Download Pulmonary veins in the normal lung and pulmonary - AJP-Lung

Am J Physiol Lung Cell Mol Physiol 305: L725–L736, 2013.
First published September 13, 2013; doi:10.1152/ajplung.00186.2013.
Pulmonary veins in the normal lung and pulmonary hypertension due to left
heart disease
James M. Hunt,1 Brian Bethea,2 Xiang Liu,3,4 Aneta Gandjeva,1 Pradeep P. A. Mammen,5
Elvira Stacher,6,7 Marina R. Gandjeva,1 Elisabeth Parish,8 Mario Perez,1 Lynelle Smith,1
Brian B. Graham,1 Wolfgang M. Kuebler,3,9 and Rubin M. Tuder1
1
Submitted 15 July 2013; accepted in final form 8 September 2013
Hunt JM, Bethea B, Liu X, Gandjeva A, Mammen PP, Stacher
E, Gandjeva MR, Parish E, Perez M, Smith L, Graham BB,
Kuebler WM, Tuder RM. Pulmonary veins in the normal lung and
pulmonary hypertension due to left heart disease. Am J Physiol Lung
Cell Mol Physiol 305: L725–L736, 2013. First published September
13, 2013; doi:10.1152/ajplung.00186.2013.—Despite the importance
of pulmonary veins in normal lung physiology and the pathobiology
of pulmonary hypertension with left heart disease (PH-LHD), pulmonary veins remain largely understudied. Difficult to identify histologically, lung venous endothelium or smooth muscle cells display no
unique characteristic functional and structural markers that distinguish
them from pulmonary arteries. To address these challenges, we
undertook a search for unique molecular markers in pulmonary veins.
In addition, we addressed the expression pattern of a candidate
molecular marker and analyzed the structural pattern of vascular
remodeling of pulmonary veins in a rodent model of PH-LHD and in
lung tissue of patients with PH-LHD obtained at time of placement on
a left ventricular assist device. We detected urokinase plasminogen
activator receptor (uPAR) expression preferentially in normal pulmonary veins of mice, rats, and human lungs. Expression of uPAR
remained elevated in pulmonary veins of rats with PH-LHD; however,
we also detected induction of uPAR expression in remodeled pulmonary arteries. These findings were validated in lungs of patients with
PH-LHD. In selected patients with sequential lung biopsy at the time
of removal of the left ventricular assist device, we present early data
suggesting improvement in pulmonary hemodynamics and venous
remodeling, indicating potential regression of venous remodeling in
response to assist device treatment. Our data indicate that remodeling
of pulmonary veins is an integral part of PH-LHD and that pulmonary
veins share some key features present in remodeled yet not normotensive pulmonary arteries.
pulmonary hypertension; pulmonary circulation; left heart failure;
pulmonary veins; vessel remodeling
ALTHOUGH THE ARTERIAL and venous pulmonary circulations
form a communicating, integrated vascular system, pulmonary
veins are developmentally, physiologically, and structurally
distinct from pulmonary arteries. Pulmonary veins develop
Address for reprint requests and other correspondence: R. M. Tuder,
Program in Translational Lung Research, Division of Pulmonary Sciences and
Critical Care Medicine, Univ. of Colorado Denver, Anschutz Medical Campus,
Research 2 — 9th floor, Rm. 9001; Mail stop C-272, 12700 East 19th Ave.,
Aurora, CO 80045 (e-mail: [email protected]).
http://www.ajplung.org
chronologically after the initial segments of large pulmonary
arteries form and are often identifiable in a perpendicular
arrangement to that of the pulmonary arteries in the bronchoarterial sheath (5). Pulmonary veins respond physiologically to
the vasodilator prostacyclin and to vasoconstrictors, such as
platelet activating factor, to a larger extent than that observed
with pulmonary arteries (9, 34). These anatomic and physiological distinctions are carried over to the histological level
since pulmonary veins lack distinct double internal and external elastic laminae and have only a thin medial muscular layer
(as compared with similar diameter arteries) (32). However,
this more refined histological distinction does not persist in
diseases characterized by pulmonary hypertension (PH), because pulmonary veins become more arterial in appearance,
acquiring a distinct double elastic layer flanking a hypertrophic
media (33).
Although veins in the lung parenchyma lack unique distinguishing features, the location of larger veins in interlobular
septa (alongside pulmonary lymphatics) provides for a unique
anatomic landmark that aids in their histological recognition.
Indeed, venous pathology within interlobular septa serves as
the diagnostic criterion of a rare but characteristic form of PH
due to thrombotic venous occlusion, veno-occlusive disease
(VOD) (21, 25). The rarity of VOD contrasts with PH due to
left heart disease (PH-LHD), which is the most common cause
of PH in the United States and affects over 250,000 Americans
and perhaps as many as 2 million people worldwide (11, 26,
27). Despite its high prevalence and significant impact on
morbidity and mortality, the venous pathology and pathophysiology of PH-LHD are poorly understood, and insights that can
guide proper recognition and translational investigations are
lacking. These shortcomings also apply to the venous involvement in pulmonary vascular disease caused by the autoimmune
disease scleroderma, thought to contribute to the severity of the
disease (6, 7).
A critical limitation to the study of pulmonary venous
disease is the current inability to identify parenchymal pulmonary veins; as outlined above, these vessels lack clear histological or distinct molecular markers. A recent publication
showed that the pulmonary vein endothelium in adult mice
expresses the ephrin B4 receptor (EphB4), which was, however, not reactive in adult human pulmonary veins (4, 12);
1040-0605/13 Copyright © 2013 the American Physiological Society
L725
Downloaded from http://ajplung.physiology.org/ by 10.220.33.3 on May 6, 2017
Program in Translational Lung Research, Division of Pulmonary Sciences and Critical Care Medicine, Anschutz Medical
Campus, Aurora, Colorado; 2University of Texas Southwestern Medical Center, Department of Cardiothoracic and Vascular
Surgery, Dallas, Texas; 3Institute of Physiology, Charité — Universitätsmedizin Berlin and German Heart Institute, Berlin,
Germany; 4Department of Anesthesia, Shanghai East Hospital, China; 5University of Texas Southwestern Medical Center,
Division of Cardiology, Dallas, Texas; 6Institute of Pathology, Medical University of Graz, Graz, Austria; 7Ludwig
Boltzmann Institute for Lung Vascular Research, Graz, Austria; 8University of Texas Southwestern Medical Center, School of
Medicine, Dallas, Texas; and 9The Keenan Research Centre, St. Michael’s Hospital, Toronto, Ontario, Canada
L726
PULMONARY VEINS AND LEFT HEART DISEASE
MATERIALS AND METHODS
Reagents. We purchased commercial antibodies to uPAR (Santa
Cruz Biotechnology, Santa Cruz, CA), anti-smooth muscle actin
(anti-␣-SMA) (Abcam, Cambridge, MA), heavy chain cardiac myosin
(HCCM) (mouse monoclonal) IgG1 (Abcam), and EphB4 (R&D
Systems, Minneapolis, MN). Commercial kits were used for all
immunohistochemistry and immunofluorescence (Vector Labs, Burlingame, CA), and manufacturer’s instructions were followed. A
commercial kit for the Russell-Movat pentachrome stain was purchased and the manufacturer’s instructions were followed (American
MasterTech Scientific, Lodi, CA).
Animals. Animal studies were conducted in accordance with the
Guide for the Care and Use of Laboratory Animals, 9th Edition
(2011), and all protocols were approved by the University of Colorado’s Institutional Animal Care and Use Committee. C57BL/6 male
wild-type mice were purchased from The Jackson Laboratory (Bar
Harbor, ME), and 2- to 3-wk-old male Sprague Dawley rats were
purchased from Harlan Laboratories (Denver, CO).
Humans. Study subjects were prospectively enrolled patients referred to a tertiary cardiac hospital for LVAD placement due to
PH-LHD from February 2010 to June 2011. The study was approved
by the University of Texas Southwestern Institutional Review Board
(IRB 092010-093), and all samples were deidentified by assignment
of a code at the site of collection. Briefly, wedge biopsies of the left
or right lower lung were taken at the time of LVAD placement. Three
patients had additional wedge biopsies collected at the time of LVAD
removal and heart transplantation. Tissue was immediately fixed in
4% paraformaldehyde, processed, and embedded in paraffin blocks.
Clinical data were obtained and matched to the coded lung samples
(Table 1).
Control subjects consisted of 20 randomly chosen failed organ
donors enrolled in the Pulmonary Hypertension Breakthrough Initiative from April 2006 to August 2011 (31). Deidentified clinical data
and lung tissue samples were available. The study was approved by
the Colorado Multiple Institution Review Board (COMIRB 10-1440
and 11-0135).
Venous backfilling. Venous backfilling was performed in anesthetized animals ventilated at tidal volumes of 6 ml/kg. A median
thoracotomy was performed and the pulmonary circulation was
flushed with heparin and phosphate-buffered saline until clear. The
aorta was cross-clamped with a hemostat and a 1:10 bead-1% agarose
mixture (3 ml for rats, 250 ml for mice) was injected under constant
pressure (20 mmHg) into the left ventricle, backfilling the left atria
and pulmonary veins. The beads were 1 ␮m in diameter and were
either red fluosphere polystyrene microspheres (Molecular Probes,
Eugene, OR), to be later used in fresh frozen sections, or green silica
Table 1. Demographic and hemodynamic details in control
subjects and patients with PH-LHD
No.
Age, yr
Sex (male), no. (%)
Race, no. (%)
W
AA
Ever smoker, %
BMI, kg/m2
CHF diagnosis, no. (%)
Ischemic
Nonischemic
Viral
Idiopathic DCM
Echocardiogram, no. (%)
Severe LV
Dysfunction
RV Dilatation
Normal
Mild
Moderate
Severe
RHC hemodynamics
RAP, mmHg
mPAP, mmHg
PAOP, mmHg
TPG, mmHg
CO, l/min
PVR, Woods units
NYHA class (%)
Class I
Class II
Class III
Class IV
PH-LHD
Control
18*
57.4 ⫾ 12.9 (30–78)
13 (72)
19
34.5 ⫾ 17.8 (12–62)
14 (73)
18 (100)
18 (95)
1 (5)
NA
NA
12 (67)
28.6 ⫾ 6.3
8 (44)
8 (44)
1 (6)
1 (6)
NA
18 (100)
3 (16)
5 (28)
6 (33)
1 (6)
13.2 ⫾ 6.4
41.6 ⫾ 12.3
24.0 ⫾ 6.9
18.6 ⫾ 6.7
4.2 ⫾ 1.1
4.7 ⫾ 2.4
0
0
4 (23)
14 (77)
NA
NA
NA
Data are shown as ⫾SD. AA, African American; BMI, body mass index;
CHF, congestive heart failure; CO, cardiac output; DCM, dilated cardiomyopathy; LV, left ventricle; NYHA, New York Heart Association; PH-LHD,
pulmonary hypertension with left heart disease; mPAP, mean pulmonary
arterial pressure; PAOP, pulmonary arterial occlusion pressure; PVR, pulmonary vascular resistance; RAP, right atrial pressure; RV right ventricle; TPG,
transpulmonary gradient; W, White; NA, not available. *Clinical data were not
available for 4 cases of PH-LHD.
particles (Kisker Biotech, Steinfurt, Germany) for use in formaldehyde-fixed paraffin-embedded tissue.
Aortic banding model of PH-LHD. PH-LHD was induced in juvenile rats (92 ⫾ 9.7 g in weight) by supracoronary aortic banding
(AOB) as previously described (35). Isoflurane was used as the
anesthetic for all procedures. Briefly, anesthetized rats underwent a
left thoracotomy and the ascending aorta was partially occluded by a
titanium clip (Hemoclip, Weck Closure System, Research Triangle
Park, NC) with an internal diameter of 0.8 mm. Sham-operated rats
(with implantation of the clip onto the thymus) of similar body weight
and age served as controls.
Hemodynamic monitoring. Nine weeks after AOB or sham operations, rats underwent invasive hemodynamic monitoring. PH-LHD or
control rats were anesthetized and ventilated with a tidal volume of 6
ml/kg body wt. Polyvinyl catheters with an internal diameter of 0.58
mm were introduced via the left carotid artery and the right jugular
vein for continuous monitoring of mean arterial and central venous
pressure, respectively. Measurement of pulmonary arterial pressure
was performed through the right jugular catheter after it was advanced
into the pulmonary artery via right atria and ventricle. Following a
median thoracotomy the pericardium was opened and a 1.6-Fr solidstate catheter (Transonic Systems, Ithaca, NY) was introduced into the
left ventricle to measure left ventricular end-diastolic pressure
AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00186.2013 • www.ajplung.org
Downloaded from http://ajplung.physiology.org/ by 10.220.33.3 on May 6, 2017
furthermore, rats lack an annotated EphB4 gene analog. Consequently, molecular tools to systematically study the pulmonary venous circulation and pulmonary venous disease have
been largely lacking to date.
Reliant on fluorescent backcasting, which is allied to laser
capture microdissection (LCM) enrichment of vein cells from
normal lungs, we report on a set of molecules that are preferentially expressed in normal pulmonary veins. When applied to
both a rodent model of PH due to left heart dysfunction and
human lung samples of patients with the disease, one of these
markers, urokinase plasminogen activator receptor (uPAR),
became shared with pulmonary arteries, consistent with a
structural and molecular convergence of arterial and venous
phenotypes at in pulmonary hypertensive states. Importantly,
these novel observations were validated in lungs of patients
with PH-LHD, in whom the PH-LHD was attenuated after
placement of a left ventricular assist device (LVAD).
L727
PULMONARY VEINS AND LEFT HEART DISEASE
Table 2. PCR array with the 19 top genes preferentially expressed in veins compared with arteries⫹veins
Average ⌬Ct
Position in
PCR
Array
E10
E03
D03
F04
C10
C03
F02
D12
C12
E12
D10
F01
G04
F11
D04
E11
D01
Description
Plasminogen
activator, urokinase
Matrix
metallopeptidase 9
Interleukin 6
Sphingosine kinase 1
Hepatocyte growth
factor
Fibroblast growth
factor 6
Serine (or cysteine)
peptidase inhibitor,
clade F, member 1
Midkine
Interferon gamma
Plexin domain
containing 1
Leptin
Prostaglandinendoperoxide
synthase 1
Tissue inhibitor of
metalloproteinase 1
Transforming growth
factor, beta 2
Integrin alpha V
Plasminogen
Insulin-like growth
factor 1
Gene
Symbol
Gene Name
Veins
Control
group
artery⫹
parenchyma
2⫺⌬Ct
Fold Up- or
Downregulation
Group 1
veins
Control
group
artery/
parenchyma
Group 1 veins
vs. control
group artery/
parenchyma
u-PA/uPA
Plau
⫺0.62
6.71
1.536875
0.009552
160.9
AW\?\43869/\?\MM P9/Clo4b/
MMPIl-6
1110006G24Rik/Sk1/Spk1
C230052L06Rik/HGF/SF/NK1/
NK2/SF/HGF
Fgf-6/HSTF-2
Mmp9
⫺0.25
6.71
1.189207
0.009552
124.5
Il6
Sphk1
Hgf
0.01
0.14
0.06
6.71
6.66
6.52
0.993092
0.907519
0.959264
0.009552
0.009889
0.010896
103.97
91.77
88.03
Fgf6
0.8
6.71
0.574349
0.009552
60.13
AI195227/EPC-1/Pedf/Pedfl/Sdf3
Serpinf1
0.03
5.92
0.97942
0.016459
59.51
MK/Mek
IFN-g/Ifg
2410003I07Rik/AI848450/
MGC130377/Tem7
ob/obese
COX1/Cox-1/Cox-3/Pghs1
Mdk
Ifng
Plxdc1
0.74
1.11
0.58
6.62
6.71
6.09
0.598739
0.463294
0.668964
0.010132
0.009552
0.01468
59.1
48.5
45.57
Lep
Ptgs1
1.28
1.05
6.71
6.23
0.411796
0.482968
0.009552
0.013276
43.11
36.38
Clgi/MGC7143/TIMP-1/Timp
Timp1
1.63
6.71
0.323088
0.009552
33.82
BB105277/Tgf-beta2/Tgfb-2
Tgfb2
1.22
6.28
0.429283
0.012869
33.36
1110004F14Rik/2610028E01Rik/
CD51/D430040G12Rik
AI649309/Pg
C730016P09Rik/Igf-1/Igf-1
Itgav
⫺0.51
4.45
1.42405
0.045753
31.12
1.95
1.61
6.71
6.07
0.258816
0.327598
0.009552
0.014833
27.1
22.09
Plg
Igf1
The full set of gene list containing 96 genes can be provided upon request.
AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00186.2013 • www.ajplung.org
Downloaded from http://ajplung.physiology.org/ by 10.220.33.3 on May 6, 2017
10 min, followed by 40 cycles of denaturation at 95°C for 15 s and
annealing at 60°C for 1 min. Software available through the manufacturer’s website (http://pcrdataanalysis.sabiosciences.com/pcr/
arrayanalysis.php) was used to analyze the RT2 PCR data. We
calculated relative abundance of mRNA expression in each RT-PCR
sample as 2⫺⌬⌬Ct (Table 2). The full list of genes and the fold
increase/decrease in veins vs. arteries⫹parenchyma can be provided
upon request.
Immunohistochemistry and immunofluorescence. We performed
immunohistochemistry and immunofluorescence on 4-␮m-thick paraffin-embedded sections of agarose-inflated lungs from mice or rats as
previously described, or on 4-␮m-thick paraffin-embedded sections of
human lung tissue. Primary antibodies included rabbit polyclonal
antibody to human uPAR (1:25, sc-10815, Santa Cruz Biotechnology), mouse monoclonal antibody to ␣-SMA (1:100, clone 1A4,
Abcam), mouse monoclonal to HCCM (1:100, clone 3– 48, Abcam),
and goat polyclonal antibody to mouse EphB4 (1:100, R&D Systems
AF446). Primary antibodies were incubated at 4°C overnight with
tissue sections. Isotype antibodies served as negative controls. We
performed image quantification using Metamorph as previously described (28).
Quantification of vascular remodeling and density. To determine
volume density of venous and arterial intima, media, and adventitia in
human lung samples, slides were first stained with Russell-Movat
pentachrome stain. Slides were then scanned with the Aperio ImageScope system (Vista), and the free stereological software Stepanizer
(http://www.stepanizer.com) was used to overlay a 256-point grid (for
assessment of the vascular structures) with a 16-point (coarse) grid for
subsampling alveolar septae as previously described (31).
(LVEDP). Cardiac output was measured either by an ultrasonic flow
probe (Transonic, Transonic Systems) or by intravenous injection of
cardiogreen dye (Sigma, St. Louis, MO) and measured by densitometry
(3). Systemic arterial, central venous, pulmonary arterial, LVEDP, and
cardiac output were continuously registered by use of the software
package LabChart 7. Pulmonary vascular resistance was calculated by
the standard equation: (mean pulmonary arterial pressure ⫺ left
ventricular end-diastolic pressure)/cardiac output.
Laser capture microdissection. Fresh lung samples were inflated
with OCT compound (Sakura, Torrance, CA) and immediately flash
frozen in liquid nitrogen. Individual 15-␮m sections were cut by
cryostat at ⫺20°C, adhered to LCM membrane slides, stained with
crystal violet and eosin for contrast, and immediately dissected by use
of LCM (Molecular Machines and Industries, Zurich, Switzerland).
Approximately 20 veins, arteries, and parenchymal samples were
obtained per slide.
RNA purification and RT-PCR. RNA was purified from LCM
dissected lung tissue by use of an Arcturus PicoPure kit and following
the manufacturer’s instructions (Applied Biosystems, Grand Island,
NY). Isolated RNA was further purified by DNase digestion by using
a commercial kit and following the manufacturer’s instructions (Qiagen). cDNA was made by using RT First strand kit (Qiagen) for later
use in the RT2 PCR arrays, or reverse-transcription iScript cDNA
synthesis kit (Bio-Rad, Hercules, CA) for RT-PCR. RT2 PCR array
(Mouse Angiogenesis, PAMM-024A) (SABiosciences, Valencia, CA)
or gene-targeted RT-PCR using commercial primer sets to uPAR or
EphB4 (Invitrogen, CA) and cyclophilin A (Applied Biosystems)
were performed on an Applied Biosystems 7300 real-time PCR
system. RT-PCR conditions included initial denaturation at 95°C for
L728
PULMONARY VEINS AND LEFT HEART DISEASE
In rat (sham and AOB) and human (control and PH-LHD) lung,
tissue morphometric analyses were applied to obtain media and
venous cardiac myocyte sheath total wall fractional thickness as
previously described (10). These measurements were performed on
circular vascular profiles by use of immunofluorescent staining to
␣-SMA, HCCM, and uPAR. The external and internal media radii and
intima radius were calculated by using r ⫽ A/␲ based on the measured
area within each perimeter. For each case, 3 to 11 arteries were
examined (median 6, interquartile range 4 –9).
Statistics. Statistical analyses were performed with Sigma Stat
Version 2.03 (IBM, Armonk, NY). Data are presented as means ⫾ SD
for normally distributed data. Differences between groups were assessed by Student’s t-test. Correlation between two variables was
determined by using the Pearson’s product-moment correlation coefficient. P values less than 0.05 were considered statistically significant.
Backfilling of pulmonary veins. To reliably identify parenchymal pulmonary veins, we modified a previously described
fluorescent microangiography technique (8) by retrogradely
infusing fluorescein-tagged beads through the left heart of mice
and rats (Fig. 1A). This approach led to venous casting extending from large to postcapillary small veins and venules, without leakage into capillaries or pulmonary arteries. We confirmed the accuracy of this technique by simultaneous immunostaining for EphB4, a known and reliable marker of mouse
pulmonary vein endothelium (but not of rat or human veins)
(Fig. 1B). The bead marking of pulmonary veins highly correlated with expression of EphB4 (Pearson’s correlation coefficient ⫽ 0.9). The venous backfilling technique was also
adapted with similar efficacy to rat lungs (Fig. 1C). The
Fig. 1. A: backfilling pulmonary veins with fluorescent tags is accomplished by first flushing the pulmonary circulation with heparin and PBS. The aorta is then
clamped and a mixture of 1% agarose and 1-␮m fluorescent beads is injected in to the left ventricle under ⱕ20 mmHg pressure. B: mouse vein (ve; arrows)
backfilled with fluorescent beads and counterstained for EphB4 (arrows) (N ⫽ 5). C: rat lungs. Scale bars ⫽ 100 ␮m. Green fluorescence ⫽ beads backfilled
into the veins; blue ⫽ DAPI.
AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00186.2013 • www.ajplung.org
Downloaded from http://ajplung.physiology.org/ by 10.220.33.3 on May 6, 2017
RESULTS
cardiomyocyte sheath extends proximally from the left atrium
around pulmonary veins and envelops the ␣-SMA-positive
venous smooth muscle cells of the larger veins; we relied on
the identification of this sheath to aid in confirmation of venous
specific casting. The extent of this sheath depends on the
species and size of animal studied, but in general it extends out
to vessels of 100 –150 ␮m in diameter in rats (16, 20, 24).
Laser capture microdissection and identification of pulmonary vein markers. To identify candidate molecular markers of
pulmonary veins, we used LCM to specifically isolate pulmonary vein mRNA, once the veins were identified by venous
backfilling in mice (Fig. 2A). Samples of pulmonary arteries
and alveolar septa that contain capillaries were processed in
parallel to the pulmonary veins. Notwithstanding that mRNA
extraction procedures and preservation were optimized in fresh
frozen lung samples, the quality of the returned lung mRNA
did not comply with the requirement for reproducible microarray analyses. It could nevertheless be used for accurate mRNA
quantification by RT-PCR. We therefore employed the RT2
PCR array (SABiosciences) to screen for expressed candidate
genes. Though technically challenging and laborious, this approach led us to identify a number of genes that were differentially expressed by pulmonary veins when compared with
arteries/parenchyma obtained from selected lung samples. Importantly, we were able to detect an increased venous expression of EphB4 and, particularly, the urokinase plasminogen
activator (uPA), whereas pulmonary arteries and alveolar septae expressed preferentially ephrinB2 (Fig. 2B and Table 2)
(18). These results were confirmed on the basis of specific
mRNA RT-PCR using target-specific primers sets. Indeed, we
also found uPAR upregulated in pulmonary veins compared
PULMONARY VEINS AND LEFT HEART DISEASE
L729
with arteries or lung parenchyma harvested from selected lungs
(Fig. 2C).
Subsequent immunofluorescence staining of uPAR in mouse
lungs confirmed its specificity for the pulmonary veins over
arteries (Fig. 3A). uPAR staining pattern was remarkably
similar between the mouse, rat, and human lung tissues (Fig.
3B). As previously described, uPAR expression was observed
in the pulmonary bronchial epithelium and in scattered cells
throughout the alveolar spaces. In regard to the pulmonary
circulation, uPAR was largely absent from pulmonary arteries,
but strongly expressed in pulmonary veins (Fig. 3B). Moreover, uPAR expression in pulmonary veins was also strongly
present in the cardiomyocyte sheath of large veins and in the
intima (Fig. 3B, right top) and media of more distal veins in
both rats and humans (Fig. 3B). Quantification of uPAR immunofluorescence intensity confirmed the preferential expression of uPAR in veins over arteries and was similar between
human and rat lung tissue (Fig. 3C). With a leading molecular
candidate to identify pulmonary veins, we then interrogated
whether uPAR expression was preferentially restricted to pulmonary veins in experimental and human PH-LHD.
Rat aortic banding and pulmonary vascular remodeling.
The model based on AOB-induced PH-LHD has been used to
describe the pulmonary venous and arterial remodeling and to
identify mechanisms involved in the pathogenesis of PH following left ventricular dysfunction in rats (13, 14, 35). We
therefore performed AOB or sham operations in rats, followed,
9 wk later, by invasive hemodynamic measurements and morphometric/morphological studies. AOB rats developed PH
with left heart dysfunction, as indicated by significantly increased mean pulmonary arterial pressure, LVEDP, and biventricular enlargement (Fig. 4A, D–G) compared with sham
animals. Overall, AOB rats closely approximated the hemodynamic characteristics observed in cohorts human PH-LHD
patients, including our own patient lung samples (Table 1).
To quantify the extent of pulmonary vascular remodeling,
AOB and sham rats also underwent pulmonary venous backfilling at the time of tissue harvest. Marked vascular remodeling was observed in the lungs of AOB rats, specifically media
thickening of pulmonary arteries and veins (Fig. 5A). Quantification of arterial and venous media fractional thickness confirmed these observations (Fig. 5, B and C). Notably, the
venous cardiomyocyte sheath fractional thickness was not
significantly increased in AOB animals (Fig. 5D).
Given uPAR’s previously described role in vascular remodeling and cellular proliferation, we assessed uPAR expression
AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00186.2013 • www.ajplung.org
Downloaded from http://ajplung.physiology.org/ by 10.220.33.3 on May 6, 2017
Fig. 2. A: mouse lung demonstrating pulmonary venous backfilling (top, left), allowing reliable identification of pulmonary veins (top, right). Identified veins
are outlined (top, right) then excised and isolated by laser capture microdissection (bottom sequence). Note absence of beads in bronchioles (br) and arteries (ar).
Scale bar ⫽ 100 ␮m. Green and red fluorescence ⫽ beads backfilled into the veins; blue ⫽ DAPI. B: qRT2 PCR array comparing mouse pulmonary veins to
a composite of arteries and parenchyma (n ⫽ 1 each). C: RT-PCR of urokinase plasminogen activator receptor (uPAR) and EphB4 expression in mouse
pulmonary veins vs. arteries and parenchyma (N ⫽ 2 each). uPA, urokinase plasminogen activator; EphB4, venous developmental marker; EphrinB2, arterial
developmental marker.
L730
PULMONARY VEINS AND LEFT HEART DISEASE
in the pulmonary vasculature of AOB rats. Increased uPAR
staining of pulmonary arteries was observed in AOB arteries
compared with controls (Fig. 6, A and B). Quantification of
immunofluorescent staining intensity confirmed these observations and also demonstrated that overall uPAR expression
appeared unchanged in the pulmonary veins of AOB compared
with sham controls (Fig. 6B). PCNA staining was significantly
increased in the pulmonary veins of AOB rats and associated
with uPAR expression, suggesting that uPAR, despite not
changing in expression in pulmonary veins, appears associated
with increased cellular proliferation and possible vascular remodeling in PH-LHD (Fig. 6, C and D). Similar results were
obtained with assessment of proliferating vascular arterial cells
(Fig. 6E).
Vascular remodeling in PH-LHD. To expand on the relevance of our experimental data with AOB rats, we collected
lungs from patients with PH-LHD when subjected to LVAD
placement. Patient characteristics are described in detail in
Table 1.
To determine the nature of vascular remodeling in patients
with PH-LHD, we evaluated volume of media, intima, and
adventitia relative to alveolar septae (used as reference space)
in pulmonary arteries and veins of PH-LHD and control subjects (Fig. 7). We identified pulmonary veins using their typical
topographical location in interlobular septa and in parenchymal
branches communicating with interlobular vessels. We were
not able to use uPAR expression in these human samples to
specifically identify pulmonary veins since, as observed in
AOB-rats, uPAR was similarly expressed between remodeled
pulmonary veins and arteries in the setting of PH-LHD (Fig. 7,
A and B). Media volume density of control cases was significantly higher in arteries than veins (Fig. 7C, P ⬍ 0.05). Intima
and adventitia volume density, however, did not appear significantly different in veins and arteries of controls (Fig. 7, D and
E). Media, intima, and adventitia volume density was significantly increased in both arteries and veins of PH-LHD cases
compared with controls (Fig. 7, C–E). Notably, there were no
significant differences in volume densities of media, intima, or
adventitia in veins compared with arteries of PH-LHD patients.
Pulmonary arteries of PH-LHD patients typically demonstrated
increased medial and intimal thickness, nearly obliterating the
vessel lumen in selected cases (Fig. 8, A and B).
Venous histopathological patterns in PH-LHD lungs typified
“arterialization”; i.e., greater delineation of the medial, intimal,
and adventitial layers, presence of a double elastic lamina,
muscularization of the vessel, and increased medial and intimal
thickness (Fig. 8, C and D). Three PH-LHD/LVAD subjects
went on to receive a second lung biopsy at the time of heart
transplantation. Pre/post LVAD hemodynamics and vascular
remodeling parameters for these three patients are summarized
AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00186.2013 • www.ajplung.org
Downloaded from http://ajplung.physiology.org/ by 10.220.33.3 on May 6, 2017
Fig. 3. A: quantification of uPAR-positive veins and arteries in normal mouse lungs. B: representative images of uPAR immunofluorescence in lung vessels of
control rats (top row) and humans (middle and bottom rows). uPAR expression (arrows) is similar between rat and human samples and is present in bronchiolar
epithelium and veins but absent in arteries (A). Detailed high magnification shown in bottom row for human lungs, with uPAR in red, CD31 or SMA in green,
and DAPI in blue. C: uPAR IF intensity in arteries and veins of sham-operated rats (N ⫽ 4) and control human tissue (N ⫽ 4) (*P ⬍ 0.05). SMA, ␣-smooth
muscle actin. Scale bar: 100 ␮m.
PULMONARY VEINS AND LEFT HEART DISEASE
L731
in Fig. 9. These preliminary data suggest that, in some patients,
improvement in hemodynamic parameters upon treatment may
parallel decreases of venous remodeling.
DISCUSSION
In the present study, we found that pulmonary veins can be
reliably identified by venous backfilling. On the basis of this
methodological tool and using laser capture microdissection, we
isolated venous RNA for identification of vein markers; this
approach led us to identify and subsequently confirm the expression of uPAR as a novel vascular marker of normal mouse, rat,
and human pulmonary veins. To study uPAR and venous remodeling in pulmonary vascular disease, we evaluated vascular remodeling and uPAR expression in lung tissue samples from
PH-LHD patients and the AOB rat model of PH-LHD. Unlike in
control tissues, uPAR expression was increased in pulmonary
arteries, similar to the level in pulmonary veins, of both PH-LHD
and AOB lungs. Although uPAR expression was similar in
control and AOB veins, it was significantly associated with
PCNA-positive cells, suggesting a possible role of uPAR in
vascular remodeling. Intima, media, and adventitia were significantly remodeled in veins and arteries to similar degrees, in both
human PH-LHD and rat AOB lungs.
The study of pulmonary veins has been limited by the
difficulty of their reliable identification, through histological,
anatomic, or molecular means, in the lung parenchyma. Recently, EphB4, a developmental venous marker, has been
shown to identify pulmonary veins in adult mice (4). However,
this finding may not be generalizable to other organisms:
careful embryological studies in humans have demonstrated
overlap of EphB4 expression between pulmonary veins and
arteries (12), whereas EphB4 does not appear to be present in
the pulmonary veins of the rat (data not shown, and personal
communication with Slaven Crnkovic, Ludwig Boltzmann Institute for Lung Vascular Research, Graz, Austria). Cardiomyocyte markers, such as heavy chain cardiac myosin, are specific
to the venous cardiomyocyte sheath, but this structure does not
extend deep into the parenchyma of larger animals, especially
humans (2, 24). We were also unable to detect protein of
CoupTF2, an orphan nuclear receptor that suppresses Notch
AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00186.2013 • www.ajplung.org
Downloaded from http://ajplung.physiology.org/ by 10.220.33.3 on May 6, 2017
Fig. 4. Pulmonary hemodynamics and cardiac parameters of aortic banded (AOB) rats. Mean pulmonary arterial pressures (mPAP; A), cardiac output (CO; B), pulmonary
vascular resistance (PVR; C), left ventricular end-diastolic pressure (LVEDP; D), right ventricle weight/body weight (LV/BW; E), and left ventricle weight/body weight
(F). *P ⬍0.05, **P ⬍ 0.01; N ⫽ 4 – 6 per group. G: a typical heart and lungs 9 wk after aortic banding (arrow). Catheter courses through the pulmonary artery. Note
the biventricular enlargement (top and top right arrowheads) and marked left (left arrowhead) and right (bottom right arrowhead) atrial dilation.
L732
PULMONARY VEINS AND LEFT HEART DISEASE
signaling and establishes venous identity during development
(36), in the pulmonary veins of adult animals (data not shown).
Consequently, we relied on an imaging with fluorescent backfilling of veins and embarked on a screening approach to
identify novel pulmonary venous molecular markers.
Candidate markers were identified by LCM and RT2-PCR
arrays and confirmed by RT-PCR of pulmonary veins compared with arteries and parenchyma obtained from selected
lungs. Several of these candidate markers, such as pigment
epithelium-derived factor (PEDF), CoupTF2, uPA, and uPAR,
have known biological significance in the vasculogenesis and
remodeling of veins. For instance, PEDF, a secreted serpin, has
antiangiogenic properties that have been described in umbilical
venous endothelial cells (1). Preliminary experiments, however, indicated that uPAR protein was much more abundant in
pulmonary veins compared with arteries and was consequently
further investigated as a marker of pulmonary veins. In control
mice, rats, and human lung tissues, uPAR expression could be
detected by immunofluorescence primarily in the media and
less so in the intima and was specific to pulmonary veins.
Knockout of uPAR has been shown to attenuate PH due to
hypoxia (23). Given the known role of uPAR in smooth muscle
and endothelial cell signaling promoting proliferation, migra-
tion, and vascular remodeling, we investigated uPAR expression patterns in PH-LHD (19, 22, 30).
PH-LHD is the most common cause of PH in the developed
world and several orders of magnitude more common than
pulmonary arterial hypertension (PAH) (27, 29). It results in
significant morbidity and mortality, and yet there are no available treatments that target the underlying pulmonary vascular
disease. Experimental models of PH-LHD have indicated increased vascular resistance of both the pulmonary arterial and
venous beds (15). Consequently, the failure of PAH medications in PH-LHD may, in part, be due to the presence of venous
remodeling and the relative importance of pathogenic cellular
signaling cascades in both the arterial and venous systems.
Consistent with this hypothesis, and with the aforementioned
observations of increased pulmonary vascular resistance in the
arterial and venous compartments, we observed significant and
similar remodeling in the pulmonary veins and arteries of AOB
rats and patients with PH-LHD. We also found that uPAR
expression remained elevated in pulmonary veins and was
significantly associated with cellular proliferation. PhosphoERK 1/2 was also elevated in both the pulmonary arteries and
veins of AOB and PH-LHD patients (data not shown), possibly
serving as an intermediary signaling relay between uPAR
AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00186.2013 • www.ajplung.org
Downloaded from http://ajplung.physiology.org/ by 10.220.33.3 on May 6, 2017
Fig. 5. Pulmonary vascular remodeling in AOB rats compared with sham animals. A: media thickening (arrows) in pulmonary arteries and pulmonary veins of
AOB animals. Vessels from sham animals are below for comparison. Assessments of pulmonary artery media fractional thickness (B) and pulmonary vein media
fractional thickness (C) in AOB vs. sham animals. N ⫽ 6 per group. HCCM, heavy chain cardiac myosin (**P ⬍ 0.01, ***P ⬍ 0.005). D: assessment of venous
cardiomyocyte sheath fractional thickness in sham and AOB rats. There was no significant difference between the 2 groups [not significant (NS); P ⫽ 0.28]. N ⫽
4 – 6 per group. Scale bar: 100 ␮m.
PULMONARY VEINS AND LEFT HEART DISEASE
L733
expression and vascular remodeling, consistent with its previously described roles in uPAR signal transduction (22).
Unlike in control lungs, uPAR expression was not exclusive
to the pulmonary veins, being also elevated in the pulmonary
arteries of AOB rats and PH-LHD patients. Interestingly, we
noted in a selected group of patients who underwent sequential
biopsy that improvement in hemodynamics with LVAD placement correlated with decreased arterial and venous remodeling,
hence suggesting that these changes can be reversible. When
considered together, these data suggest that a convergence of
pathogenic processes may be involved in both pulmonary vein
and arterial remodeling in PH-LHD. Future investigations to
characterize the role of uPAR in this vascular remodeling may
employ small molecule inhibitors to uPAR, such as IPR-803
(17), or related molecules involved in signaling and uPAR
signal transduction, such as uPA, plasminogen activator inhibitor-1 (PAI-1), or ERK 1/2 (22).
Given the limitations in the knowledge of pulmonary veins
and of the pathology and pathobiology of venous PH, our study
focused largely on discovery and validation of molecular
expression pattern and structural alterations. These findings
relied on integrating methods to visualize pulmonary veins,
methods for assessment of gene expression, modeling of PHLHD in rats, and, finally, for the first time, a stereological
assessment of PH-LHD in a unique cohort of patient samples.
We identified a number of potential candidate molecular markers of pulmonary veins and confirmed uPAR’s specificity to
pulmonary veins in control mouse, rat, and human lungs. We
also confirmed the prior observations that vascular remodeling
in both the arteries and veins, and so-called arterialization of
the veins, in patients with advanced PH-LHD. Furthermore, we
found that the arteries and veins share similarities in the degree
of remodeling of the intima, media, and adventitia. We anticipate that the present work will form the basis of future
mechanistic interventions and further delineation of molecular
targets to therapeutically target pulmonary veins remodeling
and pulmonary venous hypertension.
GRANTS
This research was funded by The Cardiovascular Medical Research and
Education Fund and RC1 HL 100849 (to R. M. Tuder), HL 102478 (to P. P. A.
Mammen), and KO8 HL 105536 and Parker B. Francis Fellowship award (to
B. B. Graham).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the
author(s).
AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00186.2013 • www.ajplung.org
Downloaded from http://ajplung.physiology.org/ by 10.220.33.3 on May 6, 2017
Fig. 6. A: representative images of uPAR IF (arrows) in lung arteries and veins of AOB and sham rats. B: quantification of uPAR IF intensity in sham and AOB
rat vessels. N ⫽ 3– 4 per group. C: representative image of proliferating cell nuclear antigen (PCNA) immunofluorescence (arrows) in rat lung veins. Note the
high number of PCNA-positive nuclei among the HCCM negative cells. Quantification of PCNA-positive cells, or PCNA- and uPAR-positive cells, divided by
total DAPI-positive cells in both sham and AOB rat pulmonary veins (D) and arteries (E) (N ⫽ 4). *P ⬍ 0.01 in D; P ⬍ 0.5 in E. Scale bar ⫽ 100 ␮m.
L734
PULMONARY VEINS AND LEFT HEART DISEASE
Fig. 8. Characteristic histopathological findings in
control subjects (A and C) and patients with PHLHD (B and D) (Russel-Movat pentachrome stains).
A: pulmonary arteries (arrows) with normal intima
and media in a control subject. B: arterial intima
(arrowhead) and media (arrow) thickening with luminal narrowing in PH-LHD. C: pulmonary vein in
a control subject with typical poorly organized media (arrows) and thin intima (arrowhead). D: venous
intima (arrowheads) and media (arrows) thickening.
Note “arterialization” of vein with double elastic
lamina. Scale bars ⫽ 100 ␮m.
AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00186.2013 • www.ajplung.org
Downloaded from http://ajplung.physiology.org/ by 10.220.33.3 on May 6, 2017
Fig. 7. A: representative images of uPAR IF (arrows) in lung arteries and veins of control subjects and patients with pulmonary hypertension with left heart disease
(PH-LHD). B: uPAR IF intensity in PH-LHD patients and controls. *P ⬍ 0.05. Scale bar ⫽ 100 ␮m. Assessment of media (C), intima (D), and adventitia (E)
volume density relative to septa of both arteries and veins of control subjects (N ⫽ 19) and patients with PH-LHD (N ⫽ 22). Student’s t-test, *P ⬍ 0.05,
***P ⬍ 0.001.
PULMONARY VEINS AND LEFT HEART DISEASE
L735
AUTHOR CONTRIBUTIONS
J.M.H., B.B.G., W.M.K., and R.M.T. conception and design of research;
J.M.H., B.B., X.L., A.G., P.P.M., E.S., M.R.G., E.P., M.P., L.S., B.B.G., and
R.M.T. performed experiments; J.M.H., B.B.G., and W.M.K. analyzed data;
J.M.H., B.B.G., W.M.K., and R.M.T. interpreted results of experiments;
J.M.H., B.B.G., W.M.K., and R.M.T. prepared figures; J.M.H., B.B.G.,
W.M.K., and R.M.T. drafted manuscript; J.M.H., B.B.G., W.M.K., and R.M.T.
edited and revised manuscript; J.M.H., B.B., X.L., A.G., P.P.M., E.S., E.P.,
M.P., L.S., B.B.G., W.M.K., and R.M.T. approved final version of manuscript.
REFERENCES
1. Aparicio S, Sawant S, Lara N, Barnstable CJ, Tombran-Tink J.
Expression of angiogenesis factors in human umbilical vein endothelial
cells and their regulation by PEDF. Biochem Biophys Res Commun 326:
387–394, 2005.
2. Arnstein C. Zur Kenntnis der quergestreiften Muskulatur in den Lungenvenen. Zentbl Med Wiss 692–694, 1877.
3. Coleman TG. Cardiac output by dye dilution in the conscious rat. J Appl
Physiol 37: 452–455, 1974.
4. Crnkovic S, Hrzenjak A, Marsh LM, Olschewski A, Kwapiszewska G.
Origin of neomuscularized vessels in mice exposed to chronic hypoxia.
Respir Physiol Neurobiol 179: 342–345, 2011.
5. Demello DE, Reid LM. Embryonic and early fetal development of human
lung vasculature and its functional implications. Pediatr Dev Pathol 3:
439 –449, 2000.
6. Dorfmuller P, Humbert M, Perros F, Sanchez O, Simonneau G,
Muller KM, Capron F. Fibrous remodeling of the pulmonary venous
system in pulmonary arterial hypertension associated with connective
tissue diseases. Hum Pathol 38: 893–902, 2007.
7. Dorfmuller P, Montani D, Humbert M. Beyond arterial remodelling:
pulmonary venous and cardiac involvement in patients with systemic
sclerosis-associated pulmonary arterial hypertension. Eur Respir J 35:
6 –8, 2010.
8. Dutly AE, Kugathasan L, Trogadis JE, Keshavjee SH, Stewart DJ,
Courtman DW. Fluorescent microangiography (FMA): an improved tool
to visualize the pulmonary microvasculature. Lab Invest 86: 409 –416,
2006.
9. Gao Y, Zhou H, Ibe BO, Raj JU. Prostaglandins E2 and I2 cause greater
relaxations in pulmonary veins than in arteries of newborn lambs. J Appl
Physiol 81: 2534 –2539, 1996.
10. Graham BB, Mentink-Kane MM, El-Haddad H, Purnell S, Zhang L,
Zaiman A, Redente EF, Riches DW, Hassoun PM, Bandeira A,
Champion HC, Butrous G, Wynn TA, Tuder RM. Schistosomiasisinduced experimental pulmonary hypertension: role of interleukin-13
signaling. Am J Pathol 177: 1549 –1561, 2010.
11. Guazzi M, Arena R. Pulmonary hypertension with left-sided heart disease. Nat Rev Cardiol 7: 648 –659, 2010.
12. Hall SM, Hislop AA, Haworth SG. Origin, differentiation, and maturation of
human pulmonary veins. Am J Respir Cell Mol Biol 26: 333–340, 2002.
13. Hentschel T, Yin N, Riad A, Habbazettl H, Weimann J, Koster A,
Tschope C, Kuppe H, Kuebler WM. Inhalation of the phosphodiesterase-3 inhibitor milrinone attenuates pulmonary hypertension in a rat model
of congestive heart failure. Anesthesiology 106: 124 –131, 2007.
14. Hoffmann J, Yin J, Kukucka M, Yin N, Saarikko I, Sterner-Kock A,
Fujii H, Leong-Poi H, Kuppe H, Schermuly RT, Kuebler WM. Mast
cells promote lung vascular remodelling in pulmonary hypertension. Eur
Respir J 37: 1400 –1410, 2011.
15. Huang W, Kingsbury MP, Turner MA, Donnelly JL, Flores NA,
Sheridan DJ. Capillary filtration is reduced in lungs adapted to chronic
heart failure: morphological and haemodynamic correlates. Cardiovasc
Res 49: 207–217, 2001.
16. Jones WK, Sanchez A, Robbins J. Murine pulmonary myocardium: developmental analysis of cardiac gene expression. Dev Dyn 200: 117–128, 1994.
17. Khanna M, Wang F, Jo I, Knabe WE, Wilson SM, Li L, Bum-Erdene
K, Li J, Sledge W, Khanna R, Meroueh SO. Targeting multiple
conformations leads to small molecule inhibitors of the uPAR. uPA
protein-protein interaction that block cancer cell invasion. ACS Chem Biol
6: 1232–1243, 2011.
18. Kim YH, Hu H, Guevara-Gallardo S, Lam MT, Fong SY, Wang RA.
Artery and vein size is balanced by Notch and ephrin B2/EphB4 during
angiogenesis. Development 135: 3755–3764, 2008.
19. Kiyan J, Kiyan R, Haller H, Dumler I. Urokinase-induced signaling in
human vascular smooth muscle cells is mediated by PDGFR-beta. EMBO
J 24: 1787–1797, 2005.
20. Klika E, Zajicova A, Votavova B. [Participation of the myocardium in
the formation of the pulmonary vein wall of small mammals (a quantitative study)]. Arkh Anat Gistol Embriol 82: 81–83, 1982.
21. Lantuejoul S, Sheppard MN, Corrin B, Burke MM, Nicholson AG.
Pulmonary veno-occlusive disease and pulmonary capillary hemangiomatosis: a clinicopathologic study of 35 cases. Am J Surg Pathol 30:
850 –857, 2006.
AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00186.2013 • www.ajplung.org
Downloaded from http://ajplung.physiology.org/ by 10.220.33.3 on May 6, 2017
Fig. 9. Right atrial pressures (RAP; A) and mPAP (B) prior to left ventricular assist device (LVAD) placement and 2 days post-LVAD placement in the 3 PH-LHD
patients who received a second lung biopsy at time of transplantation. Volume density of venous media (C) and intima (D) and arterial media (E) and intima
(F) relative to alveoli both before LVAD placement and at time of subsequent transplant (P ⫽ NS, paired t-test). Lines represent the same individual throughout
graphs: light gray, 38-yr-old male transplanted at 27 days post-LVAD; medium gray, 60-yr-old female transplanted at 41 days post-LVAD; black, 60-yr-old
female transplanted 7 mo post-LVAD.
L736
PULMONARY VEINS AND LEFT HEART DISEASE
30.
31.
32.
33.
34.
35.
36.
Langleben D, Nakanishi N, Souza R. Updated clinical classification of
pulmonary hypertension. J Am Coll Cardiol 54: S43–S54, 2009.
Smith HW, Marshall CJ. Regulation of cell signalling by uPAR. Nat Rev
Mol Cell Biol 11: 23–36, 2010.
Stacher E, Graham BB, Hunt JM, Gandjeva A, Groshong SD,
McLaughlin VV, Jessup M, Grizzle WE, Aldred MA, Cool CD, Tuder
RM. Modern age pathology of pulmonary arterial hypertension. Am J
Respir Crit Care Med 186: 261–272, 2012.
Wagenvoort CA, Wagenvoort N. Normal circulation of the lungs. In:
Pathology of Pulmonary Hypertension, edited by Wagenvoort CA,
Wagenvoort N. New York: Wiley, 1977, p. 1–8.
Wagenvoort CA, Wagenvoort N. Pulmonary venous hypertension. In:
Pathology of Pulmonary Hypertension, edited by Wagenvoort CA,
Wagenvoort N. New York: Wiley, 1977, p. 177–216.
Walch L, Labat C, Gascard JP, de Montpreville V, Brink C, Norel X.
Prostanoid receptors involved in the relaxation of human pulmonary
vessels. Br J Pharmacol 126: 859 –866, 1999.
Yin J, Kukucka M, Hoffmann J, Sterner-Kock A, Burhenne J, Haefeli
WE, Kuppe H, Kuebler WM. Sildenafil preserves lung endothelial
function and prevents pulmonary vascular remodeling in a rat model of
diastolic heart failure. Circ Heart Fail 4: 198 –206, 2011.
You LR, Lin FJ, Lee CT, DeMayo FJ, Tsai MJ, Tsai SY. Suppression
of Notch signalling by the COUP-TFII transcription factor regulates vein
identity. Nature 435: 98 –104, 2005.
AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00186.2013 • www.ajplung.org
Downloaded from http://ajplung.physiology.org/ by 10.220.33.3 on May 6, 2017
22. LaRusch GA, Mahdi F, Shariat-Madar Z, Adams G, Sitrin RG,
Zhang WM, McCrae KR, Schmaier AH. Factor XII stimulates ERK1/2
and Akt through uPAR, integrins, and the EGFR to initiate angiogenesis.
Blood 115: 5111–5120, 2010.
23. Levi M, Moons L, Bouche A, Shapiro SD, Collen D, Carmeliet P.
Deficiency of urokinase-type plasminogen activator-mediated plasmin
generation impairs vascular remodeling during hypoxia-induced pulmonary hypertension in mice. Circulation 103: 2014 –2020, 2001.
24. Ludatscher RM. Fine structure of the muscular wall of rat pulmonary
veins. J Anat 103: 345–357, 1968.
25. Montani D, Kemp K, Dorfmuller P, Sitbon O, Simonneau G, Humbert
M. Idiopathic pulmonary arterial hypertension and pulmonary venoocclusive disease: similarities and differences. Semin Respir Crit Care
Med 30: 411–420, 2009.
26. Murali S. Pulmonary hypertension in heart failure patients who are
referred for cardiac transplantation. Adv Pulm Hypertens 5: 30 –35, 2006.
27. Oudiz RJ. Pulmonary hypertension associated with left-sided heart disease. Clin Chest Med 28: 233–241, x, 2007.
28. Petrache I, Natarajan V, Zhen L, Medler TR, Richter AT, Cho C,
Hubbard WC, Berdyshev EV, Tuder RM. Ceramide upregulation
causes pulmonary cell apoptosis and emphysema-like disease in mice. Nat
Med 11: 491–498, 2005.
29. Simonneau G, Robbins IM, Beghetti M, Channick RN, Delcroix M,
Denton CP, Elliott CG, Gaine SP, Gladwin MT, Jing ZC, Krowka MJ,