terminal bronchi- and this variability is associated with differ-
olus are muscular, whereas the intra-acinar ences in the activation of protein kinase C in-
arteries (i.e., those accompanying respiratory tracellular signaling pathways (13).
bronchioli) are either partially muscular (sur- Studies by several groups have related
rounded by a spiral of muscle) or nonmuscu- changes in vascular development to specific
lar. Arteries at the alveolar duct and wall lev- alterations in growth factors and extracellular
els are nonmuscular. The immediate postnatal matrix molecules that govern the processes of
period is characterized by rapid recruitment of migration, proliferation, and differentiation

small alveolar duct and wall vessels, which ap- of endothelial cells, SMCs, and fibroblasts,
pear to be functionally and structurally closed and that regulate branching morphogenesis.
in the prenatal period (1). Progressive di- For example, in the lung, tenascin-C is critical
latation of muscular arteries also occurs; that for endothelial cell growth and differentiation
is, the smallest muscular arteries (less than in the lung, leading to angiogenesis and vas-
250 μm) dilate and their walls thin to adult lev- culogenesis (14). A variety of growth factors—
els within a few days, and, by four months, this including vascular endothelial growth fac-
process has included the largest pulmonary ar- tor (VEGF) (15), acidic and basic fibroblast
teries at the hilum. As the external diameter of growth factor (16), and angiopoietins (17)—
the intra-acinar arteries at the various airway interacting with specific cell surface tyrosine
levels increases with age, muscle appears to kinase receptors such as flt-1 and flk-1, tek,
extend peripherally in that it is observed in ar- and tie (18–20) are also responsible for the
teries located more distally within the acinus. orderly growth and branching morphogene-
Although most alveolar duct arteries become sis of blood vessels. The balance between pro-
partially muscularized, most alveolar wall ar- teases and antiproteinases regulates the con-
teries remain nonmuscular even in the adult stant turnover of matrix and the availability of
(2). The process of distal muscularization ap- growth factor receptors.
pears to be related to the differentiation of Recently, a number of studies have shown
pericytes, as well as to the recruitment of fi- that VEGF orchestrates the development of
broblasts (3, 4). the distal vasculature and in fact coordinates
Precapillary (alveolar duct and alveolar alveolar and vascular development (21). The
wall) arteries proliferate through the neonatal effect of VEGF appears to be regulated by
period and early infancy, accompanying the a number of different factors. These include
proliferation of alveoli; the ratio of alveoli to transcription factors such as the hypoxia-
arteries can therefore be used as a measure of inducible transcription factor (22) and oth-
arterial growth. The ratio of alveoli to arter- ers such as forkhead box m1 (23). The Wnt
ies decreases from the newborn value of 20:1 signaling pathway also likely plays an impor-
to the value of 6–8:1, which is first achieved tant role in lung vascular development, but
in early childhood and persists through this has been investigated only recently (23,
adulthood. 24). Vasoactive peptides regulate pulmonary
Experimental studies indicate that the nor- vascular development; for example, endothe-
mal adaptation to postnatal life is regulated by lin production is associated with SMC prolif-
remodeling of the smooth muscle cell (SMC) eration, and nitric oxide (NO) production is
370 Rabinovitch
associated with the suppression of SMC endothelial cells and pericytes. Grades A and
growth (reviewed in References 25 and 26). B are refinements of Heath-Edwards grade I
NO also plays a key role in regulating alveo- (medial hypertrophy). Grade C may be found
larization and lung vascular development (21, with Heath-Edwards grade I, is common with
27). grade II (cellular neointimal formation), and is
invariable with grade III (occlusive neointimal
formation with fibrosis). In fact, when grade
Congenital Heart Defects III is seen, arterial concentration is generally
with Pulmonary Hypertension half that of normal, or less. In general, features
Lung biopsy studies from patients with con- of grade IIIC—or more severe abnormalities
genital heart defects, and analyses of tis- such as Heath-Edwards grade IV (dilatation
sue from experimental animals in which complexes), grade V angiomata formation, or
pulmonary arterial hypertension (PAH) was grade VI (fibrinoid necrosis)—correlate with

induced, have suggested how structural al- severe elevation in pulmonary vascular resis-
terations in the pulmonary arteries produce tance of greater than 8 units × m2 , which is
hemodynamic changes (28). The first abnor- frequently refractory to acute vasodilation in
mality detected is the extension of muscle response to oxygen, prostacyclin, or NO.
into peripheral, normally nonmuscular, arter- Our previous studies suggested that in a
ies (morphometric grade A) (3). With grade B, patient with a congenital heart defect, re-
as in grade A, there is increased extension of versibility of the vascular disease usually oc-
muscle, and, in addition, there is more-severe curs when a surgical repair is performed in
medial hypertrophy of normally muscular ar- the first six months of life. Most often, how-
teries. When medial wall thickness is greater ever, the vascular disease had not progressed
than 1.5 but less than 2 times that of nor- beyond grade IIIC. Beyond two years of age,
mal (mild grade B), pulmonary hypertension the loss of arteries and associated intimal
is usually present; when medial wall thickness formation usually results in some residual
is more than 2 times that of normal (severe hemodynamic impairment (29). Regression of
grade B), pulmonary hypertension is invari- medial hypertrophy and muscularization of
ably present, and usually the pressure value distal vessels following pressure off-loading
is greater than half that of the systemic level. has been shown in experimental studies (30).
The medial thickness is due to hypertrophy Note that the initial response to high flow in-
and hyperplasia of preexisting SMCs, and also duced experimentally from the time of birth
to an increase in the intercellular connective appears to be angiogenic, with high levels of
tissue proteins. VEGF and its receptors. However, later, in as-
With grade C, in addition to the findings sociation with the decline in arterial number,
of severe grade B, arterial concentration and, the resistance is elevated (31).
usually, artery size are reduced. Patients with Our immunohistochemical studies carried
these changes usually have a pulmonary vascu- out in lung biopsy tissue from patients with
lar resistance (mean pulmonary artery – mean congenital heart defects showed that the de-
left atrial pressure to pulmonary flow normal- position of tenascin-C and fibronectin in the
ized to body surface area) ratio of greater than media and neointima increases progressively
3.5 units × m2 . When the artery number is with the severity of the lesion (32). We related
less than half that of normal (severe grade C), increased expression of tenascin-C to vascu-
pulmonary vascular resistance values are often lar SMC proliferation (33), and fibronectin
greater than 6 units × m2 . The cause of grade to increased SMC migration associated with
C is likely the failure to develop new vessels, neointimal formation (Figure 1) (34). Stud-
as well as loss of arteries through apoptosis of ies by other investigators found evidence that
www.annualreviews.org • Pathobiology of Pulmonary Hypertension 371

there is increased activity of the serotonin

transporter (SERT). These findings are in
keeping with studies indicating that a poly-
morphism causing increased SERT activity is
highly prevalent in patients with PAH (41).
Chronic Hypoxia-Induced
Pulmonary Hypertension
Chronic hypoxia induces PAH and vascular
changes that, based upon experimental stud-
ies, consist of muscularization of distal ves-
sels, medial hypertrophy of muscular arteries,

and a reduction in the number of peripheral

Figure 1
arteries. These features are largely reversible
Photomicrographs showing immunoperoxidase staining for tenascin-C with the restoration of a normoxic environ-
(a, d, g), proliferating cell nuclear antigen (PCNA) (b, e, h), and epidermal
growth factor (EGF) (c, f, i ) in graded lung biopsy tissue sections.
ment. However, researchers have learned a
(a–c) Vessel showing a typical grade IA lesion. (d–f ) Vessel showing a great deal regarding the pathobiology of PAH
typical grade IC lesion. (g–i ) Vessel showing a typical grade IIIC lesion. through studying the impact of chronic hy-
In low-grade lesions (a), modest tenascin-C immunostaining was evident poxia on the pulmonary circulation of experi-
in the adventitia. With medial hypertrophy, tenascin-C immunoreactivity mental animals. Interestingly, the loss of pre-
became more prominent in the periendothelium (d ), with the most
intense immunostaining apparent within the neointima of high-grade
capillary arteries occurs despite the increase
lesions showing occlusive neointimal formation ( g ). In the lowest grade in capillaries; the latter finding is likely due to
of lesion, PCNA was negative (b), despite foci of EGF in the media. With an increase in hypoxia-inducible transcription
medial hypertrophy, PCNA was expressed in the media (e), together with factors (42).
foci of EGF ( f ). With the development of higher-grade occlusive lesions, Stenmark et al. (43) took newborn calves to
tenascin-C (g), PCNA (h), and EGF (i ) colocalized to the neointimal cell
layers. Note that tenascin-C and PCNA staining were performed on
a simulated high altitude of 4300 m and ob-
serial sections, whereas EGF detection was carried out on similar vessels served severe PAH with right-to-left shunt-
within the same biopsy. Original magnification × 40. Reproduced with ing due to the rapid development of suprasys-
permission from Reference 32. temic levels of pulmonary artery pressure.
There was striking medial hypertrophy and
the neointimal lesions in PAH are accompa- remarkable proliferation of a dense adventi-
nied by increased expression of transforming tial sheath that, in large vessels, sometimes ex-
growth factor-beta (TGF-β) (35) and procol- hibited neovascularization. Hypoxia-induced
lagen (36, 37). Researchers have also proposed adventitial fibroblast proliferation has been
that endothelial cell proliferation and a form related to protein kinase C activation (zeta
of angiogenesis can be observed with plexi- isoform) (44). Further studies showed strik-
form lesions (38). ing synthesis of elastin in the pulmonary ar-
More recently, we described an increase teries of these neonatal calves (8, 9). Many
in the expression of the calcium-binding pro- recent studies have implicated fibrocytes as
tein S100A4/Mts1 in advanced lesions from key contributors to the remodeling of the pul-
patients with congenital heart defects caus- monary vasculature. These fibrocytes are pu-
ing PAH, as well as in those with idiopathic tative “stem cells” with characteristics of both
PAH (IPAH) (Figure 2) (39). S100A4/Mts1 fibroblasts and leukocytes (45), and they may
can induce vascular SMC migration and pro- migrate into the vessel wall through the an-
liferation, is produced in response to sero- giomata located in the expanding adventitia
tonin stimulation (40), and is enhanced when (Figure 3) (46).
372 Rabinovitch
A variety of studies attempted to show

how acute vasoconstriction or a direct hy-
poxic “injury” initiates the structural changes
observed in the pulmonary arteries. Neona-
tal lamb pulmonary artery SMCs in cul-
ture show a decrease in the production of
prostacyclin in response to acute hypoxic ex-
posure (47), as do bovine endothelial cells
(48). Endothelin receptor blockade appears
to be the most selective agent in reversing
acute hypoxia-induced pulmonary vasocon-
striction, as well as in the remodeling as-
sociated with chronic exposure (49, 50). In

addition, hypoxia inhibits endothelin recep-

tor B–mediated NO synthesis (51). Impaired
NO synthesis and vasodilatation in chronic
hypoxia are also associated with impaired Figure 2
cyclic guanosine monophosphate–dependent Photomicrographs of human lung biopsy tissue after immunoperoxidase
mechanisms (52). Agents also shown to be staining for S100A4/Mts1. (a) Vessel from a patient with a normal
effective in the treatment of chronic hy- pulmonary artery with no immunodetectable S100A4/Mts1. (b) Vessel
poxic pulmonary hypertension in rats include showing a typical grade IB lesion, with severe medial hypertrophy but
without immunoreactivity for S100A4/Mts1. (c) An artery from a patient
inhibitors of 5-lipoxygenase-activating pro- with grade IVC disease, with occlusive neointimal proliferation and strong
tein (53), as well as those of cyclic 3 ,5 positive staining for S100A4/Mts1, particularly in the intima but also in the
guanosine monophosphate–specific phospho- media of the vessel. S100A4/Mts1 was not detected in all cells and appears
diesterase (54), and continuous inhalation of to be localized in a subpopulation of intimal cells. (d) A plexogenic lesion
NO (55). Additional strategies are aimed at from a patient with grade IVC disease, with staining of the smooth muscle
cells and sparing of the endothelial cells (arrows). Immunoreactivity for
preventing acute hypoxic vasoconstriction by S100A4/Mts1 was present in the lung parenchyma at a similar level in all
blocking potassium channels (56, 57). grades of pulmonary vascular disease. Original magnification × 40.
Recent studies in transgenic mice suggest Reproduced with permission from Reference 39.
that genetic factors might modulate the re-
sponse to chronic hypoxia. For example, in (66), as does haploinsufficiency of bone mor-
the absence of hemoxygenase 1, there is re- phogenetic protein receptor II (BMP-RII)
duced production of CO and its associated va- (67) or dominant negative BMP-RII (68),
sodilatory effects (58). Prostacyclin synthetase although in both the latter cases the degree
overexpression is protective against the hemo- of structural remodeling is relatively unim-
dynamic and vascular changes of pulmonary pressive. Haploinsufficiency of BMP-RII in
hypertension (59). Serotonin has been impli- cultured SMCs and in transgenic mice makes
cated in the increased vasoreactivity of the them more sensitive to the proliferative
fawn-hooded rat (60), and there is attenuated effects of serotonin (Figure 5) (69).
severity of pulmonary vascular disease in mice Mice overexpressing the calcium-binding
lacking the SERT gene (61). Most recently, protein S100A4/Mts1 have pulmonary hy-
Rho kinase inhibitors such as fasudil have pertension under room air conditions. Val-
proven effective, even by inhalation, in pre- ues for pulmonary arterial pressure are in-
venting pulmonary hypertension and struc- creased over control mice in chronic hypoxia,
tural changes associated with chronic hypoxia but the remodeling response also appears to
(Figure 4) (62–65). be mitigated. We related this to increased
Overexpression of SERT worsens production of fibulin-5 by S100A4/Mts1 and
hypoxia-induced pulmonary hypertension to the consequent thickening of the elastic

binding growth factors and by decreasing

smooth muscle hyperplasia. There is also
evidence that heparin might inhibit SMC
growth by increasing the activity of the Na/H
antiporter (73). Other studies have shown
that calcitonin gene–related peptide admin-
istered by gene transfer not only attenu-
ates hypoxia-induced vasoconstriction and re-
modeling, but also can enhance the effects
of phosphodiesterase inhibitors (74). We also
showed that exogenous or endogenous ser-
ine elastase inhibitors can effectively reduce
chronic hypoxia-induced pulmonary hyper-

tension (75, 76). In another strategy, acti-

vation of voltage-gated K channels (Kv2.1),
by gene transfer or by a metabolic acti-
vator, inhibited chronic hypoxic pulmonary
hypertension. (77, 78).
Collagen Vascular Disease,

Inflammation, and Pulmonary
Hypertension
Pulmonary hypertension is a frequent com-
Figure 3 plication in certain collagen vascular diseases
Immunohistochemistry (brown peroxidase signal) revealed a greater
(e.g., it occurs commonly in scleroderma);
number of c-kit+ cells in the vessel wall of distal (a) and proximal (b, c) in CREST (calcinosis cutis, Raynaud’s phe-
arteries from hypoxic animals, compared with control (d, e). The c-kit+ nomenon, esophageal dysfunction, sclero-
cells (arrows) were localized contiguous to vasa in proximal vessels (b, c) dactyly, and telangiectasias) syndrome (79,
and contiguous to, and within, the vessel wall of vasa located in the 80); and, more rarely, in systemic lupus ery-
adventitia ( f ). Scale bar represents 100 μm in panels a, d, and e, and 50
μm in panels b, c, and f. Reproduced with permission from Reference 46.
thematosus (81), rheumatoid arthritis (82),
Takayasu’s arteritis (83), polymyositis (84),
and dermatomyositis (85). Thromboembolic
laminae (70). Thus, overexpression of genes phenomena have also been described, par-
that might worsen hypoxia-induced pul- ticularly in association with the antiphos-
monary hypertension appears to invoke com- pholipid syndrome (86). However, in that
pensatory mechanisms that protect against setting, it is generally thought that an im-
the remodeling response. Understanding why mune/inflammatory vasculitis is the initiat-
these compensatory mechanisms fail may ing event. Also, high levels of circulating
be critical in appreciating why some pa- endothelin-1 have been described in subsets
tients develop rapidly progressive pulmonary of patients with systemic sclerosis (87). Au-
hypertension. toantibodies against B23, a cleavage product
Thompson et al.’s (71) and Benitz et al.’s of granzyme B, distinguish the subset of pa-
(72) experimental studies in vitro first sug- tients with scleroderma who also have PAH
gested that vascular smooth muscle growth (88, 89). Unfortunately, the results of ther-
inhibitors may prevent PAH. Heparin infu- apy, including intravenous prostacyclin, have
sion can decrease the severity of hypoxia- not appreciably improved long-term survival
induced vascular changes, presumably by in this subset of patients (90).
374 Rabinovitch
CH-SDR FHR MCT
-5
ΔMPAP (mm Hg)
* *
-10
-15
*
-20
-25
BL 100 BL 100 BL 100
Fasudil (mM)
Figure 4
Effect of inhaled fasudil on mean pulmonary arterial pressures in chronically hypoxic Sprague Dawley
rats (CH-SDR), fawn-hooded rats (FHR), and monocrotaline-treated Sprague Dawley rats (MCT).
Exposure to fasudil was performed under conditions of spontaneous breathing in CH and FHR, and
during mechanical ventilation in MCT. Values are means plus or minus standard error; n = 5 each; ∗ ,
p < 0.05 versus baseline. MPAP, mean pulmonary artery pressure. Reproduced with permission from
Reference 63.
Inflammation has been linked to the de- Neointimal formation associated with in-
velopment of PAH, and both the chemokine flammatory processes in systemic arteries is
receptor CX3CR1 and the chemokine also accompanied by an increase in serine
fractalkine appear to be elevated in PAH elastase activity (96) and by fibronectin pro-
(91). Severe PAH has been associated with duction (97, 98). In the experimental setting,
acquired immunodeficiency syndrome, even Song et al. (99) showed that haploinsufficiency
in the absence of lung parenchymal disease of BMP-RII is associated with an increase in
(92), and has been reproduced experimen- PAH in response to an inflammatory stimu-
tally (93). Moreover, the development of lus. Other experimental models of chronic in-
PAH in the subset of patients with human flammation, such as repeated injections of en-
immunodeficiency virus infection may be a dotoxin (100) or tumor necrosis factor (101),
function of the immunogenetic background can result in the development of pulmonary
(i.e., human leukocyte antigen class II alleles) vascular changes.
(94). More recently, a correlation has been
identified between the expression of human
herpesvirus 8 associated with Kaposi sarcoma Toxins and Pulmonary Hypertension
and IPAH (95), although this association PAH has been associated with the inges-
has not been confirmed in all populations tion of substances, including aminorex—
studied. which resembles epinephrine in its chemical

a b
+/+ Wild type *
750 * 40,000
+/– BMPR2+/–
incorporation (cpm)
H-thymidine
30,000
Cells/well
*
500
*
20,000
*
3
250
10,000
0 0
0.0 0.1 1.0

Day 0 Day 3
Serotonin (μm)
+5-HT
c
1000 –5-HT
+5-HT *
incorporation (cpm)
750 *
H-thymidine
500
3
250
0
Control SB Ketanserin Fluoxetine
BMPR2 +/–
Figure 5
Proliferation to serotonin in pulmonary artery smooth muscle cells (PASMCs). (a) Cells derived from
wild-type and BMPR2+/− mice, showing increased 3 H-thymidine incorporation measured in counts per
minute (cpm) in cells derived from BMPR2+/− mice in 0.1% fetal bovine serum. (b) Cell counts in
wild-type and BMPR2+/− cells exposed to serotonin for three days, showing increased proliferation in
BMPR2+/− cells. (c) 3 H-thymidine incorporation in response to serotonin (5-HT; 1 μmol/L) in
BMPR2+/− PASMCs, and the effect of co-incubation with selective antagonists of the 5-HT2B serotonin
receptor (SB; SB215505, 1 μmol/L), 5-HT2A serotonin receptor (ketanserin, 1 μmol/L), and the
serotonin transporter 5-HTT receptor (fluoxetine, 1 μmol/L). Data are representative of experiments
performed with cells from five wild-type and five BMPR2+/– mice; ∗ , p < 0.05. Reproduced with
permission from Reference 69.
structure, suppresses appetite, and causes have suggested that the PAH that develops in
symptoms of right-sided heart failure in 10% such settings resembles primary PAH in terms
of patients within 6 to 12 months of initial of monoclonal expansion of endothelial cell
administration—and the fenfluramine com- populations (103). As previously mentioned,
pounds, e.g., dexfenfluramine, a serotonin our studies have linked serotonin to increased
antagonist (102). Pathophysiological studies production and secretion of S100A4/Mts1, a
376 Rabinovitch
gene associated with advanced clinical and loids, such as bush tea, causes hepatic veno-
experimental PAH (39, 40). Certain toxic occlusive disease, and a similar compound,
oils, such as rapeseed oil, have been im- monocrotaline, causes severe pulmonary vas-
plicated in the development of malignant cular disease when ingested by animals
PAH (104). Ingestion of pyrrolizidine alka- (105).
Figure 6
Proposed cellular mechanism responsible for the reversal of pulmonary artery muscularity. Elastase
inhibition arrests tenascin-C accumulation and proliferation and induces apoptosis of smooth muscle
cells (SMCs) and loss of extracellular matrix proteins (including elastin). (a–p) Days after injection of
monocrotaline. (a–d) Saline-perfused pulmonary arteries stained with Movat pentachrome. (e–h)
Pulmonary arteries after tenascin-C immunohistochemistry. Arrows indicate positive (brown peroxidase
signal) staining. (i–l) In situ TUNEL (terminal transferase dUTP nick end labeling) assays identifying
apoptosis. Arrows indicate TUNEL-positive vascular cells. (m–p) Proliferating vascular cells, shown by
immunohistochemistry for proliferating cell nuclear antigen (PCNA); dark nuclei are PCNA-positive
cells. (q, r) Percent of SMCs that are TUNEL positive (q) or PCNA positive (r). (s) Densitometric
quantification of elastin. Graphed data represent the mean plus standard error of the mean of n = 4;
scale bars represent 5 μm; ∗ , p < 0.05, compared with †; p < 0.05, compared with results on day 21 in rats
treated with monocrotaline (M) and inhibitor (I) (IZ , ZD0892; IM , M249314); U, untreated; V, vehicle
treated. Adapted with permission from Reference 112.

Rats that ingest the toxin monocrotaline an increase in pulmonary vascular resistance
develop pulmonary arterial changes that can occurs secondary to both a further rise in
be correlated with hemodynamic evidence of pulmonary artery pressure and a drop in
progressive PAH, including increased pul- cardiac output, there is a reduction in the
monary vascular resistance (106–111). Ini- number of peripheral pulmonary arteries,
tially, there is an increase in cardiac out- and “ghost,” or disappearing, vessels can be
put associated with extension of muscle into seen.
peripheral arteries that are normally non- Ultrastructural studies have demonstrated
muscular. After a few weeks, there is an injury to the vascular endothelium within sev-
increase in pulmonary artery pressure, and eral hours of monocrotaline injection; inflam-
medial hypertrophy of normally muscular ar- matory cells and edema are apparent after one
teries is apparent. After three weeks, when week. Studies carried out in our laboratory
q r
* * *
TUNEL-positive SMCs (%)
4 40
PCNA-positive SMCs (%)

*
3 30
*
2 20
1 10
0 0
U U U V IZ IM U U U V
Saline Monocrotaline Saline Monocrotaline
Day 21 Day 28 Day 21 Day 28
s
*
(densitometric units x 10)
10.0 *
*
7.5
Elastin
5.0 * *
2.5
0
U U U V IZ IM IM
Saline Monocrotaline
Day 21 Day 28 Day 35
Figure 6
(Continued )
378 Rabinovitch
comparing the response to monocrotaline in pulmonary hypertension secondary to chronic

neonatal, infant, and adult rats suggested that, hypoxia, there is an early increase in enzy-
in the neonate, transient inflammation causes matic activity only two days after injection
a severe decrease in the number of alveoli. of the toxin, but there is also a further in-
The development of vascular changes is sim- crease in elastase activity observed with ma-
ilar in the three groups. In the infant rat, lignant progression of the disease in adult rats.
however, the abnormalities regress between Furthermore, elastase inhibitors are effective
two and four weeks after injection, whereas in in reducing the pulmonary hypertension and
the adult rat, they progress (106). Our stud-
ies showed that increased elastase activity not a PKI166, daily, (n = 8) No Tx, (n = 9)
only initiated but also contributed to the pro- PKI166, 3x/week, (n = 13) Vehicle, (n = 8)
gression of the vascular changes. Similar to
100
−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→
80
Survival (%)
Figure 7
60
Survival, hemodynamic assessment, and
morphometric analysis after epidermal growth 40
factor receptor blockade. (a) Kaplan-Meier
survival curve of monocrotaline-injected rats that 20 21 days after
monocrotaline
received either no treatment (no Tx), daily
administration of vehicle, or daily or thrice-weekly 0
0 2 4 6 8 10 12 14 32 42
administration of epidermal growth factor
receptor tyrosine kinase inhibitor, PKI166. Days since starting treatment
Saline-injected rats receiving identical treatment
were used as controls (data not shown). (b) Mean b
pulmonary artery pressure (PAP) measurements in 60
saline-injected (n = 12) and surviving
Mean PAP (mm Hg)
monocrotaline-injected, PKI166-treated rats 50

(n = 7) four weeks after cessation of therapy. PAP 40 *
in vehicle- and monocrotaline-treated rats was
measured just before euthanasia (n = 5). (c) Ratio 30
of right ventricle to left-ventricle-plus-septum
weight (RV/LV+S) as an index of right ventricular 20
hypertrophy (RVH) in PKI166-treated animals.
10
(d, e) Representative light microscopic
photographs of Movat pentachrome–stained lung 0
tissue, showing a monocrotaline-induced increase Saline Vehicle PKI166
in arterial muscularization (e) compared with Monocrotaline
saline control (d). Arrows denote alveolar
duct–associated arteries. ( f ) Percentage of
muscularized alveolar duct (Musc. AD) arteries.
c
80
( g, h) Representative light microscopic
photograph of Movat pentachrome–stained lung
*
RVH (RV/LV+S)
tissue, showing monocrotaline-induced decrease in 60

arterial number (g) compared with saline control
(h). Arrows denote alveolar duct and wall arteries. 40
(i) Morphometric analysis of fully and partially
muscularized alveolar duct and wall pulmonary
arteries relative to 100 alveoli. Bars represent 20
mean value plus or minus standard error or the
mean; p < 0.01 compared with vehicle-treated rats;
0
p < 0.05 compared with saline control rats. Saline Vehicle PKI166
Reproduced with permission from Reference Monocrotaline
115.

vascular changes (109). Elastase inhibitors can even one month after cessation of treatment.
effectively reverse the pulmonary hyperten- More recently, the use of a PDGF receptor
sion that results from monocrotaline toxicity, blocker to reverse PAH in this experimental
resulting in values similar to those in con- model (Figure 8) (116) prompted a case re-
trol animals (Figure 6) (112). Reversibility of port showing the successful use of a tyrosine
disease was associated with coordinated apop- kinase inhibitor, Imitibmab (Gleevec), in a pa-
tosis of SMCs and degradation of the ex- tient with advanced pulmonary vascular dis-
cess extracellular matrix (113), with regen- ease (117). This report has prompted several
eration of normal endothelial channels and clinical trials using Gleevec for patients with
precapillary vessels. It appears that the cas- PAH. Other experimental therapies that in-
pases released by apoptosing cells can de- duce regression of pulmonary vascular disease
grade elastin and other extracellular matrix have included simvastatin (118), endothelial
proteins. NO synthase gene therapy in association with

Because the effect of elastase was critical endothelial progenitor cell administration
in maintaining survival signals through epi- (Figure 9) (21, 119), gene therapy with sur-
dermal growth factor receptors (114), we re- vivin (Figure 10) (120), angiopoietin-1 (17),
cently blocked epidermal growth factor re- inhibition of SERT (121), adrenomedullin
ceptors (Figure 7) (115). In these studies, we (122), or the Rho kinase inhibitor fasudil.
showed sustained reversal of progressive PAH Potassium channel dysfunction is implicated
Figure 7
(Continued )
380 Rabinovitch
a b
1.0 110
* Treatment
0.8 * 100
RV/LV+S
90
Survival (%)
0.6
0.4 80
0.2 70
MCT
60 STI571, 1mg/kg/d
0.0
STI571, 10 mg/kg/d
MCT – + + + + + + 50
STI571, 50 mg/kg/d
STI571, 1mg/kg/d – – – – + – –
40
STI571, 10 mg/kg/d – – – – – + – 26 28 30 32 34 36 38 40 42
STI571, 50 mg/kg/d – – – – – – + Day

Day Day Day 42

14 28
c
0.5
*
0.4 *
RV/LV+S
0.3
0.2
0.1
0.0
Hypoxia – + + + +
STI571, 50mg/kg/d – – – + –
STI571, 100 mg/kg/d – – – – +
Day 21 Day 35
Figure 8
Effects of STI571 on right-heart hypertrophy and survival. (a) Ratio of right ventricle to
left-ventricle-plus-septum weight (RV/LV+S), and (b) survival rates of STI571-treated versus
sham-treated [monocrotaline (MCT)-treated control] rats are shown. Treatment dosages of 1, 10, and
50 mg/kg/d began at day 28 after MCT injection; the different doses are indicated. (c) RV/LV+S values of
chronically hypoxic mice. STI571 was applied in dosages of 50 and 100 mg/kg/d by gavage from day 21
through day 35. ∗ , p < 0.05 versus control; p < 0.05 versus MCT-treated at day 28 or hypoxia at day 21;
p < 0.05 versus MCT at day 42 or hypoxia at day 35. Reproduced with permission from Reference 116.
in the pathogenesis of pulmonary vascular SMC and fibroblast proliferative response,

disease (124), and gene therapy restoring K and of the initial endothelial cell susceptibility
channel function has been used effectively to apoptosis (126–129).
to suppress pulmonary vascular disease (78),
but this has not been tested in efforts to re-
verse the process. Our future ability to prevent Unexplained Pulmonary
progression or reverse advanced pulmonary Hypertension: Novel Insights
vascular disease in patients and restore the through Genetics
normal growth of blood vessels (125) may re- After all known causes of PAH have been ruled
quire further identification of the cause of the out, the patient is then diagnosed with IPAH.

This unexplained disease, in which a struc-

tural abnormality is always found either in the
arteries or in the veins, occurs in both chil-
dren and adults and sometimes with a familial
tendency (130–132). A mutation in the BMP-
RII gene is associated with 60% of familial
cases of PAH (130–132), but the penetrance
is only approximately 20%. That is, 80%
of family members that carry the mutation
never develop PAH. Moreover, mutations in
BMP-RII have been described in 20% of spo-
radic cases of PAH (133, 134). In addition,
other BMP-TGF-β receptor family members

such as ALK-1 and endoglin, which were first
←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−
Figure 9
(a) Representative confocal projection of sections
of lungs perfused with fluorescent microspheres
b (green) suspended in agarose (i.e., fluorescent
Prevention Reversal micro-angiography) and immunostained for
0.7 α-smooth muscle actin (red). Normal filling of the
0.6 microvasculature was observed in control rats (i),
whereas rats treated with monocrotaline (MCT)
Perfusion index
0.5 showed a marked loss of microvascular perfusion

0.4 and widespread precapillary occlusion 21 days (ii)
* and 35 days (iii) after MCT injection. In the
0.3 * * prevention model, animals receiving
0.2 endothelial-like progenitor cells (ELPCs)
0.1 displayed improved microvascular perfusion and
preserved continuity of the distal vasculature (iv).
0 In the reversal model, endothelial nitric oxide
Control MCT ELPC ELPC/
eNOS synthase (eNOS)-transduced ELPCs dramatically
improved the appearance of the pulmonary
microvasculature (vi), whereas progenitor cells
alone resulted in more modest increases in
perfusion and little noticeable reduction in
arteriolar muscularization (v). Calibration
c NM PM FM bar = 100 μm. In the prevention model, animals
100 receiving ELPCs displayed improved
microvascular perfusion and preserved continuity
% muscularization
80 of the distal vasculature, and similar findings were

observed in the reversal model. (b) Summary data
60 * * for pulmonary microvascular perfusion for animals
treated in the prevention and reversal protocols (6
40 to 7 animals per group). (c) Proportion of small
* pulmonary arterioles (<30 μm) that are
20 nonmuscularized (NM), partially (PM), or fully
muscularized (FM) in the various treatment
0 groups of the reversal protocol (9 to 13 animals
Control MCT ELPC ELPC/eNOS
per group). ∗ p < 0.05 versus control; p < 0.05
versus MCT saline. Reproduced with permission
from Reference 119.
382 Rabinovitch
80
60
mm Hg
40
20
2 seconds
0
Control MCT +Ad-GFP +Ad-GFP-S-M
Control MCT MCT+Ad-GFP MCT+Ad-GFP-S-M

(mm Hg x g x min ml–1)
(mm Hg x g x min ml–1)

300
120
200
PVRi
SVRi
80
*
100
40
0 0
Figure 10
Gene therapy of rat monocrotaline (MCT)-pulmonary arterial hypertension (PAH) with adenovirus
green fluorescent protein survivin mutant (Ad-GFP-S-M) improves hemodynamics, reduces remodeling
of the resistance pulmonary arteries (PAs), and prolongs survival. (a) Representative high-fidelity PA
pressure tracings and mean data show that Ad-GFP-S-M, but not Ad-GFP, therapy reduces PA pressure
and pulmonary vascular resistance index (PVRi) without altering systemic hemodynamics. SVRi,
systemic vascular resistance index; PVRi, pulmonary valve resistance index. (b, c) Ad-GFP-S-M, but not
Ad-GFP, reduces the right ventricular (RV) thickness measured in parasternal short axis and preserves
the normal round shape of the left ventricle (LV). Similarly, Ad-GFP-S-M reduces pulmonary artery
acceleration time (PAAT). Resistance PA remodeling, as measured by percent medial thickness, is
reduced by treatment with Ad-GFP-S-M. RVOT, right ventriclular outflow tract; AV, aortic valve. Panel
c, original magnification × 40. (d) Targeting survivin with inhaled gene therapy in MCT-PAH
significantly prolongs survival within the study period. ∗ p < 0.05 versus MCT; p < 0.05 versus Ad-GFP.
Reproduced with permission from Reference 120.
identified as mutated in hemorrhagic telang- independent of a mutation in BMP-RII, all

iectasia, are also occasionally mutated in IPAH patients have reduced BMP-RII pro-
patients with PAH (135, 136). Focusing ad- tein expression, as do, to some extent, pa-
ditional evidence on this family of recep- tients with secondary PAH (138). In addi-
tors, a microsatellite instability in the TGF-β tion, a reduction in the coreceptor BMP-RIa
receptor II—resulting in its reduced expres- is frequently observed in patients with IPAH
sion and function—can also be observed in (139). A number of laboratories are address-
patients with IPAH (137). ing the functional consequence of reduced
Although the penetrance is low, the func- or absent BMP-RII in endothelial cells or
tional link between mutations in BMP-RII SMCs. Studies by Morrell et al. (140) reported
and PAH is reinforced by the fact that, that SMCs from patients with IPAH (with or

Figure 10
(Continued )
without a known BMP-RII mutation) prolifer- apoptosis (144). Recent studies have shown
ate in response to TGF-β1 and BMP2. This that BMP4 induces differentiation of fetal
is in contrast to the inhibition of proliferation lung fibroblasts into SMCs and inhibits their
observed with these ligands in SMCs from pa- proliferation (145), suggesting that lack of
tients with normal pulmonary artery pressure BMP-RII might expand this population of
(141–143). The latter is in part linked to SMC cells in disease. In addition, BMP4 normally
384 Rabinovitch
Figure 10
(Continued )
suppresses proximal pulmonary artery SMC each contribute to the development of PAH
proliferation and promotes distal pulmonary (148).
artery SMC proliferation, but in patients with Impaired Smad 1/5 signaling has been
PAH, proximal pulmonary artery SMCs also described in association with a variety of
proliferate in response to BMP4 (146). In BMP-RII mutations (149, 150). There is also
a recent experimental study, deletion of BMP- evidence that signaling through a Smad-
RII in cultured pulmonary artery SMC re- independent, p38-mediated pathway may be
sulted in gain-of-function signaling through abnormal when there are alterations in BMP-
activin receptor IIa, in addition to reduced RII and BMP-RIa oligomerization (146, 151).
BMP2 and −4 and increased BMP6 and Most of the functional data on BMP-RII sig-
−7 signaling (147). Thus, changes in the naling come from studies in SMCs rather
expression of BMP-RII and BMP-RIa, in than in endothelial cells. Although much at-
the availability of ligands such as BMP2, tention has focused on changes in signal-
−4, or −7 or in downstream signaling ing patterns in response to BMPs when
events that influence gene expression, may BMP-RII is dysfunctional, few studies have

specifically addressed the transcription fac- TGF-β, matrix proteins, and macrophages
tors and genes that are subsequently upregu- (159), as well as the molecules observed
lated or suppressed. One candidate supported with advanced lesions in congenital heart de-
by our unpublished studies is the transcrip- fect patients, i.e., fibronectin, tenascin-C, and
tion factor peroxisome proliferator-activated S100A4/Mts1.
receptor gamma (PPARγ). BMP2 can induce The lack of expression of NO synthase
PPARγ transcriptional activity in adipocytes (160), coupled with increased expression of
(152), and TGF-β also causes a rapid increase endothelin (161) in the vessels of patients with
in expression of PPARγ (153). IPAH, suggested that treatment with NO, l-
In young children, the pathobiology sug- arginine, phosphodiesterase inhibitors such as
gests failure of the neonatal vasculature to sildenafil (162, 163), or endothelin receptor
open and a striking reduction in arterial num- blockers might also be beneficial. Recent re-
ber (154). In older children, intimal hyper- sults with both the dual endothelin receptor
plasia and occlusive changes are found in the antagonist (164), as well as with a more selec-
pulmonary arteries, as well as plexiform le- tive antagonist (165, 166), particularly in pa-
sions. In adults, in addition to the latter tients with less severe symptomatology (167,
changes, ghost arteries have also been re- 168), show alleviation of symptoms and slow-
ported. An electron microscopic study (155), ing or even arrest in the progression of dis-
performed on a lung biopsy of an adult ease. There is also increasing experience with
patient, showed severe endothelial injury, sildenafil, used mainly thus far as adjunctive
which was speculated to be the initial site therapy but with promising results in primary
of damage. In some patients, defective von as well as in secondary PAH resulting from
Willebrand factor (156) and fibrinolysis (157) scleroderma (169), sickle cell disease (170),
have been described. Additional features in- thromboembolism [at least based upon exper-
clude thickening of the pulmonary adventitia imental data (171)], congenital heart disease
and venous hypertrophy, as well as endothe- (163), and persistent PAH of the newborn
lial cell hyperplasia (158). Immunohistochem- (172). Of interest are observations that silde-
ical changes include increased expression of nafil can improve PAH in patients refractory
−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→
Figure 11
(a) We speculate, on the basis of our studies in cultured cells and in experimental animals, as to how a
stimulus could induce activity of an elastolytic enzyme and how this might stimulate the remodeling
process. In response to an injurious stimulus, the first casualty is the endothelial cell. As a result of
structural and functional alterations in endothelial cells, some of the barrier function would be lost,
allowing a leak into the subendothelium of a serum factor normally excluded from this region. The
serum factor could induce activity of an endogenous vascular elastase. This enzyme, released from
precursor or mature smooth muscle cells (SMCs), would activate growth factors normally stored in the
extracellular matrix in an inactive form, which are known to induce smooth muscle hypertrophy and
proliferation and increase in connective tissue protein (e.g., collagen and elastin) synthesis. The growth
factors could also result in the differentiation of precursor cells into mature SMCs and thereby
contribute to the muscularization of normally nonmuscular small peripheral arteries. Elastase activity
could also contribute to the loss of small arteries. (b) As a result of elastase activity, a proteolytic cascade
could be amplified through the activation of matrix metalloproteinases. We have shown that proteolysis
of the matrix leads to the induction of tenascin-C, which, in clustering to β3 integrins, results in the
clustering and activation of growth factor receptors that send important survival as well as growth signals
to the cells. Continued elastase activity would cause migration of SMCs in several ways. The elastin
peptides, or degradation products of elastin, can stimulate fibronectin, a glycoprotein that is pivotal in
altering SMC shape and in switching SMCs to the motile phenotype. Inhibition of elastase activity could
lead to SMC apoptosis and regression of pulmonary vascular disease (PVD).
386 Rabinovitch
to intravenous prostacyclin (173). The direct UNRESOLVED

comparison of sildenafil with a dual endothe- CONTROVERSIES
lin receptor antagonist as adjunctive therapies In IPAH, whether the plexiform lesion arises
in combination with anticoagulants showed de novo as a perturbed angiogenic response
comparable results of the two therapies (174). or as a result of remodeling of the vessel
a Elastase-mediated pulmonary vascular disease

Endothelial cell Endothelial injury Degradation SMC proliferation

first casualty serum leak of matrix migration
Elastase Release of
growth factors
Muscularization
Loss of
arteries
Apoptosis
b
Elastase inhibition and regression of PVD
Elastase
Fibronectin
Growth Metalloproteinases
factors
Tenascin-C /
β3 integrin
SMC proliferation Growth factor SMC apoptosis

receptor activation
Progression Regression

Genetic Injury or Inflammation

abnormality
• Increased asymmetric dimethyl arginine, endothelin, chemokines
BMP-RII or
TGF-β family • Reduced prostacyclin, nitrous oxide, vascular endothelial growth factor
member
• Increased platelet-derived growth factor, epidermal growth factor signal
Serotonin
transporter • Impaired voltage-gated potassium channel activity
• Increased intracellular calcium
• Increased elastase, matrix metalloproteinases,
• Increased tenascin-C, fibronectin, Mts1
• Decreased survivin
• Activation of Rho kinase

Impaired endothelial survival, resistance to

apoptosis of smooth muscle cells, enhanced
smooth muscle cell proliferation and migration,
dysregulated antiogenesis
Figure 12
Outline of current processes and future targets of therapy. Those in blue represent vasoactive targets
currently in clinical use, and one growth factor target and one cytoskeletal target in which clinical trials
are ongoing. Those in red represent future potential targets.
following occlusion remains to be established. ogy via release of growth factors (178) and via
Experimental models showing the evolution tenascin-mediated activation of growth factor
of these changes will be most helpful in assess- receptors. Continued release of elastase might
ing the efficacy of future therapies. It will also promote SMC migration by inducing further
be important in the future to link, in a more changes in the extracellular matrix that sig-
unifying hypothesis, the diverse receptor signals the SMCs to produce a new constella-
naling pathways with effectors implicated in tion of gene products, including fibronectin
the pathobiology of PAH. We have tried to (179, 180). In this hypothesis, suppression of
link the BMP-RII/1a pathway with the patho- elastase activity can then lead to regression
biology of PAH by showing that loss of down- of disease by inducing SMC apoptosis and
stream signaling from these receptors might by restoring normal vessels (Figure 11b). In
influence activation of the transcription factor Figure 12, we show how new knowledge in
AML-1, which we have described as critical PAH is producing a variety of new therapeu-
to the production of elastase by SMCs (175– tic targets in this setting. Some of those suc-
177). In Figure 11a and b, we present our cessful in the experimental setting are finding
proposed model for the mechanism by which their way into clinical application, and others
elastase activity could lead to vascular pathol- are close behind.
LITERATURE CITED
1. Hall SM, Haworth SG. 1986. Normal adaptation of pulmonary arterial intima to ex-
trauterine life in the pig: ultrastructural study. J. Pathol. 149:55–66
388 Rabinovitch
2. Hislop M, Reid L. 1973. Pulmonary arterial development during childhood: branching

pattern and structure. Thorax 28:129–35
3. Meyrick B, Reid L. 1980. Ultrastructural findings in lung biopsy material from children
with congenital heart defects. Am. J. Pathol. 101:527–37
4. Jones R. 1993. Ultrastructural analysis of contractile cell development in lung microves-
sels in hyperoxic pulmonary hypertension: fibroblasts and intermediate cells selectively
reorganize nonmuscular segments. Am. J. Pathol. 141:1491–505
5. Hall SM, Gorenflo M, Reader J, Lawson D, Haworth SG. 2000. Neonatal pulmonary hy-
pertension prevents reorganisation of the pulmonary arterial smooth muscle cytoskeleton
after birth. J. Anat. 196(Pt. 3):391–403
6. Wojciak-Stothard B, Tsang LY, Haworth SG. 2005. Rac and Rho play opposing roles
in the regulation of hypoxiareoxygenation-induced permeability changes in pulmonary
artery endothelial cells. Am. J. Physiol. Lung. Cell Mol. Physiol. 288:L749–60
7. Bailly K, Ridley AJ, Hall SM, Haworth SG. 2004. RhoA activation by hypoxia in pul-
monary arterial smooth muscle cells is age and site specific. Circ. Res. 94:1383–91
8. Prosser IW, Stenmark KR, Suthar M, Crouch EC, Mecham RP, Parks WC. 1989. Re-
gional heterogeneity of elastin and collagen gene expression in intralobar arteries in
response to hypoxic pulmonary hypertension as demonstrated by in situ hybridization.
Am. J. Pathol. 135:1073–88
9. Durmowicz AG, Parks WC, Hyde DM, Mecham RP, Stenmark KR. 1994. Persistence,
re-expression, and induction of pulmonary arterial fibronectin, tropoelastin, and type
I procollagen mRNA expression in neonatal hypoxic pulmonary hypertension. Am. J.
Pathol. 145:1411–20
10. Allen K, Haworth SG. 1988. Human postnatal pulmonary arterial remodeling. Ultra-
structural studies of smooth muscle cell and connective tissue maturation. Lab. Invest.
59:702–9
11. Frid MG, Moiseeva EP, Stenmark KR. 1994. Multiple phenotypically distinct smooth
muscle cell populations exist in the adult and developing bovine pulmonary arterial media
in vivo. Circ. Res. 75:669–81
12. Das M, Stenmark KR, Dempsey EC. 1995. Enhanced growth of fetal and neonatal pul-
monary artery adventitial fibroblasts is dependent on protein kinase C. Am. J. Physiol.
269:L660–67
13. Wohrley JD, Frid MG, Moiseeva EP, Orton EC, Belknap JK, Stenmark KR. 1995. Hy-
poxia selectively induces proliferation in a specific subpopulation of smooth muscle cells
in the bovine neonatal pulmonary arterial media. J. Clin. Invest. 96:273–81
14. Ihida-Stansbury K, McKean DM, Gebb SA, Martin JF, Stevens T, et al. 2004. Paired-
related homeobox gene Prx1 is required for pulmonary vascular development. Circ. Res.
94:1507–14
15. Drake CJ, Little CD. 1995. Exogenous vascular endothelial growth factor induces mal-
formed and hyperfused vessels during embryonic neovascularization. Proc. Natl. Acad. Sci.
USA 92:7657–61
16. Klein S, Giancotti FG, Presta M, Albelda SM, Buck CA, Rifkin DB. 1993. Basic fibroblast
growth factor modulates integrin expression in microvascular endothelial cells. Mol. Biol.
Cell 4:973–82
17. Zhao YD, Campbell AI, Robb M, Ng D, Stewart DJ. 2003. Protective role of
angiopoietin-1 in experimental pulmonary hypertension. Circ. Res. 92:984–91
18. Dumont DJ, Fong GH, Puri MC, Gradwohl G, Alitalo K, Breitman ML. 1995. Vascu-
larization of the mouse embryo: a study of flk-1, tek, tie, and vascular endothelial growth
factor expression during development. Dev. Dyn. 203:80–91

19. Shalaby F, Rossant J, Yamaguchi TP, Gertsenstein M, Wu XF, et al. 1995. Failure of
blood-island formation and vasculogenesis in FLK-1 deficient mice. Nature 376:62–66
20. Fong GH, Rossant J, Gertsenstein M, Breitman ML. 1995. Role of the Flt-1 receptor
tyrosine kinase in regulating the assembly of vascular endothelium. Nature 376:66–70
21. Zhao L, Wang K, Ferrara N, Vu TH. 2005. Vascular endothelial growth factor co-
ordinates proper development of lung epithelium and vasculature. Mech. Dev. 122:877–86
22. Asikainen TM, Ahmad A, Schneider BK, Ho WB, Arend M, et al. 2005. Stimulation of
HIF-1α, HIF-2α, and VEGF by prolyl 4-hydroxylase inhibition in human lung endothe-
lial and epithelial cells. Free Radic. Biol. Med. 38:1002–13
23. Kim IM, Ramakrishna S, Gusarova GA, Yoder HM, Costa RH, Kalinichenko VV. 2005.
The forkhead box m1 transcription factor is essential for embryonic development of
pulmonary vasculature. J. Biol. Chem. 280:22278–86
24. Wang Z, Shu W, Lu MM, Morrisey EE. 2005. Wnt7b activates canonical signaling in
epithelial and vascular smooth muscle cells through interactions with Fzd1, Fzd10, and
LRP5. Mol. Cell. Biol. 25:5022–30
25. Ziegler JW, Ivy DD, Kinsella JP, Abman SH. 1995. The role of nitric oxide, endothelin,
and prostaglandins in the transition of the pulmonary circulation. Clin. Perinatol. 22:387–
403
26. Halbower AC, Tuder RM, Franklin WA, Pollock JS, Forstermann U, Abman SH. 1994.
Maturation-related changes in endothelial nitric oxide synthase immunolocalization in
developing ovine lung. Am. J. Physiol. 267:L585–91
27. Balasubramaniam V, Tang JR, Maxey A, Plopper CG, Abman SH. 2003. Mild hypoxia
impairs alveolarization in the endothelial nitric oxide synthase-deficient mouse. Am. J.
Physiol. Lung. Cell Mol. Physiol. 284:L964–71
28. Rabinovitch M, Haworth SG, Castaneda AR, Nadas AS, Reid LM. 1978. Lung biopsy
in congenital heart disease: a morphometric approach to pulmonary vascular disease.
Circulation 58:1107–22
29. Rabinovitch M, Keane JF, Norwood WI, Castaneda AR, Reid L. 1984. Vascular structure
in lung tissue obtained at biopsy correlated with pulmonary hemodynamic findings after
repair of congenital heart defects. Circulation 69:655–67
30. O’Blenes S, Fischer S, McIntyre B, Keshavjee S, Rabinovitch M. 2000. Hemodynamic
unloading leads to regression of pulmonary vascular disease in rats. J. Thorac. Cardiovasc.
Surg. 121(2):279–89
31. Mata-Greenwood E, Meyrick B, Steinhorn RH, Fineman JR, Black SM. 2003. Alterations
in TGF-β1 expression in lambs with increased pulmonary blood flow and pulmonary
hypertension. Am. J. Physiol. Lung. Cell Mol. Physiol. 285:L209–21
32. Jones P, Cowan K, Rabinovitch M. 1997. Progressive pulmonary vascular disease is char-
acterized by a proliferative response related to deposition of tenascin-C and is preceded
by subendothelial accumulation of fibronectin. Am. J. Pathol. 150:1349–60
33. Jones P, Rabinovitch M. 1996. Tenascin-C is induced with progressive pulmonary vascular
disease in rats is functionally related to increased smooth muscle cell proliferation. Circ.
Res. 79:1131–42
34. Clausell N, Rabinovitch M. 1993. Upregulation of fibronectin synthesis by interleukin-
1 β in coronary artery smooth muscle cells is associated with the development of the
postcardiac transplant arteriopathy in piglets. J. Clin. Invest. 92:1850–58
35. Botney MD, Bahadori L, Gold LI. 1994. Vascular remodeling in primary pulmonary
hypertension. Potential role for transforming growth factor-β. Am. J. Pathol. 144:286–95
390 Rabinovitch
36. Botney MD, Liptay MJ, Kaiser LR, Cooper JD, Parks WC, Mecham RP. 1993. Active
collagen synthesis by pulmonary arteries in human primary pulmonary hypertension. Am.
J. Pathol. 143:121–29
37. Botney MD, Kaiser LR, Cooper JD, Mecham RP, Parghi D, et al. 1992. Extracellular
matrix protein gene expression in atherosclerotic hypertensive pulmonary arteries. Am.
J. Pathol. 140:357–64
38. Tuder RM, Groves B, Badesch DB, Voelkel NF. 1994. Exuberant endothelial cell growth
and elements of inflammation are present in plexiform lesions of pulmonary hypertension.
Am. J. Pathol. 144:275–85
39. Greenway S, van Suylen RJ, Du M Sarvaas G, Kwan E, et al. 2004. S100A4/Mts1 produces
murine pulmonary artery changes resembling plexogenic arteriopathy and is increased in
human plexogenic arteriopathy. Am. J. Pathol. 164:253–62
40. Lawrie A, Spiekerkoetter E, Martinez EC, Ambartsumian N, Sheward WJ, et al. 2005.
Interdependent serotonin transporter and receptor pathways regulate S100A4/Mts1, a

gene associated with pulmonary vascular disease. Circ. Res. 97(3):227–35
41. Eddahibi S, Chaouat A, Morrell N, Fadel E, Fuhrman C, et al. 2003. Polymorphism
of the serotonin transporter gene and pulmonary hypertension in chronic obstructive
pulmonary disease. Circulation 108:1839–44
42. Brusselmans K, Compernolle V, Tjwa M, Wiesener MS, Maxwell PH, et al. 2003. Het-
erozygous deficiency of hypoxia-inducible factor-2α protects mice against pulmonary
hypertension and right ventricular dysfunction during prolonged hypoxia. J. Clin. Invest.
111:1519–27
43. Stenmark KR, Fasules J, Hyde DM, Voelkel NF, Henson J, et al. 1987. Severe pulmonary
hypertension and arterial adventitial changes in newborn calves at 4,300 m. J. Appl. Physiol.
62:821–30
44. Das M, Dempsey EC, Bouchey D, Reyland ME, Stenmark KR. 2000. Chronic hypoxia
induces exaggerated growth responses in pulmonary artery adventitial fibroblasts: po-
tential contribution of specific protein kinase c isozymes. Am. J. Respir. Cell Mol. Biol.
22:15–25
45. Stenmark KR, Davie NJ, Reeves JT, Frid MG. 2005. Hypoxia, leukocytes, and the pul-
monary circulation. J. Appl. Physiol. 98:715–21
46. Davie NJ, Crossno JTJ, Frid MG, Hofmeister SE, Reeves JT, et al. 2004. Hypoxia-
induced pulmonary artery adventitial remodeling and neovascularization: contribution
of progenitor cells. Am. J. Physiol. Lung. Cell Mol. Physiol. 286:L668–78
47. Rabinovitch M, Boudreau N, Vella G, Coceani F, Olley P. 1992. Oxygen-related
prostaglandin synthesis in ductus arteriosus and other vascular cells. Pediatr. Res. 32:285
48. Madden MC, Vender RL, Friedman M. 1986. Effect of hypoxia on prostacyclin produc-
tion in cultured pulmonary artery endothelium. Prostaglandins 31:1049–62
49. Li H, Chen SJ, Chen YF, Meng QC, Durand J, et al. 1994. Enhanced endothelin-1 and
endothelin receptor gene expression in chronic hypoxia. J. Appl. Physiol. 77:1451–59
50. Chen SJ, Chen YF, Meng QC, Durand J, DiCarlo VS, Oparil S. 1995. Endothelin-
receptor antagonist bosentan prevents and reverses hypoxic pulmonary hypertension in
rats. J. Appl. Physiol. 79:2122–31
51. Sato K, Rodman DM, McMurtry IF. 1999. Hypoxia inhibits increased ETB receptor-
mediated NO synthesis in hypertensive rat lungs. Am. J. Physiol. 276:L571–81
52. Berkenbosch JW, Baribeau J, Perrault T. 2000. Decreased synthesis and vasodilation
to nitric oxide in piglets with hypoxia-induced pulmonary hypertension. Am. J. Physiol.
Lung. Cell Mol. Physiol. 278:L276–83

53. Voelkel NF, Tuder RM, Wade K, Hoper M, Lepley RA, et al. 1996. Inhibition of 5-
lipoxygenase-activating protein (FLAP) reduces pulmonary vascular reactivity and pul-
monary hypertension in hypoxic rats. J. Clin. Invest. 97:2491–98
54. Cohen AH, Hanson K, Morris K, Fouty B, McMurty IF, et al. 1996. Inhibition of cyclic 3 -
5 -guanosine monophosphate-specific phosphodiesterase selectively vasodilates the pul-
monary circulation in chronically hypoxic rats. J. Clin. Invest. 97:172–79
55. Kouyoumdjian C, Adnot S, Levame M, Eddahibi S, Bousbaa H, Raffestin B. 1994. Contin-
uous inhalation of nitric oxide protects against development of pulmonary hypertension
in chronically hypoxic rats. J. Clin. Invest. 94:578–84
56. Weir EK, Archer SL. 1995. The mechanism of acute hypoxic pulmonary vasoconstriction:
the tale of two channels. FASEB J. 9:183–89
57. Cornfield DN, Reeve HL, Tolarova S, Weir EK, Archer S. 1996. Oxygen causes fetal
pulmonary vasodilation through activation of a calcium-dependent potassium channel.
Proc. Natl. Acad. Sci. USA 93:8089–94

58. Yet SF, Perrella MA, Layne MD, Hsieh CM, Maemura K, et al. 1999. Hypoxia induces
severe right ventricular dilatation and infarction in heme oxygenase-1 null mice. J. Clin.
Invest. 103:R23–29
59. Geraci M, Gao B, Shepherd DC, Moore MD, Westcott JY, et al. 1999. Pulmonary
prostacyclin synthase overexpression in transgenic mice protects against development of
hypoxic pulmonary hypertension. J. Clin. Invest. 103:1509–15
60. Le Cras TD, Kim DH, Markham NE, Abman AS. 2000. Early abnormalities of pulmonary
vascular development in the fawn-hooded rat raised at Denver’s altitude. Am. J. Physiol.
Lung. Cell Mol. Physiol. 279:L283–91
61. Eddahibi S, Hanoun N, Lanfumey L, Lesch KP, Raffestin B, et al. 2000. Attenuated
hypoxic pulmonary hypertension in mice lacking the 5-hydroxytryptamine transporter
gene. J. Clin. Invest. 105:1555–62
62. Fagan KA, Oka M, Bauer NR, Gebb SA, Ivy DD, et al. 2004. Attenuation of acute hypoxic
pulmonary vasoconstriction and hypoxic pulmonary hypertension in mice by inhibition
of Rho-kinase. Am. J. Physiol. Lung. Cell Mol. Physiol. 287:L656–64
63. Nagaoka T, Fagan KA, Gebb SA, Morris KG, Suzuki T, et al. 2005. Inhaled Rho kinase
inhibitors are potent and selective vasodilators in rat pulmonary hypertension. Am. J.
Respir. Crit. Care. Med. 171:494–99
64. Stenmark KR, McMurtry IF. 2005. Vascular remodeling versus vasoconstriction in
chronic hypoxic pulmonary hypertension: a time for reappraisal? Circ. Res. 97:95–98
65. Hyvelin JM, Howell K, Nichol A, Costello CM, Preston RJ, McLoughlin P. 2005. Inhibi-
tion of Rho-kinase attenuates hypoxia-induced angiogenesis in the pulmonary circulation.
Circ. Res. 97:185–91
66. MacLean MR, Deuchar GA, Hicks MN, Morecroft I, Shen S, et al. 2004. Overexpression
of the 5-hydroxytryptamine transporter gene: effect on pulmonary hemodynamics and
hypoxia-induced pulmonary hypertension. Circulation 109:2150–55
67. Beppu H, Ichinose F, Kawai N, Jones RC, Yu PB, et al. 2004. BMPR-II heterozygous
mice have mild pulmonary hypertension and an impaired pulmonary vascular remodel-
ing response to prolonged hypoxia. Am. J. Physiol. Lung. Cell Mol. Physiol. 287:L1241–
47
68. West J, Fagan K, Steudel W, Fouty B, Lane K, et al. 2004. Pulmonary hypertension in
transgenic mice expressing a dominant-negative BMPRII gene in smooth muscle. Circ.
Res. 94(8):1109–14
69. Long L, MacLean MR, Jeffery TK, Morecroft I, Yang X, et al. 2006. Serotonin increases
susceptibility to pulmonary hypertension in BMPR2-deficient mice. Circ. Res. 98:818–27
392 Rabinovitch
70. Merklinger SL, Wagner RA, Spiekerkoetter E, Hinek A, Knutsen RH, et al. 2005. In-
creased fibulin-5 and elastin in S100A4/Mts1 mice with pulmonary hypertension. Circ.
Res. 97:596–604
71. Thompson BT, Spence CR, Janssens SP, Joseph PM, Hales CA. 1994. Inhibition of hy-
poxic pulmonary hypertension by heparins of differing in vitro antiproliferative potency.
Am. J. Respir. Crit. Care Med. 149:1512–17
72. Benitz WE, Lesser DS, Coulson JC, Bernfield M. 1986. Heparin inhibits proliferation
of fetal vascular smooth muscle cells in the absence of platelet-derived growth factor. J.
Cell. Physiol. 127:1–7
73. Quinn DA, Dahlberg CG, Bonventre JP, Scheid CR, Honeyman T, et al. 1996. The
role of Na+ /H+ exchange and growth factors in pulmonary artery smooth muscle cell
proliferation. Am. J. Respir. Cell Mol. Biol. 14:139–45
74. Champion HC, Bivalacqua TJ, Toyoda K, Heistad DD, Hyman AL, Kadowitz PJ. 2000.
In vivo gene transfer of prepro-calcitonin gene-related peptide to the lung attenuates

chronic hypoxia-induced pulmonary hypertension in the mouse. Circulation 101:923–

30
75. Maruyama K, Ye CL, Woo M, Venkatacharya H, Lines LD, et al. 1991. Chronic hypoxic
pulmonary hypertension in rats and increased elastolytic activity. Am. J. Physiol. 261(Pt.
2):H1716–26
76. Zaidi SHE, You XM, Ciura S, Husain M, Rabinovitch M. 2002. Overexpression of the
serine elastase inhibitor elafin protects transgenic mice from hypoxic pulmonary hyper-
tension. Circulation 105:516–21
77. Michelakis ED, Dyck JR, McMurtry MS, Wang S, Wu XC, et al. 2001. Gene transfer
and metabolic modulators as new therapies for pulmonary hypertension. Increasing ex-
pression and activity of potassium channels in rat and human models. Adv. Exp. Med. Biol.
502:401–18
78. Michelakis ED, McMurtry MS, Wu XC, Dyck JR, Moudgil R, et al. 2002. Dichloroac-
etate, a metabolic modulator, prevents and reverses chronic hypoxic pulmonary hyperten-
sion in rats: role of increased expression and activity of voltage-gated potassium channels.
Circulation 105:244–50
79. Sacks DG, Okano Y, Steen VD, Curtiss E, Shapiro LS, Medsger TAJ. 1996. Isolated
pulmonary hypertension in systemic sclerosis with diffuse cutaneous involvement: asso-
ciation with serum anti-U3RNP antibody. J. Rheumatology 23:639–42
80. Damuth TE, Bower JS, Cho K, Dantzker DR. 1980. Major pulmonary artery stenosis
causing pulmonary hypertension in sarcoidosis. Chest 78:888–91
81. Winslow TM, Ossipov MA, Faxio GP, Simonson JS, Redberg RF, Schiller NB. 1995.
Five-year follow-up study of the prevalence and progression of pulmonary hypertension
in systemic lupus erythematosus. Am. Heart J. 129:510–15
82. Sharma V, Vaccharajani A, Mandke J. 1990. Severe pulmonary hypertension in rheuma-
toid arthritis. Int. J. Cardiol. 26:220–22
83. Lande A, Bard R. 1976. Takayasu’s arteritis: an unrecognized cause of pulmonary hyper-
tension. Angiology 27:114–21
84. Bunch TW, Tancredi RG, Lie JT. 1981. Pulmonary hypertension in polymyositis. Chest
79:105–7
85. Caldwell IW, Aitchison JD. 1956. Pulmonary hypertension in dermatomyositis. Br. Heart
J. 18:273–76
86. Sandoval J, Amigo MC, Barragan R, Izauirre R, Reyes PA, et al. 1996. Primary an-
tiphospholipid syndrome presenting as chronic thromboembolic pulmonary hyperten-
sion. Treatment with thromboendarterectomy. J. Rheumatol. 23:772–75

87. Vancheeswaran R, Magoulas T, Efrat G, Wheeler-Jones C, Olsen I, et al. 1994. Circu-

lating endothelin-1 levels in systemic sclerosis subsets—a marker of fibrosis or vascular
dysfunction? J. Rheumatol. 21:1838–44
88. Ulanet DB, Wigley FM, Gelber AC, Rosen A. 2003. Autoantibodies against B23, a nu-
cleolar phosphoprotein, occur in scleroderma and are associated with pulmonary hyper-
tension. Arthritis Rheum. 49:85–92
89. Ulanet DB, Flavahan NA, Casciola-Rosen L, Rosen A. 2004. Selective cleavage of nu-
cleolar autoantigen B23 by granzyme B in differentiated vascular smooth muscle cells:
insights into the association of specific autoantibodies with distinct disease phenotypes.
Arthritis Rheum. 50:233–41
90. Sanchez D, Ganfornina MD, Martinez S. 2002. Expression pattern of the lipocalin
apolipoprotein D during mouse embryogenesis. Mech. Dev. 110:225–29
91. Balabanian K, Foussat A, Dorfmuller P, Durand-Gasselin I, Capel F, et al. 2002. CX3 C

chemokine fractalkine in pulmonary arterial hypertension. Am. J. Respir. Crit. Care Med.
165:1419–25
92. Rhodes J, Schiller MS, Montoya CH, Fikrig S. 1992. Severe pulmonary hypertension
without significant pulmonary parenchymal disease in a pediatric patient with acquired
immunodeficiency syndrome. Clin. Pediatr. (Phila) 31:629–31
93. Gillespie MN, Hartsfield CL, O’Connor WN, Cohen DA. 1994. Pulmonary hyperten-
sion in a murine model of the acquired immunodeficiency syndrome. Am. J. Respir. Crit.
Care Med. 150:194–99
94. Morse JH, Barst RJ, Itescu S, Flaster ER, Sinha G, et al. 1996. Primary pulmonary
hypertension in HIV infection: an outcome determined by particular HLA class II alleles.
Am. J. Respir. Crit. Care Med. 153:1299–301
95. Cool CD, Rai PR, Yeager ME, Hernandez-Saavedra D, Serls AE, et al. 2003. Expression
of human herpesvirus 8 in primary pulmonary hypertension. N. Engl. J. Med. 349:1113–22
96. Oho S, Rabinovitch M. 1994. Post-cardiac transplant arteriopathy in piglets is associated
with fragmentation of elastin and increased activity of a serine elastase. Am. J. Pathol.
145:202–10
97. Molossi S, Clausell N, Rabinovitch M. 1993. Coronary artery endothelial interleukin-1β
mediates enhanced fibronectin production related to postcardiac transplant arteriopathy
in piglets. Circulation 88:248–56
98. Molossi S, Clausell N, Sett S, Rabinovitch M. 1995. ICAM-1 and VCAM-1 expression
in accelerated cardiac allograft arteriopathy and myocardial rejection are influenced dif-
ferently by cyclosporine A and tumor necrosis factor-α blockade. J. Pathol. 176:175–82
99. Song Y, Jones JE, Beppu H, Keaney JFJ, Loscalzo J, Zhang YY. 2005. Increased sus-
ceptibility to pulmonary hypertension in heterozygous BMPR2-mutant mice. Circulation
112:553–62
100. Meyrick B, Brigham K. 1986. Repeated Escherichia coli endotoxin-induced pulmonary
inflammation causes chronic pulmonary hypertension in sheep. Lab. Invest. 55:164–76
101. Stevens T, Janssen PL, Tucker AD. 1992. Acute and long-term TNF-α administration
increases pulmonary vascular reactivity in isolated rat lungs. J. Appl. Physiol. 73:708–12
102. Abenhaim L, Moride Y, Brenot F, Rich S, Benichou J, et al. 1996. Appetite-suppressant
drugs and the risk of primary pulmonary hypertension. International Primary Pulmonary
Hypertension Study Group. N. Engl. J. Med. 335:609–16
103. Tuder R, Radisavljevic Z, Shroyer K, Polak J, Voelkel N. 1998. Monoclonal endothelial
cells in appetite suppressant-associated pulmonary hypertension. Am. J. Respir. Crit. Care
Med. 185:1999–2001
394 Rabinovitch
104. James TN. 1994. The toxic oil syndrome. Clin. Cardiol. 17:463–70
105. Meyrick B, Gamble W, Reid L. 1980. Development of Crotalaria pulmonary hyperten-
sion: hemodynamic and structural study. Am. J. Physiol. 239:H692–702
106. Todd L, Mullen M, Olley P, Rabinovitch M. 1985. Pulmonary toxicity of monocrotaline
differs at critical periods of lung development. Pediatr. Res. 19:731–37
107. Todorovich-Hunter L, Ranger P, Johnson D, Keeley F, Rabinovitch M. 1988. Altered
elastin and collagen synthesis associated with progressive pulmonary hypertensin induced
by monocrotaline. A biochemical and ultrastructural study. Lab. Invest. 58:184–95
108. Todorovich-Hunter L, Dodo H, Ye C, McCready L, Keeley FW, Rabinovitch M. 1992.
Increased pulmonary artery elastolytic activity in adult rats with monocrotaline-induced
progressive hypertensive pulmonary vascular disease compared with infant rats with non-
progressive disease. Am. Rev. Respir. Dis. 146:213–23
109. Ye C, Rabinovitch M. 1991. Inhibition of elastolysis by SC-37698 reduces development

and progression of monocrotaline pulmonary hypertension. Am. J. Physiol. 261:H1255–

67
110. Zhu L, Wigle D, Hinek A, Kobayashi J, Ye C, et al. 1994. The endogenous vascular
elastase that governs development and progression of monocrotaline-induced pulmonary
hypertension in rats is a novel enzyme related to the serine proteinase adipsin. J. Clin.
Invest. 94:1163–71
111. Rosenberg HC, Rabinovitch M. 1988. Endothelial injury and vascular reactivity in
monocrotaline pulmonary hypertension. Am. J. Physiol. 255:H1484–91
112. Cowan KN, Heilbut A, Humpl T, Lam C, Ito S, Rabinovitch M. 2000. Complete reversal
of fatal pulmonary hypertension in rats by a serine elastase inhibitor. Nat. Med. 6:698–
702
113. Cowan KN, Leung WC, Mar C, Bhattacharjee R, Zhu YH, Rabinovitch M. 2005. Cas-
pases from apoptotic myocytes degrade extracellular matrix: a novel remodeling paradigm.
FASEB J. 19(13):1848–50
114. Jones P, Crack J, Rabinovitch M. 1997. Regulation of tenascin-C, a vascular smooth
muscle cell survival factor that interacts with the αvβ3 integrin to promote epidermal
growth factor receptor phosphorylation and growth. J. Cell Biol. 139:279–93
115. Merklinger SL, Jones PL, Martinez EC, Rabinovitch M. 2005. Epidermal growth factor
receptor blockade mediates smooth muscle cell apoptosis and improves survival in rats
with pulmonary hypertension. Circulation 112:423–31
116. Schermuly RT, Dony E, Ghofrani HA, Pullamsetti S, Savai R, et al. 2005. Reversal of
experimental pulmonary hypertension by PDGF inhibition. J. Clin. Invest. 115:2811–21
117. Ghofrani HA, Seeger W, Grimminger F. 2005. Imatinib for the treatment of pulmonary
arterial hypertension. N. Engl. J. Med. 353:1412–13
118. Nishimura T, Vaszar LT, Faul JL, Zhao G, Berry GJ, et al. 2003. Simvastatin rescues rats
from fatal pulmonary hypertension by inducing apoptosis of neointimal smooth muscle
cells. Circulation 108:1640–45
119. Zhao YD, Courtman DW, Deng Y, Kugathasan L, Zhang Q, Stewart DJ. 2005. Rescue
of monocrotaline-induced pulmonary arterial hypertension using bone marrow-derived
endothelial-like progenitor cells: efficacy of combined cell and eNOS gene therapy in
established disease. Circ. Res. 96:442–50
120. McMurtry MS, Archer SL, Altieri DC, Bonnet S, Haromy A, et al. 2005. Gene therapy tar-
geting survivin selectively induces pulmonary vascular apoptosis and reverses pulmonary
arterial hypertension. J. Clin. Invest. 115:1479–91

121. Marcos E, Adnot S, Pham MH, Nosjean A, Raffestin B, et al. 2003. Serotonin transporter
inhibitors protect against hypoxic pulmonary hypertension. Am. J. Respir. Crit. Care Med.
168:487–93
122. Nagaya N, Mori H, Murakami S, Kangawa K, Kitamura S. 2005. Adrenomedullin: an-
giogenesis and gene therapy. Am. J. Physiol. Regul. Integr. Comp. Physiol. 288:R1432–37
123. Abe K, Shimokawa H, Morikawa K, Uwatoku T, Oi K, et al. 2004. Long-term treatment
with a Rho-kinase inhibitor improves monocrotaline-induced fatal pulmonary hyperten-
sion in rats. Circ. Res. 94:385–93
124. Weir EK, Reeve HL, Johnson G, Michelakis ED, Nelson DP, Archer SL. 1998. A role
for potassium channels in smooth muscle cells and platelets in the etiology of primary
pulmonary hypertension. Chest 114:S200–4
125. Winkler F, Kozin SV, Tong RT, Chae SS, Booth MF, et al. 2004. Kinetics of vascular
normalization by VEGFR2 blockade governs brain tumor response to radiation: role of

oxygenation, angiopoietin-1, and matrix metalloproteinases. Cancer Cell 6:553–63

126. Voelkel NF, Cool C, Taraceviene-Stewart L, Geraci MW, Yeager M, et al. 2002. Janus
face of vascular endothelial growth factor: the obligatory survival factor for lung vascular
endothelium controls precapillary artery remodeling in severe pulmonary hypertension.
Crit. Care Med. 30:S251–56
127. Taraseviciene-Stewart L, Kasahar Y, Alger L, Hirth P, McMahon G, et al. 2001. Inhibition
of the VEGF receptor 2 combined with chronic hypoxia causes cell death-dependent
pulmonary endothelial cell proliferation and severe pulmonary hypertension. FASEB J.
15:427–38
128. Taraseviciene-Stewart L, Gera L, Hirth P, Voelkel NF, Tuder RM, Stewart JM. 2002. A
bradykinin antagonist and a caspase inhibitor prevent severe pulmonary hypertension in
a rat model. Can. J. Physiol. Pharmacol. 80:269–74
129. Kanmogne GD, Primeaux C, Grammas P. 2005. Induction of apoptosis and endothelin-
1 secretion in primary human lung endothelial cells by HIV-1 gp120 proteins. Biochem.
Biophys. Res. Commun. 333:1107–15
130. Nichols W, Koller D, Slovis B, Foroud T, Terry V, et al. 1997. Localization of the
gene for familial primary pulmonary hypertension to chromosome 2q31–32. Nat. Genet.
15:277–80
131. Lane KB, Machado RD, Pauciulo MW, Thomson JR, Loyd JE III, et al. 2000. Heterozy-
gous germline mutations in BMPR2, encoding a TGF-β receptor, cause familial primary
pulmonary hypertension. Nat. Genet. 26:81–84
132. Deng Z, Morse JH, Slager SL, Cuervo N, Moore KJ, et al. 2000. Familial primary
pulmonary hypertension (gene PPH1) is caused by mutations in the bone morphogenetic
protein receptor-II gene. Am. J. Hum. Genet. 67:737–44
133. Machado R, Pauciulo M, Thomson J, Lane K, Morgan N, et al. 2001. BPR2 haploin-
sufficiency as the inherited molecular mechanism for primary pulmonary hypertension.
Am. J. Hum. Genet. 68:92–102
134. Thomson JR, Machado RD, Pauciulo MW, Morgan NV, Humbert M, et al. 2000. Spo-
radic primary pulmonary hypertension is associated with germline mutations of the gene
encoding BMPR-II, a receptor member of the TGF-β family. J. Med. Genet. 37:741–
45
135. Harrison RE, Flanagan JA, Sankelo M, Abdalla SA, Rowell J, et al. 2003. Molecular and
functional analysis identifies ALK-1 as the predominant cause of pulmonary hypertension
related to hereditary hemorrhagic telangiectasia. J. Med. Genet. 40:865–71
136. Chaouat A, Coulet F, Favre C, Simonneau G, Weitzenblum E, et al. 2004. Endoglin
396 Rabinovitch
germline mutation in a patient with hereditary hemorrhagic telangiectasia and dexfen-

fluramine associated pulmonary arterial hypertension. Thorax 59:446–48
137. Yeager ME, Halley GR, Golpon HA, Voelkel NF, Tuder RM. 2001. Microsatellite insta-
bility of endothelial cell growth and apoptosis genes within plexiform lesions in primary
pulmonary hypertension. Circ. Res. 88:E2–E11
138. Atkinson C, Stewart S, Upton PD, Machado R, Thomson J, et al. 2002. Primary pul-
monary hypertension is associated with reduced pulmonary vascular expression of type
II bone morphogenetic protein receptor. Circulation 105:1672–78
139. Du L, Sullivan CC, Chu D, Cho AJ, Kido M, et al. 2003. Signaling molecules in nonfa-
milial pulmonary hypertension. N. Engl. J. Med. 348:500–9
140. Morrell NW, Yang X, Upton PD, Jourdan KB, Morgan N, et al. 2001. Altered growth
responses of pulmonary artery smooth muscle cells from patients with primary pulmonary
hypertension to transforming growth factor-β1 and bone morphogenetic proteins. Cir-

culation 104:790–95
141. Dorai H, Vukicevic S, Sampath TK. 2000. Bone morphogenetic protein-7 (osteogenic
protein-1) inhibits smooth muscle cell proliferation and stimulates the expression of
markers that are characteristic of SMC phenotype in vitro. J. Cell Physiol. 184:37–45
142. Nakaoka T, Gonda K, Ogita T, Otawara-Hamamoto Y, Okabe F, et al. 1997. Inhibition
of rat vascular smooth muscle proliferation in vitro and in vivo by bone morphogenetic
protein-2. J. Clin. Invest. 100:2824–32
143. Willette RN, Gu JL, Lysko PG, Anderson KM, Minehart H, Yue T. 1999. BMP-2 gene
expression and effects on human vascular smooth muscle cells. J. Vasc. Res. 36:120–25
144. Zhang S, Fantozzi I, Tigno DD, Yi ES, Platoshyn O, et al. 2003. Bone morphogenetic
proteins induce apoptosis in human pulmonary vascular smooth muscle cells. Am. J.
Physiol. Lung. Cell Mol. Physiol. 285(3):L740–54
145. Jeffery TK, Upton PD, Trembath RC, Morrell NW. 2005. BMP4 inhibits proliferation
and promotes myocyte differentiation of lung fibroblasts via Smad1 and JNK pathways.
Am. J. Physiol. Lung. Cell Mol. Physiol. 288:L370–78
146. Yang X, Long L, Southwood M, Rudarakanchana N, Upton PD, et al. 2005. Dysfunc-
tional Smad signaling contributes to abnormal smooth muscle cell proliferation in familial
pulmonary arterial hypertension. Circ. Res. 96:1053–63
147. Yu PB, Beppu H, Kawai N, Li E, Bloch KD. 2005. Bone morphogenetic protein (BMP)
type II receptor deletion reveals BMP ligand-specific gain of signaling in pulmonary
artery smooth muscle cells. J. Biol. Chem. 280:24443–50
148. Bae SC, Lee KS, Zhang YW, Ito Y. 2001. Intimate relationship between TGF-β/BMP
signaling and runt domain transcription factor, PEBP2/CBF. J. Bone Joint Surg. Am.
83(Pt. 1):S48–55
149. Nishihara A, Watabe T, Imamura T, Miyazono K. 2002. Functional heterogeneity of bone
morphogenetic protein receptor-II mutants found in patients with primary pulmonary
hypertension. Mol. Biol. Cell 13:3055–63
150. Rudarakanchana N, Flanagan JA, Chen H, Upton PD, Machado R, et al. 2002. Functional
analysis of bone morphogenetic protein type II receptor mutations underlying primary
pulmonary hypertension. Hum. Mol. Genet. 11:1517–25
151. Nohe A, Hassel S, Ehrlich M, Neubauer F, Sebald W, et al. 2002. The mode of bone
morphogenetic protein (BMP) receptor oligomerization determines different BMP-2
signaling pathways. J. Biol. Chem. 277:5330–38
152. Zehentner BK, Leser U, Burtscher H. 2000. BMP-2 and sonic hedgehog have contrary
effects on adipocyte-like differentiation of C3H10T1/2 cells. DNA Cell Biol. 19:275–81

153. Fu M, Zhang J, Lin Y, Zhu X, Zhao L, et al. 2003. Early stimulation and late inhibi-
tion of peroxisome proliferator-activated receptor gamma (PPAR gamma) gene expres-
sion by transforming growth factor beta in human aortic smooth muscle cells: role of
early growth-response factor-1 (Egr-1), activator protein 1 (AP1) and Smads. Biochem. J.
370:1019–25
154. Robertson B. 1971. Idiopathic pulmonary hypertension in infancy and childhood. Mi-
croangiographic and histological observations in five cases. Acta Pathol. Microbiol. Scand.
[A] 79:217–27
155. Meyrick B, Clarke SW, Symons C, Woodgate DJ, Reid L. 1974. Primary pulmonary
hypertension: a case report including electronmicroscopic study. Br. J. Dis. Chest 68:11–
20
156. Geggel RL, Carvalho AC, Hoyer LW, Reid LM. 1987. von Willebrand factor abnormal-
ities in primary pulmonary hypertension. Am. Rev. Respir. Dis. 135:294–99

157. Tubbs RR, Levin RD, Shirey EK, Hoffman GC. 1979. Fibrinolysis in familial pulmonary
hypertension. Am. J. Clin. Pathol. 71:384–87
158. Chazova I, Loyd JE, Zhdanov VS, Newman JH, Belenkov Y, Meyrick B. 1995. Pulmonary
artery adventitial changes and venous involvement in primary pulmonary hypertension.
Am. J. Pathol. 146:389–97
159. Liptay MJ, Parks WC, Mecham RP, Roby J, Kaiser LR, et al. 1993. Neointimal
macrophages colocalize with extracellular matrix gene expression in human atheroscle-
rotic pulmonary arteries. J. Clin. Invest. 91:588–94
160. Giaid A, Saleh D. 1995. Reduced expression of endothelial nitric oxide synthase in the
lungs of patients with pulmonary hypertension. N. Engl. J. Med. 333:214–21
161. Giaid A, Yanagisawa M, Langleben D, Michel RP, Levy R, et al. 1993. Expression of
endothelin-1 in the lungs of patients with pulmonary hypertension. N. Engl. J. Med.
238:1732–39
162. Preston IR, Klinger JR, Houtches J, Nelson D, Farber HW, Hill NS. 2005. Acute and
chronic effects of sildenafil in patients with pulmonary arterial hypertension. Respir. Med.
99(12):1501–10
163. Humpl T, Reyes JT, Holtby H, Stephens D, Adatia I. 2005. Beneficial effect of oral
sildenafil therapy on childhood pulmonary arterial hypertension: twelve-month clinical
trial of a single-drug, open-label, pilot study. Circulation 111:3274–80
164. Sitbon O, Badesch DB, Channick RN, Frost A, Robbins IM, et al. 2003. Effects of
the dual endothelin receptor antagonist bosentan in patients with pulmonary arterial
hypertension: a 1-year follow-up study. Chest 124:247–54
165. Horn EM, Widlitz AC, Barst RJ. 2004. Sitaxsentan, a selective endothelin-A receptor
antagonist for the treatment of pulmonary arterial hypertension. Expert. Opin. Investig.
Drugs 13:1483–92
166. Barst RJ, Langleben D, Frost A, Horn EM, Oudiz R, et al. 2004. Sitaxsentan therapy for
pulmonary arterial hypertension. Am. J. Respir. Crit. Care Med. 169:441–47
167. McLaughlin VV, Sitbon O, Badesch DB, Barst RJ, Black C, et al. 2005. Survival with first-
line bosentan in patients with primary pulmonary hypertension. Eur. Respir. J. 25:244–49
168. Rosenzweig EB, Ivy DD, Widlitz A, Doran A, Claussen LR, et al. 2005. Effects of long-
term bosentan in children with pulmonary arterial hypertension. J. Am. Coll. Cardiol.
46:697–704
169. Hayakawa I, Shirasaki F, Hirano T, Oishi N, Hasegawa M, et al. 2005. Successful treat-
ment with sildenafil in systemic sclerosis patients with isolated pulmonary arterial hyper-
tension: two case reports. Rheumatol. Int. 26(3):270–73
398 Rabinovitch
170. Machado RF, Martyr S, Kato GJ, Barst RJ, Anthi A, et al. 2005. Sildenafil therapy in
patients with sickle cell disease and pulmonary hypertension. Br. J. Haematol. 130:445–53
171. Dias-Junior CA, Souza-Costa DC, Zerbini T, da Rocha JB, Gerlach RF, Tanus-Santos
JE. 2005. The effect of sildenafil on pulmonary embolism-induced oxidative stress and
pulmonary hypertension. Anesth. Analg. 101:115–20
172. Juliana AE, Abbad FC. 2005. Severe persistent pulmonary hypertension of the newborn
in a setting where limited resources exclude the use of inhaled nitric oxide: successful
treatment with sildenafil. Eur. J. Pediatr. 164(10):626–29
173. Kataoka M, Satoh T, Manabe T, Anzai T, Yoshikawa T, et al. 2005. Oral sildenafil
improves primary pulmonary hypertension refractory to epoprostenol. Circ. J. 69:461–
65
174. Wilkins MR, Paul GA, Strange JW, Tunariu N, Gin-Sing W, et al. 2005. Sildenafil versus
Endothelin Receptor Antagonist for Pulmonary Hypertension (SERAPH) study. Am. J.

Respir. Crit. Care Med. 171:1292–97

175. Wigle DA, Thompson KE, Yablonsky S, Zaidi SHE, Coulber C, et al. 1998. AML1-like
transcription factor induces serine elastase activity in ovine pulmonary artery smooth
muscle cells. Circulation Res. 83:252–63
176. Kobayashi J, Rabinovitch M. 1994. Elastin-bound serum factor and endothelial cell factor
induce pulmolnary artery smooth muscle cell elastolytic activity. Circulation 90(4, part
II):I–417
177. Kobayashi J, Wigle D, Childs T, Zhu L, Keeley FW, Rabinovitch M. 1994. Serum-
induced vascular smooth muscle cell elastolytic activity through tyrosine kinase intracel-
lular signaling. J. Cell. Physiol. 160:121–31
178. Thompson K, Rabinovitch M. 1996. Exogenous leukocyte and endogenous elastases
can mediate mitogenic activity in pulmonary artery smooth muscle cells by release of
extracellular-matrix bound basic fibroblast growth factor. J. Cell Physiol. 166:495–505
179. Hinek A, Boyle J, Rabinovitch M. 1992. Vascular smooth muscle cell detachment from
elastin and migration through elastic laminae is promoted by chondroitin sulfate-induced
“shedding” of the 67-kDa cell surface elastin binding protein. Exp. Cell Res. 203:344–53
180. Hinek A, Molossi S, Rabinovitch M. 1996. Functional interplay between interleukin-1
receptor and elastin binding protein regulates fibronectin production in coronary artery
smooth muscle cells. Exp. Cell Res. 225:122–31

Contents ARI 13 December 2006 2:13
Annual Review
of Pathology:
Mechanisms of
Disease
Contents
Volume 2, 2007
Frontispiece
Henry C. Pitot p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p xii

Adventures in Hepatocarcinogenesis
Henry C. Pitot p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 1
Endocrine Functions of Adipose Tissue
Hironori Waki and Peter Tontonoz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p31
Endometrial Carcinoma
Antonio Di Cristofano and Lora Hedrick Ellenson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p57
Muscle Diseases: The Muscular Dystrophies
Elizabeth M. McNally and Peter Pytel p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p87
Pathobiology of Neutrophil Transepithelial Migration: Implications in
Mediating Epithelial Injury
Alex C. Chin and Charles A. Parkos p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 111
von Hippel-Lindau Disease
William G. Kaelin, Jr. p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 145
Cancer Stem Cells: At the Headwaters of Tumor Development
Ryan J. Ward and Peter B. Dirks p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 175
Neurofibromatosis
Andrea I. McClatchey p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 191
Malaria: Mechanisms of Erythrocytic Infection and Pathological
Correlates of Severe Disease
Kasturi Haldar, Sean C. Murphy, Dan A. Milner, Jr., and Terrie E. Taylor p p p p p p p p p p p p 217
VEGF-A and the Induction of Pathological Angiogenesis
Janice A. Nagy, Ann M. Dvorak, and Harold F. Dvorak p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 251
In Vivo Pathology: Seeing with Molecular Specificity and Cellular
Resolution in the Living Body
Christopher H. Contag p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 277
vi
Contents ARI 13 December 2006 2:13
Cell-Based Therapy for Myocardial Ischemia and Infarction:

Pathophysiological Mechanisms
Michael A. Laflamme, Stephan Zbinden, Stephen E. Epstein,
and Charles E. Murry p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 307
Cystic Disease of the Kidney
Patricia D. Wilson and Beatrice Goilav p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 341
Pathobiology of Pulmonary Hypertension
Marlene Rabinovitch p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 369
Body Traffic: Ecology, Genetics, and Immunity in Inflammatory
Bowel Disease
Jonathan Braun and Bo Wei p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 401

Contents vii

Pathobiology of Pulmonary Hypertension: Marlene Rabinovitch

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Pathobiology of Pulmonary Hypertension: Marlene Rabinovitch

Uploaded by

Copyright:

Available Formats

ANRV295-PM02-14 ARI 13 December 2006 18:31

Department of Pediatrics, Stanford University School of Medicine, Stanford,

Annu. Rev. Pathol. Mech. Dis. 2007. Key words

DEVELOPMENT OF THE actin cytoskeleton (5), which likely involves

period is characterized by rapid recruitment of migration, proliferation, and differentiation

pulmonary arterial hypertension (PAH) was grade VI (ﬁbrinoid necrosis)—correlate with

www.annualreviews.org • Pathobiology of Pulmonary Hypertension 371

there is increased activity of the serotonin

sels, medial hypertrophy of muscular arteries,

and a reduction in the number of peripheral

A variety of studies attempted to show

sociated with chronic exposure (49, 50). In

addition, hypoxia inhibits endothelin recep-

www.annualreviews.org • Pathobiology of Pulmonary Hypertension 373

binding growth factors and by decreasing

chronic hypoxia-induced pulmonary hyper-

tension (75, 76). In another strategy, acti-

Collagen Vascular Disease,

CH-SDR FHR MCT

www.annualreviews.org • Pathobiology of Pulmonary Hypertension 375

0.0 0.1 1.0

www.annualreviews.org • Pathobiology of Pulmonary Hypertension 377

PCNA-positive SMCs (%)

Day 21 Day 28 Day 21 Day 28

Day 21 Day 28 Day 35

comparing the response to monocrotaline in pulmonary hypertension secondary to chronic

monocrotaline-injected, PKI166-treated rats 50

tissue, showing monocrotaline-induced decrease in 60

www.annualreviews.org • Pathobiology of Pulmonary Hypertension 379

proteins. NO synthase gene therapy in association with

STI571, 50 mg/kg/d – – – – – – + Day

Day Day Day 42

in the pathogenesis of pulmonary vascular SMC and ﬁbroblast proliferative response,

www.annualreviews.org • Pathobiology of Pulmonary Hypertension 381

This unexplained disease, in which a struc-

other BMP-TGF-β receptor family members

such as ALK-1 and endoglin, which were ﬁrst

0.5 showed a marked loss of microvascular perfusion

80 of the distal vasculature, and similar ﬁndings were

Control MCT MCT+Ad-GFP MCT+Ad-GFP-S-M

(mm Hg x g x min ml–1)

(mm Hg x g x min ml–1)

identiﬁed as mutated in hemorrhagic telang- independent of a mutation in BMP-RII, all

www.annualreviews.org • Pathobiology of Pulmonary Hypertension 383

www.annualreviews.org • Pathobiology of Pulmonary Hypertension 385

to intravenous prostacyclin (173). The direct UNRESOLVED

a Elastase-mediated pulmonary vascular disease

Endothelial cell Endothelial injury Degradation SMC proliferation

SMC proliferation Growth factor SMC apoptosis

www.annualreviews.org • Pathobiology of Pulmonary Hypertension 387

Genetic Injury or Inflammation

• Increased intracellular calcium

• Increased elastase, matrix metalloproteinases,

• Increased tenascin-C, fibronectin, Mts1

• Activation of Rho kinase

Impaired endothelial survival, resistance to

2. Hislop M, Reid L. 1973. Pulmonary arterial development during childhood: branching

www.annualreviews.org • Pathobiology of Pulmonary Hypertension 389

Interdependent serotonin transporter and receptor pathways regulate S100A4/Mts1, a

www.annualreviews.org • Pathobiology of Pulmonary Hypertension 391

Proc. Natl. Acad. Sci. USA 93:8089–94

In vivo gene transfer of prepro-calcitonin gene-related peptide to the lung attenuates

chronic hypoxia-induced pulmonary hypertension in the mouse. Circulation 101:923–