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,¶
* Department of Pathology,
Department of Medicine, Division of Pulmonary Sciences and Critical Care Medicine, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA;
SUGEN, Incorporated, South San Francisco, California 94080, USA;
§ Department of Internal Medicine II, Cardiology, Ulm University Medical Center, D-89081, Germany; and
¶ Pulmonary Hypertension Center, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA
1Correspondence: Department of Pathology, Campus Box B216, University of Colorado Health Sciences Center, 4200 E. 9th Ave., Denver, CO 80262, USA. E-mail: Rubin.Tuder{at}uchsc.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: apoptosis • survival • selection • pulmonary vascular remodeling • angiogenesis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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.
Although chronic prostacyclin infusion improves the hemodynamic status
of many patients and increases survival (2)
, there is
presently no cure for these diseases, and many patients still die
within a few years after diagnosis in part because of our limited
understanding of the pathobiology of pulmonary vascular disorders. In
contrast to systemic vascular disorders, including arteriosclerosis and
systemic vasculitis, severe pulmonary hypertension presents with unique
tumorlets of endothelial cells that obliterate medium-sized
precapillary arteries (3
, 4)
. The proliferating
endothelial cells often form structures known as plexiform lesions
(5)
and express angiogenesis factors, including vascular
endothelial growth factor (VEGF) and its receptor VEGF receptor 2
(VEGFR-2/KDR/Flk-1) (R. M. Tuder et al., unpublished results).
Because of this pattern of VEGF and VEGFR-2 expression, and the fact
that in primary pulmonary hypertension the lung endothelial cells
expand in a monoclonal pattern (6)
and contain an
inactivating mutation of the transforming growth factor receptor II
(7)
, we postulate that plexiform lesions arise from a
process of dysregulated angiogenesis that has several features in
common with that seen in neoplastic processes. The trigger of
endothelial cell proliferation in the human disease remains unknown.
In contrast to the human disease, both classical rodent models of mild
to moderate pulmonary hypertension—the chronic hypoxia and the
alkaloid monocrotaline models—lack clustered proliferated endothelial
cells in the lumen of pulmonary arteries (8
, 9)
. Pulmonary
endothelial cells constitute a stable cell population with a very low
turnover rate and, apparently, neither severe chronic hypoxia/hypoxemia
nor monocrotaline pyrrole causes the emergence of a proliferative,
dysfunctional endothelial cell phenotype (10)
. The
defining pulmonary vascular alteration in both of these models, medial
muscular thickening, is potentially reversible upon reexposure to
normoxia or with the passage of time after monocrotaline injection.
Although the physiological role of the abundantly expressed VEGF in the
lung is unknown, we reason that VEGF supports pulmonary endothelial
cell maintenance and survival (11
, 12)
. We hypothesize
that the combination of chronic blockade of VEGF or its receptor 2 and
chronic hypoxia might cause, in rats, 1) pulmonary
endothelial cell dysfunction and cell death, which 2) allows
for the selection of an apoptosis-resistant proliferating endothelial
cell phenotype, and 3) development of severe pulmonary
hypertension. Our results show that synthetic VEGFR-2 inhibitor
3-[(2,4-dimethylpyrrol-5-yl) methylidenyl]-indolin-2-one
(SU5416) (13)
causes, in chronically hypoxic rats,
pulmonary arterial endothelial cell death, followed by obliteration of
the artery lumen by proliferated endothelial cells, which is associated
with severe pulmonary hypertension. Because endothelial cell death,
cell proliferation, and the development of severe pulmonary
hypertension can be blocked by a broad spectrum caspase inhibitor, it
appears that the selection of an apoptosis-resistant endothelial cell
phenotype is the critical event responsible for pulmonary artery
endothelial cell proliferation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-tubulin (TU-02, 1/1000 for WB), and proliferating cell
nuclear antigen (PCNA, 1/50 dilution for immunohistochemistry) (Santa
Cruz Biotechnology, Santa Cruz, Calif.); c-Src (1/250), Akt-1 (1/1000),
and phospho-Akt-1 (1/150) used for WB (Upstate Biotechnology, Lake
Placid, N.Y.); factor VIII-related antigen (polyclonal antibody used at
1/250 dilution for immunohistochemistry; Dako, Carpinterio, Calif.);
smooth muscle -actin (1/150 dilution for immunohistochemistry);
sheep-horseradish peroxidase (HRP) -conjugated antibodies (Sigma, St.
Louis, Mo.), rat macrophage antibody (1/50 dilution for
immunohistochemistry) (Chemicon International, El Segundo, Calif.),
anti-mouse HRP-conjugated antibody (Vector Laboratories,
Burlingame, Calif.); goat anti-rabbit HRP- and swine
anti-goat-HRP-conjugated antibodies (BioSource International,
Camarillo, Calif.). Activated caspase 3 polyclonal antibody
(14)
was a gift from A. Srinivisan (at 1/250 dilution for
immunohistochemistry).
Experimental protocols
The experimental protocol was approved by the Animal Care and
Use Committee of the UCHSC. Adult male Sprague-Dawley rats weighing
200 g were injected subcutaneously (s.c.) with SU5416, which was
suspended in CMC [0.5% (w/v) carboxymethylcellulose sodium, 0.9%
(w/v) sodium chloride, 0.4% (v/v) polysorbate 80, 0.9% (v/v) benzyl
alcohol in deionized water]. Control rats received only diluent. The
treatment protocols consisted of 3 weekly injections of SU5416 at high
dose (200 mg/kg) or a low dose (20 mg/kg) for 3 wk or a single
injection (200 mg/kg) at the beginning of the 3 wk experiment. The
animals were exposed to chronic hypoxia (simulated altitude of
5000 m in a hypobaric chamber, with an inspired partial oxygen
pressure of 
76 mmHg), Denver altitude (1600 m, inspired partial
oxygen pressure of 124 mmHg), or sea-level oxygen (inspired partial
oxygen pressure of 150 mmHg) conditions. To study the time course of
the effect of SU5416 on pulmonary vessels, rats were treated for 1, 2,
and 3 wk with SU5416 or diluent under hypoxic conditions. To test
whether the pulmonary hypertension and the pulmonary vascular
remodeling were reversible, rats were treated with SU5416 and chronic
hypoxia for 3 wk and re-exposed to Denver altitude conditions for an
additional 3 wk. In a separate experiment, we treated rats under
normoxia (Denver altitude) with the combination of SU5416 at 20 mg/kg,
3 times/wk, and dexfenfluramine (Sigma) at 2 mg/kg for 5 days/wk, for 3
wk (n=3). Controls consisted of rats treated with SU5416
alone (n=3) or dexfenfluramine alone (n=3). To
test the effect of caspase inhibition in the development of severe
pulmonary hypertension triggered by chronic hypoxia and SU5416, rats
were treated with a single injection of SU5416 (200 mg/kg) + the broad
spectrum caspase inhibitor Z-Asp-2,6-dichlorobenzoyloxymethylketone
(Z-Asp-CH2-DCB) (Alexis, San Diego, Calif.), 2
mg/rat, intraperitoneally, dissolved in DMSO, and then immediately
before the injections diluted in phosphate buffer solution, resulting
in a final concentration of DMSO 30%, 3 times/wk for 3 wk
(n=6). We have previously determined that this dose of
Z-Asp-CH2-DCB inhibits lung cell apoptosis,
reduces the number of activated caspase 3 expressing cells, and
decreases lung caspase 3-like activity in SU5416-treated normoxic rats
(Y. Kasahara et al., unpublished results). Controls consisted of rats
exposed to chronic hypoxia and treated with Z-Asp-CH2-DCB alone
(n=2) or to SU5416 alone (n=2). Because the
findings in these latter 2 rats were identical to those in the
remaining SU5416-treated animals, we compared the results obtained in
the Z-Asp-CH2-DCB-treated animals with the whole group of animals that
received SU5416 but not the caspase inhibitor under chronic hypoxic
conditions (n=14).
Assessment of pulmonary hypertension
At the end of the treatment period, the rats were weighed, then
anesthetized with ketamine hydrochloride (60 mg/kg) and xylazine (8
mg/kg) administered intramuscularly. The pulmonary artery pressure and
the ratio of the right ventricle wall/(left ventricle plus septum
weight) were determined as previously reported (15)
.
Lung morphology
Freshly excised lungs from all animals were inflated with
low-melt agarose as described previously (16)
. Kidney,
heart, liver, spleen, and bone marrow were also examined. Each lung was
sectioned transversally through the hilum and included intermediate and
peripheral lung tissue. A second sagittal section through the
peripheral lung parenchyma was also made.
Immunohistochemistry
Histological sections were heated in 1x citrate buffer, with
times varying from 15 to 25 min, followed by
H2O2 block of endogenous
peroxidase for 15 min, avidin-biotin block for 10 min each, and block
with rabbit or mouse 10% serum for 30 min. The primary antibodies were
incubated at room temperature (except for anti-smooth muscle actin,
which was incubated at 37°C) for times varying between 15 min
(VEGFR-2) and 1 h (rat macrophage antibody), followed by Quickkit
(Vector, Burlingame, Calif.) for 10 min at room temperature. This kit
involves a second step: incubation with a universal biotinylated horse
antibody for 10 min, followed by streptavidin-peroxidase for 5 min and
development with diaminobenzidine with hydrogen peroxide, according to
the manufacturer’s protocol.
Pulmonary artery morphometry
Morphometry was performed on lung slides of two randomly chosen
animals in each treatment group. The percent medial thickness was
calculated with the formula (external diameter-internal
diameter/external diameter) x100 in slides immunostained with a smooth
muscle -actin antibody. Only vessels sectioned longitudinally or
with an approximately circular profile were analyzed for assessment of
medial thickness. The diameter of pulmonary arteries was determined by
use of ImagePro software.
Cell death assays
TUNEL was performed with TACS 2 TdT diaminobenzidine (DAB) kit
(Trevigen, Gaithersburg, Md.), following the manufacturer’s
instructions. Briefly, after deparaffinization and dehydration,
sections were digested with proteinase K at a concentration of 20
µg/ml for 20 min. Endogenous peroxidase activity was quenched with
2% (v/v) H2 O2 for 5 min.
The slides were immersed in terminal deoxynucleotidyl transferase (TdT)
buffer. TdT (1 mM Mn2+) and biotinylated dNTP in
TdT buffer were then added to cover the sections, which were incubated
in a humid atmosphere at 37°C for 60 min. The slides were washed with
phosphate-buffered saline (PBS) and incubated with
streptavidin-horseradish peroxidase for 15 min. After rinsing with PBS,
the slides were immersed in DAB solution. The slides were
counterstained for 1 min with 0.5% methyl green.
In parallel slides, we detected activated caspase 3 as described by
Srinivisan et al. (14)
. Quantitation of activated caspase
3-positive endothelial cells in precapillary pulmonary arteries was
preformed in lung sections of 2 rats randomly chosen in each
experimental group. Fifty vessels/lung were analyzed.
Caspase 3-like lung activity
Cytosolic extract was incubated at 37°C with 200 µM of
Ac-DEVD-pNA in 96-well microtiter plates (caspase 3 cellular activity
assay kit plus; Biomol Research Laboratories Inc., Plymouth Meeting,
Pa.). At different times, hydrolytic activities were determined by the
measure of absorbance of p-nitroaniline at 405 nm.
Western blot studies
Frozen rat lung tissue was pulverized in a mortar and pestle in
liquid nitrogen, resuspended in homogenization buffer HB (20 mM HEPES,
pH 7.55, 1.5 mM MgCl2, 150 mM NaCl, 10%
glycerol, 1% Triton X-100, 2 mM EDTA, 2 mM
Na3VO4, 50 mM NaF, 2
µg/ml aprotinin, 5 µg/ml leupeptin, 1 mM PMSF), and homogenized in
a glass homogenizer with 10 strokes of TeflonTM pestle. The tissue
homogenate was centrifuged at 3000 rpm for 15 min. Protein
concentration was determined by Bradford assay using Bradford reagent
from Sigma. Positive controls for VEGFR-2 consisted of porcine
endothelial cells transfected with VEGFR-2 cDNA (17)
.
Proteins (25 µg) were subjected to electrophoresis on 4%–12%
gradient Bio-Tris gels (Novex, San Diego, Calif.) and transferred to
PolyScreen PVDF Transfer membrane (NEN Life Science Products,
Frankfurt, Germany) in tris-glycine buffer containing 10–20%
methanol. Prestained molecular mass marker proteins (Bio-Rad, Hercules,
Calif.) were used as standards for the sodium dodecyl
sulfate-polyacrylamide gel electrophoresis. Western blots were
visualized using Renaissance Western blot Chemiluminescence Reagent
(NEN Life Science Products).
Proliferation assay
Assessment of smooth muscle or endothelial cell proliferation
was performed in PCNA immunostained slides and expressed as positive
endothelial or vascular smooth muscle cells/vessel. The diameter of
pulmonary vessels stained with PCNA antibody was determined with a
scaled eyepiece.
Statistical analyses
Statistical significance determined by the Student’s unpaired
t test (P<0.05). Values are expressed as
mean ± SE.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), whereas the right ventricular mass in SU5416-treated rats
was not different from that of control rats (Fig. 1B
). To
test whether the lower inspired oxygen levels at Denver altitude when
compared with oxygen levels in a sea level-like environment explained
the development of pulmonary hypertension due to treatment with SU5416,
rats were housed in an environmental chamber at simulated sea level
altitude and treated with SU5416 (200 mg/kg, s.c., n=6) or
vehicle (n=6) for 3 wk. The sea level SU5416-treated group
showed an increase in pulmonary arterial pressure (28.9 mmHg±0.6) over
sham-treated controls (18.4 mmHg±0.5), yet lower than that observed
with the Denver altitude SU5416-treated rats (32.3 mm Hg±2.1 vs. 18.3
mmHg±0.3 in controls) (Fig. 1A
).
|
SU5416-treated rats exposed to hypobaric hypoxia (n=12)
(simulated altitude of 5000 meters) for 3 wk developed severe pulmonary
hypertension (Fig. 2A
) accompanied by pronounced right atrial dilation and right
ventricular hypertrophy (Figs. 1B
, C
, D
). The high pulmonary
artery pressures in the chronically hypoxic rats treated with SU5416
were even more surprising because of the lower hematocrit in the
SU5416-treated rats (53%±1.2) when compared to that of hypoxic
control rats (n=8) (60%±0.8).
|
Vehicle- and SU5416-treated animals developed similar levels of
pulmonary artery pressures during the first (n=2) and second
week (n=2) of chronic hypoxia, but the pulmonary artery
pressure was clearly higher during the third week of exposure in the
SU5416-treated animals (n=2) compared to vehicle controls
(n=2) (Fig. 2B
). Under chronic hypoxic
conditions, the pulmonary artery pressures after a low dose of SU5416
(20 mg/kg, n=6) (46 mmHg±1.8) or a single injection of
SU5416 (at 200 mg/kg, n=4) (47 mmHg) were of a degree
comparable to that observed in rats treated 3 times/wk with the higher
dose (200 mg/kg) of SU5416.
We tested whether the pulmonary vascular alterations seen in chronically hypoxic rats treated with SU5416 were reversible upon reexposure to normoxia. Rats treated with SU5416, exposed to chronic hypoxia, and returned to Denver altitude for an additional 3 wk had high pulmonary pressures and low hematocrit, as observed with the SU5416-treated rats exposed to chronic hypoxia for 3 wk (58 mmHg±4.4, hematocrit=42%±4, n=4). One chronically hypoxic control rat that was returned to Denver altitude had a regression to normal pulmonary artery pressures.
The pulmonary artery pressure of animals treated with a combination of the anorexigen dexfenfluramine, which has been associated with primary pulmonary hypertension, and SU5416 in normoxic conditions was not different from that of SU5416-treated rats (data not shown). Dexfenfluramine alone did not cause pulmonary hypertension.
Histological and morphometric findings
Histologically, lungs from control (Fig. 3A
) and
SU5416-treated rats (Fig. 3B
), whether at sea level or at
Denver altitude, showed only thickening of the pulmonary artery media
(Figs. 3A
, B
). The pulmonary arteries were lined by a uniform
monolayer of endothelial cells in sea level or Denver altitude SU5416-
or vehicle-treated rat lungs (Fig. 3C
).
|
Lungs of chronically hypoxic SU5416-treated rats showed near-complete
lumen obliteration of medium-sized and precapillary intra-alveolar
arteries, caused by endothelial cell expansion (Fig. 3D
) as
confirmed by immunoreactivity to factor VIII-related antigen, VEGFR-2,
and VEGF antibodies (Figs. 3E
, F
, G
) and by lack of expression
of smooth muscle cell -actin (Fig. 3H
). In addition to
the endothelial cells obliterating the lumen, the intra-alveolar
arteries had acquired a smooth muscle cell media that expressed smooth
muscle -actin (Fig. 3I
). These findings became evident
between wk 2 and 3 of hypoxia exposure. Precapillary pulmonary vessels
with almost complete lumenal occlusion were more frequently observed in
lungs of rats returned to Denver altitude for an additional 3 wk of
recovery from chronic hypoxia than in those exposed to SU5416 + 3 wk of
chronic hypoxia. Rats injected with a single dose of SU5416 and exposed
to hypoxia for 3 wk had a similar irreversible occlusion of their
pulmonary arteries. The endothelial cell proliferative process was
usually seen in close proximity to shear stress-prone branching points.
We did not detect intravascular accumulation of macrophages in
SU5416-treated chronically hypoxic lungs. A small number of macrophages
was present in the alveolar spaces in lungs of rats treated with SU5416
+ chronic hypoxia for 3 wk, and in rats treated with SU5416 + chronic
hypoxia for 3 wk and reexposed to Denver altitude condition for an
additional 3 wk. These findings were observed in the lungs of all
SU5416-treated chronically hypoxic animals, irrespective of the
treatment protocol used (200 mg/kg, 3 times/wk, 20 mg/kg 3 times/wk, or
200 mg/kg at the beginning of the experiment).
Histological examination of liver, kidney, spleen, and bone marrow of all SU5416-treated rats, whether at Denver altitude or chronic hypoxia conditions, revealed no alterations in blood vessels (data not shown), indicating that the vascular effects of SU5416 induced VEGFR-2 blockade in both Denver altitude and chronic hypoxia were restricted to pulmonary arteries.
The increase in medial thickness in SU5416-treated rat lungs compared
to vehicle-treated lungs under normoxia (Denver altitude and sea level
conditions) or chronic hypoxia was confirmed morphometrically using
lung sections stained with an antibody against smooth muscle cell
-actin (Fig. 4A
, B
). Although the pulmonary artery pressures peaked after 3
wk in rats treated with the combination of chronic hypoxia and SU5416,
the vascular medial thickening increased further in lungs of
SU5416-treated rats followed for an additional 3 wk at Denver altitude
(bar graph in Fig. 4B
). There was no correlation between
pulmonary vessel size and the degree of medial thickening in all groups
examined (Pearson’s correlation coefficient ranged between -0.16 and
0.07).
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Endothelial cell death in SU5416-treated rat lungs
Since VEGF withdrawal has been reported to trigger endothelial
cell death both in vitro and in vivo (11
, 12
, 18)
, we investigated whether VEGFR-2 blockade by SU5416
resulted in endothelial cell death in precapillary pulmonary arteries.
Endothelial cells expressed activated caspase 3 immunoreactivity
infrequently in vehicle-treated lungs at Denver altitude conditions
(1.56±0.17 positive endothelial cells/vessel). When compared with
control lungs, lungs of SU5416-treated rats at Denver altitude had
increased numbers of activated caspase 3-positive endothelial cells in
precapillary pulmonary arteries (3.34±0.25).
Lung endothelial cells in SU5416-treated lungs exposed to chronic
hypoxia (Fig. 5A
, C
) were more frequently positive for activated caspase 3
expression than were endothelial cells in vehicle-treated control lungs
(Fig. 5B
, C
). The number of activated caspase 3-positive
endothelial cells in the lungs of control rats exposed to 3 wk chronic
hypoxia (2.3±0.31) was lower than that seen in lungs of rats treated
with SU5416 under Denver altitude conditions. Endothelial cells
expressing activated caspase 3 peaked in rats treated with SU5416 and
exposed to chronic hypoxia for 2 wk. Lungs of SU5416-treated rats that
recovered from chronic hypoxia for an additional 3 wk at Denver
altitude had the highest number of caspase 3-positive endothelial cells
among all groups studied. Lung caspase 3-like activity was higher in wk
1 (231 pmol/min/mg lung tissue, n=2) and 3 (227 pmol/min/mg
lung tissue, n=2) of chronic hypoxia and SU5416 treatment
when compared with chronic control lungs (131 and 25 pmol/min/mg lung
tissue, in wk 1 and 3 of chronic hypoxia exposure).
|
Endothelial cells positive for activated caspase 3 were usually located
close to the internal elastic layer rather than in the cluster of
proliferating cells protruding into the vascular lumen (Fig. 5A
). Identification of endothelial cell death by TUNEL assay
matched that obtained with the immunolocalization pattern of activated
caspase 3 (Figs. 5D
, E
). No activated caspase 3 expression
or TUNEL positivity was seen in vascular smooth muscle cells in the
lungs of control or SU5416-treated rats at Denver altitude or under
chronic hypoxia.
Expression of VEGFR-2, phosphorylated VEGFR-2, Akt, phosphorylated
Akt, and Src
Consistent with the increased endothelial cell death present in
SU5416-treated lungs exposed to chronic hypoxia, we found decreased
expression of VEGFR-2, Src, Akt, and phosphorylated (active) Akt
proteins by Western blot analysis of SU5416-treated chronically hypoxic
lungs when compared with normoxia and vehicle-treated chronic hypoxic
lungs (Fig. 6
). The levels of phosphorylated (active) VEGFR-2 were decreased by
30% in rat lungs exposed for 3 wk hypoxia and treated with SU5416
when compared with control lungs.
|
Vascular cell proliferation in control and SU5416-treated rats
We quantitated proliferating vascular smooth muscle and
endothelial cells that stained positive for PCNA by
immunohistochemistry. Denver altitude and sea level-exposed rats were
grouped together, since under these conditions each group exhibited
similar numbers of proliferating smooth muscle and endothelial cells.
Under normoxic conditions, PCNA-positive endothelial cells were found
infrequently in both control and SU5416-treated lungs, whereas there
was a 40-fold increase in the number of PCNA-positive smooth muscle
cells/pulmonary vessel in SU5416-treated normoxic lungs (Fig. 7A
, P<0.01).
|
The increase in the number of endothelial cells in pulmonary arteries
of rats treated with chronic hypoxia and SU5416 paralleled the
identification of increased PCNA-positive endothelial cells (Figs. 7B
, C
). Two weeks of hypoxia exposure resulted in a
significantly higher number of PCNA-positive lung endothelial cells in
SU5416-treated when compared to vehicle-treated rats
(P<0.01, Fig. 7C
). The number of proliferating
endothelial cells in SU5416-treated rats continued to increase through
wk 3 of hypoxia and achieved 14.5-fold normoxia levels in rats
reexposed to Denver altitude conditions for an additional 3 wk, whereas
hypoxic control lungs had a decrease in proliferating EC between wk 2
and 3 of chronic hypoxia exposure.
The number of proliferating vascular smooth muscle cells increased up
to wk 2 of hypoxia to a degree similar in both the control and
SU5416-treated groups (Fig. 7D
). The number of proliferating
vascular smooth muscle cells decreased in wk 3 in both SU5416- and
vehicle-treated lungs (Fig. 7D
). The maximum levels of
proliferating endothelial cells in chronically hypoxic SU5416-treated
rat lungs were fivefold higher than those observed with vascular smooth
muscle cells.
Effect of broad spectrum caspase inhibition with
Z-Asp-CH2-DCB on endothelial cell death, endothelial cell
proliferation, and severe pulmonary hypertension in chronically hypoxic
SU5416-treated animals
Caspase inhibition with
Z-Asp-CH2-DCB resulted in significantly lower
pulmonary artery pressures in SU5416-treated, chronically hypoxic rats
(35 mmHg±2.8, n=5) when compared with chronically hypoxic
SU5416-treated rats with no caspase inhibitor (PAP=45.4 mmHg±1.7,
n=14, P=0.01). In addition, Z-Asp-CH2-DCB
restored the polycythemia to levels seen in rats exposed to chronic
hypoxia (hematocrit=63%±1.2). Most strikingly was the finding that
Z-Asp-CH2-DCB treatment resulted in preservation
of the pulmonary endothelial cell monolayer, with an absence of
intralumenal clusters of endothelial cells (Fig. 8A
, B
). Medial muscular thickening was the main component of
pulmonary artery remodeling observed in these rats (Fig. 8C
)
to an extent similar to that present in lungs of rats treated with
chronic hypoxia alone. Z-Asp-CH2-DCB treatment resulted in a remarkable
reduction of endothelial cells positive for activated caspase 3
(0.2±0.02 positive vessels/total vessels, 19 pulmonary arteries/lung
examined) in SU5416-treated chronically hypoxic rats (Fig. 8D
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 20)
.
Chronic VEGFR-2 blockade by SU5416 caused pulmonary hypertension and
muscularization of the pulmonary resistance vessels. Since rats treated
with SU5416 at Denver altitude had thicker pulmonary vascular media
than rats treated at sea level, it is possible that the increase in
pulmonary artery medial thickening was a consequence of mild hypoxic
vasoconstriction at Denver altitude and chronic VEGFR-2 blockade, which
via decreasing endothelial cell nitric oxide (21
22
23)
or
prostacyclin production (22
, 24)
could have altered
pulmonary vascular tone. Yet because the degree of vascular smooth
muscle cell proliferation was increased by SU5416 treatment and likely
disproportionate to the pulmonary artery pressure of the normoxic rats,
we must consider a vascular pressure/resistance-independent effect of
SU5416 on pulmonary vascular smooth muscle cell growth. One concept is
that endothelial cells and vascular smooth muscle cell in small
pulmonary arteries form a syncytium and that impairment of endothelial
cell biology has consequences for vascular smooth muscle cell behavior
(1
, 25)
.
In combination with chronic hypoxia, chronic VEGFR-2 blockade caused severe pulmonary hypertension, which persisted, and in fact progressed, after the animals had been removed from the hypobaric chamber. The persistence and progression of pulmonary vascular disease and right heart failure with death of some of the animals after one of the injurious experimental modalities in this model had been removed are characteristic of human severe pulmonary hypertensive disorders. Because the severity and rapid progression of pulmonary hypertension were somewhat reminiscent of the anorexigen drug-induced form of human pulmonary hypertension, we asked whether the combination of daily dexfenfluramine administration and VEGFR-2 blockade also caused severe pulmonary hypertension. Dexfenfluramine by itself did not cause pulmonary hypertension in rats after 3 wk of treatment nor did it amplify the effect of SU5416. These results indicate to us that dexfenfluramine does not cause pulmonary endothelial cell proliferation in rats as is seen when hypoxia or hypoxic vasoconstriction interacted with VEGFR-2 blockade.
Because VEGF is a survival factor for endothelial cells
(11
, 12)
, we investigated whether VEGFR-2 blockade caused
pulmonary endothelial cell death. Using two independent tests, we
documented that vascular endothelial cell death occurred in pulmonary
arteries during the period when the vascular smooth muscle cells were
proliferating. Under normoxic conditions, the occurrence of endothelial
cell death was not sufficient to cause proliferation of pulmonary
endothelial cells. However, during chronic hypoxia, lungs of
SU5416-treated rats exhibited increased endothelial cell death prior to
and concomitant with the intralumenal growth of endothelial cells. In
addition, lungs of rats treated with SU5416 and chronic hypoxia showed
a decrease in the expression of VEGF, VEGFR-2 and phosphorylated
VEGFR-2, Src, and phosphorylated Akt, which have been implicated in the
VEGF-dependent survival of endothelial cells (12
, 26)
.
Further, using microchip array for gene expression, we found that a
lung of an animal treated with SU5416 and chronic hypoxia for 2 wk had
decreased mRNA expression of the pro-survival molecules Bcl-2
(1.8-fold), Bcl-XL (2.7-fold), and
IGF-1(7.1-fold) when compared to an untreated chronically hypoxic lung
(L. Alger et al., unpublished results). We propose that endothelial
cell death together with chronic hypoxia results in the selection of an
apoptosis-resistant, proliferating endothelial cell phenotype. Oxidant
stress induced by localized lung ischemia-reperfusion as the result of
chronic hypoxia may have contributed to the death of endothelial cells
susceptible to SU5416 (27)
. On the other hand, the
apoptosis-resistant endothelial cell phenotype may grow
VEGFR-2-independently, at precapillary arterial branching sites where
the shear stress induced by chronic hypoxia is high. That indeed cell
death is a prerequisite for endothelial cell proliferation follows from
our findings that caspase inhibition throughout the course of chronic
hypoxia and SU5416 treatment prevented endothelial cell proliferation
and the development of severe pulmonary hypertension. It is also
possible that the loss of the normal population of endothelial cells by
apoptosis interrupts a growth-inhibitory effect exerted by normal
endothelial cells on the dysfunctional endothelial cell proliferation.
Whether the action of SU5416 is exclusively via VEGFR-2 blockade
or via other effects is of a great importance for the interpretation of
our data and the mechanisms involved in the development of
pulmonary hypertension. That SU5416 did indeed block VEGFR-2 is
supported by the findings of interference with the tyrosine kinase
domain of VEGFR-2, which resulted in reduced expression of
VEGFR-2, Src, Akt, and phosphorylated Akt. SU5416 treatment also
resulted in inhibition of hypoxia-induced polycythemia, consistent with
the role of VEGF in the maturation of hematopoietic cells
(28)
. And, as already stated, endothelial cell apoptosis
is certainly a finding consistent with effective VEGFR-2 blockade
(11
, 29)
. Whereas VEGFR-2 blockade is central to the
design of this pulmonary hypertension model, we do not yet know to what
extent downstream effects associated with the disruption of the normal
VEGF/VEGFR-2 signaling contribute to the extensive pulmonary vascular
remodeling. VEGF affects the expression and action of other growth
factors and the production of endothelial cell nitric oxide, which also
promote endothelial cell survival (30
, 31)
.
It is remarkable that VEGFR-2 blockade by chronic SU5416
treatment affected only the lung and not other organs. One explanation
for this lung specific action might be that the lung endothelial cells,
which are exposed to the highest oxygen tensions in the body, are
particularly vulnerable to VEGFR-2 blockade. Further, pulmonary
endothelial cells may be a site of SU5416 metabolism and biological
activity. In contrast, the growth of rat aorta smooth muscle cells
maintained in low serum in vitro was not affected by SU5416
(R. M. Tuder, unpublished observations). Remarkably, we did not
find lung infiltration by macrophages with chronic SU5416 treatment.
Hence, chronic VEGFR-2 blockade did not induce an inflammatory response
in the lung, which might have occurred either by decreased nitric oxide
levels (32)
or as a consequence of the widespread
pulmonary endothelial cell apoptosis.
In conclusion, the combination of VEGFR-2 blockade and chronic hypoxia induces proliferation of an apoptosis resistant endothelial cell in pulmonary arteries. The endothelial cell growth, and likely the severe pulmonary hypertension, may be the result of a prior and persistent selection process of apoptosis-resistant cells. This study of drug- and hypoxia-induced severe pulmonary hypertension is the first to show the importance of endothelial cell death for the pathogenesis in the pathogenesis of an angioproliferative disorder. Inhibition of endothelial cell death may be a new treatment for severe pulmonary hypertension.
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ACKNOWLEDGMENTS |
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Received for publication May 23, 2000.
Revision received August 1, 2000.
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REFERENCES |
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