Alterations in TGF-
1 expression in lambs with increased pulmonary blood flow and pulmonary hypertension
Eugenia Mata-Greenwood,1
Barbara Meyrick,2
Robin H. Steinhorn,1
Jeffrey R. Fineman,3 and
Stephen M. Black1,4
Departments of 1Pediatrics and
4Molecular Pharmacology, Northwestern University,
Chicago, Illinois 60611-3008; 2Department of
Pathology, Vanderbilt University Medical Center, Nashville, Tennessee
37232-2650; and 3Department of Pediatrics, University
of California San Francisco, San Francisco, California 94143-0106
Submitted 3 June 2002
; accepted in final form 25 March 2003
 |
ABSTRACT
|
---|
The mechanisms responsible for pulmonary vascular remodeling in congenital
heart disease with increased pulmonary blood flow remain unclear. We developed
a lamb model of congenital heart disease and increased pulmonary blood flow
utilizing an in utero placed aortopulmonary vascular graft (shunted lambs).
Morphometric analysis of barium-injected pulmonary arteries indicated that by
4 wk of age, shunts had twice the pulmonary arterial density of controls
(P < 0.05), and their pulmonary vessels showed increased
muscularization and medial thickness at both 4 and 8 wk of age (P
< 0.05). To determine the potential role of TGF-
1 in this vascular
remodeling, we investigated vascular changes in expression and localization of
TGF-
1 and its receptors T
RI, ALK-1, and T
RII in lungs of
shunted and control lambs at 1 day and 1, 4, and 8 wk of life. Western blots
demonstrated that TGF-
1 and ALK-1 expression was elevated in shunts
compared with control at 1 and 4 wk of age (P < 0.05). In
contrast, the antiangiogenic signaling receptor T
RI was decreased at 4
wk of age (P < 0.05). Immunohistochemistry demonstrated shunts had
increased TGF-
1 and T
RI expression in smooth muscle layer and
increased TGF-
1 and ALK-1 in endothelium of small pulmonary arteries at
1 and 4 wk of age. Moreover, T
RI expression was significantly reduced in
endothelium of pulmonary arteries in the shunt at 1 and 4 wk. Our data suggest
that increased pulmonary blood flow dysregulates TGF-
1 signaling,
producing imbalance between pro- and antiangiogenic signaling that may be
important in vascular remodeling in shunted lambs.
shunted lambs
THE DEVELOPMENT OF PULMONARY hypertension and its associated
increased vascular reactivity is a common accompaniment of congenital heart
disease with increased pulmonary blood flow
(10). After birth, the
presence of a systemic to pulmonary communication results in increasing
pulmonary blood flow as pulmonary vascular resistance normally decreases. This
abnormal postnatal hemodynamic state results in progressive structural and
functional abnormalities of the pulmonary vascular bed
(22,
29). Although the vascular
morphology of pulmonary hypertension is well described, the mechanisms of
vascular remodeling remain incompletely understood. A clearer understanding of
these mechanisms would provide potential new avenues for the prevention and
treatment of this disorder.
Members of the transforming growth factor (TGF)-
superfamily play a
critical role in the regulation of cellular growth and differentiation in a
wide range of biological systems, including the vasculature
(1820,
32,
33). However, the actions of
TGF-
in vivo are complex and largely dependent on the environment of
individual target cells
(1820).
TGF-
1 has shown a crucial role in the development of various pulmonary
diseases, including fibrotic pulmonary disease due to various injuries
(28,
37). In addition, some
clinical studies have demonstrated an association between increased or
decreased expression of TGF-
1 in adult patients suffering from pulmonary
hypertension (41).
Active TGF-
1 signals through a heteromeric complex consisting of two
types of transmembrane serine/threonine kinases, known as type I and type II
receptors (18). The type II
TGF-
receptor (T
RII) is the primary receptor target for
TGF-
. On binding to TGF-
1, T
RII recruits type I TGF-
receptor (T
RI) (19,
20). T
RI, also called
activin receptor-like kinase 5 (ALK-5), is a widely expressed type I receptor
for TGF-
1 (13,
19). In addition, endothelial
cells express activin receptor-like kinase 1 (ALK-1), another type I receptor
that has recently been shown to bind TGF-
1
(16) and to be present in a
receptor complex in association with T
RII
(26). T
RI has been
attributed antiangiogenic properties, whereas ALK-1 has shown proangiogenic
effects in endothelial cells
(8).
We have developed an animal model of pulmonary hypertension by inserting an
aortopulmonary vascular graft in the late-gestational fetal lamb
(3,
4,
28). Postnatally, these lambs
have increased pulmonary blood flow and pressure. In addition, they display
pulmonary vascular remodeling characterized by increased medial wall thickness
of the small muscular pulmonary arteries and abnormal extension of muscle to
peripheral pulmonary arteries
(18,
27,
28). Last, at 4 wk of age,
these lambs have a transient increase in the number of barium-filled
intra-acinar pulmonary arteries, which may represent an early adaptive
angiogenesis and/or vessel recruitment
(28). Thus we hypothesized
that the expression of TGF-
1 and its proangiogenic receptors would be
increased in lambs with increased pulmonary blood flow. Therefore, in the
present study, we investigated the relationship between the development of
muscle and medial thickening of pulmonary arteries and changes in arterial
number with alterations in the expression of TGF-
1 and its signaling
receptors T
RII, T
RI, and ALK-1 from 1 day to 8 wk in lambs with
increased pulmonary blood flow.
 |
METHODS
|
---|
Surgical preparation and care. Forty mixed-breed, pregnant Western
ewes (137141 days gestation, term = 145 days) were operated on under
sterile conditions as previously described
(30). After spontaneous
delivery of the lambs (27 days after surgery), antibiotics
[106 units of penicillin G potassium and 25 mg of gentamicin
sulfate intramuscularly (im)] were administered for 2 days. The lambs were
weighed daily, and respiratory rate and heart rate were measured. Furosemide
(1 mg/kg im) was administered daily, and elemental iron (50 mg im) was given
weekly. At 1 day and at 1, 4, or 8 wk of age, the lambs were anesthetized with
intravenous infusions of ketamine hydrochloride
(
1mg·kg-1·min-1)
and diazepam (0.002 mg · kg-1 ·
h-1), intubated with a 5.5-mm outer diameter
endotracheal tube, and mechanically ventilated with a pediatric, time-cycled,
pressure-limited ventilator at 21% O2 (Healthdyne, Marietta, GA).
Ventilation rate was adjusted to maintain an arterial
PCO2 (Paco2) between 35 and 45
Torr. Via a midsternotomy incision, the lambs were instrumented to measure
pulmonary and systemic arterial pressure, right and left atrial pressure,
heart rate, and left pulmonary blood flow, as previously described
(31). After 60 min of
recovery, baseline hemodynamic variables and O2 saturation values
were obtained. The lambs were then euthanized with an intravenous injection of
pentobarbital sodium (Euthanasia CII; Central City Medical, Union City, CA)
and subjected to bilateral thoracotomy. An autopsy was performed to confirm
patency of the vascular graft. The lungs were removed and prepared for RNA
preparation, Western blot analysis, and immunohistochemistry. All procedures
and protocols were approved by the Committee on Animal Research of the
University of California, San Francisco and Northwestern University.
Measurements. Pulmonary and systemic arterial and right and left
atrial pressures were measured using Sorenson Neonatal Transducers (Abbott
Critical Care Systems, Chicago, IL). Mean pressures were obtained by
electrical integration. Heart rate was measured by a cardiotachometer
triggered from the phasic systemic arterial pressure pulse wave. Left
pulmonary blood flow was measured on an ultrasonic flow meter (Transonic
Systems, Ithaca, NY). All hemodynamic variables were recorded continuously on
a Gould multichannel electrostatic recorder (Gould, Cleveland, OH). Systemic
arterial blood gases and pH were measured on a Radiometer ABL5 pH/blood gas
analyzer (Radiometer, Copenhagen, Denmark). Hemoglobin concentration and
oxygen saturation were measured by a hemoximeter (270; CIBA-Corning). The
ratio of pulmonary to systemic blood flow (Qp/Qs) was
calculated using the Fick equation. Pulmonary vascular resistance was
calculated using standard formulas. It should be noted that ketamine
anesthesia increases systemic vascular resistance, which may falsely increase
the Qp/Qs.
Tissue preparation for immunohistochemistry. The heart and lungs
were removed en bloc. The lungs were dissected with care to preserve the
integrity of the vascular endothelium. Sections (23 g) from each lobe
of the lung were removed. These tissues were snap-frozen in liquid
N2 and stored at -70°C until analysis.
For immunohistochemistry, the pulmonary vascular tree was rinsed with cold
(4°C) PBS to remove blood and was fixed by perfusion with cold (4°C)
4% paraformaldehyde. The pulmonary artery was then clamped. The airways were
fixed at 20 cm of H2O pressure by filling the trachea with cold
(4°C) 4% paraformaldehyde. When the lungs were distended at this pressure,
the trachea was clamped. The lungs were fixed for 24 h at 4°C by immersion
in 4% paraformaldehyde. Representative slices from each lobe were removed,
placed in 30% sucrose until they sank, placed in optimum cutting temperature
compound, frozen on dry ice, and stored at -70°C until they were
sectioned. Five- to 10-µm sections were cut using a cryostat, transferred
to aminoalkylsilane-treated slides (Superfrost Plus; Fisher Scientific, Santa
Clara, CA), and stored at -70°C
(1).
Structural studies (morphometric analysis). The lungs, heart, and
trachea were removed intact from four shunt and four control lambs each at 1,
4, and 8 wk of age, and the pulmonary arterial bed was distended with a barium
gelatin suspension (563 ml micropaque powder, Nicholas Picker, Stoughton, MA;
50 g gelatin, Bloom 8-G, Fisher Scientific, Fairlawn, NJ; 387 ml distilled
water; and a few crystals of phenol) at 60°C from a pressure of 70 mmHg
for 2 min (11). This mixture
has been shown to not cross the capillary bed and to fill small arteries down
to a lumen of 15- to 20-µm internal diameter. Use of the hypertensive
pressure ensures that the arteries are fixed in the fully distended state,
thereby allowing application of morphometric techniques
(20a). After arterial
injection, the lungs were inflated by way of the trachea with 10%
formol-saline from a pressure of 35 cmH2O and placed in a bath of
formalin for fixation.
After fixation, the lungs were cut into longitudinal 2-cm slices, and
approximately six random blocks were taken from each lung for routine light
microscopy. Two 5-µm sections were cut from each block, one was stained
with hematoxylin and eosin, and the other was stained with Verhoff's elastin
stain, followed by van Gieson. The sections were then examined for the
characteristic structural changes of chronic pulmonary hypertension using
well-established quantitative techniques
(11). Briefly, external
diameter of at least 100 arterial profiles was measured as well as medial
thickness of the muscular and partially muscular arteries. Medial thickness
was then related to arterial size using the calculation: percent medial
thickness = 2 x medial thickness/external diameter x 100. The
structure of each artery was also noted: muscular, partially muscular, and
nonmuscular, as was the structure of the accompanying airway: bronchus,
bronchiolus, terminal bronchiolus, respiratory bronchiolus, alveolar duct, and
alveolar wall. The density of the barium-filled intra-acinar arteries was also
assessed. With the use of a x25 objective and an eyepiece reticule, the
number of barium-filled arteries of <200-µm external diameter was
counted and related to the number of alveolar profiles in these same fields.
At least 25 consecutive microscopic fields were counted for each animal.
Western blot analysis. Protein extracts (100 µg) were separated
on 420% (TGF-
1) and 12% (T
RI, T
RII, and ALK-1)
SDS-polyacrylamide gel and electrophoretically transferred to polyvinylidene
difluoride membranes (Amersham). The membranes were blocked with 5% nonfat dry
milk in Tris-buffered saline containing 0.1% Tween 20 (TBS-T). After 1 h of
blocking, the membranes were incubated at 4°C for 2 consecutive days with
1:500 (
3.4 µg/ml) dilution of rabbit polyclonal anti-human TGF-
1
antibody (2 µg/ml in blocking solution, Santa Cruz). Alternatively,
membranes were probed with goat polyclonal antibodies against human T
RI
and T
RII (1 µg/ml, Santa Cruz) and ALK-1 (1 µg/ml, R&D
Systems) for 2h at room temperature. Membranes were washed 3 x 15 min
with TBS-T and then hybridized with anti-rabbit or anti-goat horseradish
peroxidase antibody for 45 min. After 3 x 15 min washes, bands were
visualized with chemiluminescence using a Kodak Digital Science Image Station
(NEN). The following size bands were obtained: 12.5 and 25 kDa for monomeric
and dimeric TGF-
1, 50 kDa for T
RI, 62 kDa for ALK-1, and 70 kDa
for T
RII.
To compare the various protein levels obtained from controls and shunts
(n = 5, total of 10 samples per age) from various ages (1-day-old and
1-, 4-, and 8-wk-olds, total of 40 samples), four different gels were run (1
for each age containing protein samples from both control and shunted lambs).
Also, included on each gel was an internal control (a lung extract prepared
from a 1-day-old shunted lamb). Each one of the four membranes was probed for
the particular protein of interest and then reprobed the next day for
-actin. Each densitometric value was divided by its
-actin value
to obtain a relative value for TGF-
1, T
RI, T
RII, and ALK-1
protein levels. Relative values were averaged and then corrected using a
factor (relative protein level of internal control obtained from that
particular blot divided by relative protein level of internal control from the
original day 1 membrane). In this way, differences in time exposure
leading to different protein levels were observed and corrected for. Standard
deviations were also corrected by using the same factor.
Immunohistochemistry. Immunohistochemistry was performed as
previously described (1,
2). Studies were done on serial
sections of control and shunted ovine lung using rabbit polyclonal
anti-TGF-
1 (Santa Cruz) or goat polyclonal anti-T
RI, ALK-1, and
T
RII. Frozen tissue sections (7 µm) were allowed to come to room
temperature. Samples were fixed for 10 min in cold acetone and then washed
3x with PBS. To eliminate nonspecific binding of the primary antiserum
to tissue proteins, tissue sections were incubated with 1% horse serum in PBS
(blocking solution) for 1 h. The tissue sections were then incubated with
primary antibodies (5 µg/ml) in the presence of monoclonal smooth muscle
cell-actin antibody (1:400, Sigma) in blocking solution at 4°C overnight.
After three washes with PBS x 5 min, samples were visualized with a
combination of Rhodamine Red-X goat anti-rabbit and Oregon Green 488 goat
anti-mouse secondary antibodies (Molecular Probes) at a concentration of 1:400
in blocking solution for 45 min at room temperature. Alternatively, a
combination of Rhodamine Red-X rabbit anti-goat and Alexa Green 488 rabbit
anti-mouse secondary antibodies was used to localize TGF-
receptors.
After three further washes with PBS, an antifading solution was added, and
samples were visualized by fluorescence microscopy. For each tissue section, a
parallel experiment was carried out in which the primary antibody was omitted.
This served as the negative control. A minimum of three different sets of
control and shunted lung tissues were prepared and examined. Because it is
difficult to utilize immunohistochemistry for qualitative measurements on
protein expression, we used this technique only to determine protein
localization and to determine whether there were differences between shunt and
control lambs in the numbers of vessels expressing for TGF-
1, T
RI,
T
RII, and ALK-1. To carry out this procedure, small, muscularized
pulmonary arteries were visualized next to airways, and at least 30 vessels
(representing at least 10 fields) were counted as positively immunoreactive or
nonreactive for a particular protein. The number of vessels immunoreactive for
each protein as a proportion of the total vessels counted was determined.
Results were then calculated as the average number of immunoreactive vessels
± SE (n = at least 3 different lambs from each age group), and
statistical significance was calculated as described below.
Data analysis. For each age studied (1, 4, and 8 wk), the mean
value was calculated for each structural variable. Quantitation of protein
expression was performed using a Kodak image station 440CF and KDS1D imaging
software. This allows a pixel density from 1103 instead of
256 gray scale of autoradiographic film. The response is linear within this
range. In all experiments, means ± SD were calculated, and comparisons
among control and shunted lambs were made by ANOVA for repeated measures. When
differences were present among study groups, Student-Newman-Keuls post hoc
testing was performed. P < 0.05 was considered statistically
significant.
 |
RESULTS
|
---|
Functional changes. Spontaneous delivery occurred 29 days
after fetal surgery. All shunted lambs had an audible continuous murmur and an
increase in oxygen saturation between the right ventricle and the distal
pulmonary artery. In shunted lambs, mean pulmonary arterial pressure and left
pulmonary blood flow were greater than age-matched controls at all ages. Left
pulmonary vascular resistance was decreased in shunted lambs at 4 and 8 wk of
age. Biventricular cardiomegaly was present in shunted lambs aged 18 wk
and tended to increase with age (Table
1). Compared with 4-wk-old shunted lambs, mean pulmonary arterial
pressure and left pulmonary vascular resistance was significantly higher in
8-wk-old shunted lambs (P < 0.05).
Structural changes. Previous morphometric analysis showed a
significant increase in number of barium-filled peripheral arteries per unit
area in 4-wk-old shunted lambs compared with age-matched controls
(31). However, this increase
in vessel number per unit area was not yet present in 1-wk-old lambs and did
not persist in 8-wk-old lambs compared with age-matched controls
(Fig. 1A). Alveolar
number was similar in each group of shunted animals compared with age-matched
controls. However, at 4 wk, a burst of alveolar multiplication had occurred
compared with the values at 1 wk, and by 8 wk, alveolar number was reduced,
likely indicating an enlargement of the alveoli between 4 and 8 wk
(Table 2). Previously, in
4-wk-old shunted lambs, we demonstrated that the percent medial thickness of
arteries <200-µm external diameter was approximately twice that of
age-matched controls (31). In
this study, we found that medial thickness was still increased in the 8-wk-old
shunted lambs, and although values for percent medial thickness of controls
and shunted animals was less than at 4 wk, the increase remained twice that
seen in age-matched controls. At 1 wk, we found that medial thickness in
control sheep was generally less than at either 4 or 8 wk, and no significant
difference was noted between control and shunted animals at that time
(Fig. 1B). Previous
analysis of the structure of the intra-acinar arteries related to airway level
(Table 3) established the
appearance of muscle in the walls of smaller and more peripheral arteries than
normal in the 4-wk-old shunted lambs. Although arterial muscularity of the
1-wk shunted animals was similar to age-matched controls, at 8-wk, increased
muscularity of the intra-acinar arteries was still apparent, although at the
alveolar duct and alveolar wall level, this difference was not as pronounced
as at 4 wk (Table 3). Together,
these data indicate that the 4- and 8-wk shunted animals show the structural
changes of pulmonary hypertension. Since the structural changes were not
apparent in the shunted sheep at 1 wk, we did not examine 1-day-old shunted
animals.

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 1. Morphometric analysis of vascular development in the shunt model.
A: increase in blood vessel number according to age development in
control and shunted lambs. Values are means ± SE.
*P < 0.05 vs. control lambs. B: increase in
percent medial thickness in shunt modeling according to age-stage of
development. Four sets of control and shunt twins were evaluated for medial
thickness according to diameter size of blood vessel. *P
< 0.05 vs. control.
|
|
View this table:
[in this window]
[in a new window]
|
Table 3. Percentage of fully muscular, partially muscular, and nonmuscular
pulmonary arteries in shunted and control lambs
|
|
Expression and localization of TGF-
1. TGF-
1
protein levels were significantly increased in peripheral lung in the shunted
lambs at 1 wk (125%) and 4 wk (120%, Fig.
2) but were comparable to control samples at 1 day and 8 wk
(Fig. 2). In agreement with
these data, TGF-
1 mRNA expression was increased by 65% in 1-wk-old
shunted lambs and 310% in 4-wk-old shunted lambs compared with age-matched
controls (P < 0.05; Fig.
3) but was similar in shunted and control lambs at 1 day and 8 wk
(Fig. 3). Immunohistochemical
analysis revealed that TGF-
1 was highly expressed in the lung of both
control and shunted lambs. No differences were observed between shunted and
control lambs at 1 day of age. However, TGF-
1-specific staining appeared
to be more intense in both smooth muscle and endothelium of small pulmonary
arteries as well as in the smooth muscle of airways in 1- and 4-wk-old shunted
lambs compared with age-matched twin controls
(Fig. 4, AF).
At 8 wk of age, TGF-
1 stained brightly in the airway epithelium of
shunted lambs but not in control lambs. This contrasts with the Western blot
analysis of peripheral lung tissue in which TGF-
1 expression at 8 wk of
age was similar to controls.

View larger version (32K):
[in this window]
[in a new window]
|
Fig. 2. Western blot analysis for transforming growth factor (TGF)- 1 in lung
tissue from 1-day-old and 1-, 4-, and 8-wk-old lambs (control and, after
insertion of an aorta-to-pulmonary artery vascular graft in utero, shunt).
A: representative Western blots are shown from protein extracts (100
µg) prepared from lung tissue from 1-day-old and 1-, 4-, and 8-wk-old lambs
(1 control and 1 shunt from each age), separated on a 420%
SDS-polyacrylamide gradient gel, electrophoretically transferred to Hybond
membranes, and analyzed using a polyclonal rabbit anti-TGF- 1.
TGF- 1 protein expression was increased in shunted lambs only at 1 and 4
wk. Con, control; Sh, shunt. B: densitometric values for relative
TGF- 1 protein from 5 control and 5 shunted lambs at each age. In shunted
lambs, relative TGF- 1 protein is increased by 125% at 1 wk and by 120%
at 4 wk (P < 0.05). Values are means ± SE.
*P < 0.05 control vs. shunt.
|
|

View larger version (31K):
[in this window]
[in a new window]
|
Fig. 4. TGF- 1 protein expression in the lung in vivo from tissue from
1-day-old and 1-, 4-, and 8-wk-old lambs (control and, after insertion of an
aorta-to-pulmonary artery vascular graft in utero, shunt). Immunohistochemical
localization of TGF- 1 expression in the lung in vivo from 1-day-old
(A and B), 1-wk-old (C and D), 4-wk-old
(E and F), and 8-wk-old (G and H) lambs
(control: A, C, E, and G; shunted: B, D, F, and
H). Polyclonal rabbit anti-TGF- 1 and monoclonal mouse
anti-smooth muscle cell-actin antibodies were used to localize expression.
TGF- 1 expression is shown in red and smooth muscle cell-actin expression
is shown in green. Colocalization is shown in yellow. Magnification,
x200. Results are representative of 3 different sets of twin matches
(control and shunt). AW, airway; V, vessel; Sm, smooth muscle cell layer; End,
endothelium; Epi, epithelium.
|
|
TGF receptor expression. Active TGF-
1 binds to T
RII,
inducing its dimerization with either T
RI or ALK-1
(8). Because the formation of
either complex leads to different biological functions, we investigated the
changes in expression of all three receptors by immunoblotting. T
RII
expression was unchanged between shunted and control lambs over all
developmental periods (Fig. 5).
Immunohistochemical analysis localized T
RII expression mainly in the
endothelial layers of medium-sized (500200 µm), small-sized (<200
µm), and microvessels (capillaries, <10 µm), in both control and
shunt samples (Fig. 6).
Differences in T
RII localization were observed between shunt and
controls. Specifically, compared with age-matched controls, decreased
expression of T
RII was observed in the capillaries of 1-wk-old shunts
(Fig. 6, C and
D), whereas increased staining of T
RII was found in the small
pulmonary arterial endothelium of 4-wk-old shunts. Also, we observed an
increased localization of T
RII in the smooth muscle layer of vessels and
airways in 8-wk-old shunts (Fig.
6, G and H).

View larger version (74K):
[in this window]
[in a new window]
|
Fig. 6. T RII protein expression in the lung in vivo from tissue from
1-day-old and 1-, 4-, and 8-wk-old lambs (control and, after insertion of an
aorta-to-pulmonary artery vascular graft in utero, shunt). Immunohistochemical
localization of T RII expression in the lung in vivo from 1-day-old
(A and B), 1-wk-old (C and D), 4-wk-old
(E and F), and 8-wk-old (G and H) lambs
(control: A, C, E, and G; shunted: B, D, F, and
H). Polyclonal goat anti-T RII antiserum and monoclonal mouse
anti-smooth muscle cell-actin antibody were used to localize expression of
T RII. T RII expression is shown in red and smooth muscle cell-actin
expression is shown in green. Colocalization is shown in yellow.
Magnification, x200. Results are representative of at least 2 different
sets of twin matches (control and shunt). Arrows indicate pulmonary
arteries.
|
|
Although T
RI protein expression showed little change in control
animals over time, shunted lambs showed a significant decrease in expression
of T
RI in 4-wk-old shunted animals relative to age-matched controls
(226% decrease in the shunt compared with controls, P < 0.05,
Fig. 7). Studies using
immunohistochemistry showed that T
RI was highly expressed in the
endothelium of pulmonary vessels, including capillaries, of normal lambs
(Fig. 8, A, C, E, and
G). However, pulmonary vessels of shunted lambs showed a marked
absence of expression of T
RI in the endothelium of pulmonary vessels, in
particular small pulmonary arteries of less than 200-µm diameter. At 1 wk
of age, 69.4 ± 2.6% immunoreactive arteries were found in the controls
compared with only 36.3 ± 6.9% in the shunts (P < 0.05). At
4 wk of age, 56.1 ± 5.9% immunoreactive arteries were observed in the
controls compared with only 25.7 ± 6.4% in the shunts (P <
0.05, Fig. 8, D and
F). In addition, 15.2 ± 5.2% of small pulmonary arteries from
1-wk-old shunts showed increased immunoreactivity for T
RI in their
smooth muscle layer compared with only 1.5 ± 0.1% in agematched
controls (P < 0.05, Fig.
8, C and D). T
RI expression in the
endothelium of microvessels of shunted lambs was somewhat decreased in the
shunt at 1 wk of age but otherwise normal to control lambs at other
developmental ages (Fig. 8,
AH).

View larger version (31K):
[in this window]
[in a new window]
|
Fig. 7. Western blot analysis for type I TGF- receptor (T RI) in lung
tissue protein from 1-day-old and 1-, 4-, and 8-wk-old lambs (control and,
after insertion of an aorta-to-pulmonary artery vascular graft in utero,
shunt). A: representative Western blots are shown from protein
extracts (100 µg) prepared from lung tissue from 1-day-old and 1-, 4-, and
8-wk-old lambs (1 control and 1 shunt from each age), separated on a 12%
SDS-polyacrylamide gradient gel, electrophoretically transferred to Hybond
membranes, and analyzed using a specific antiserum raised against T RI.
T RI protein expression was decreased in shunted lambs only at 4 wk.
B: densitometric values for relative T RI protein from 4 control
and 4 shunted lambs at each age. In shunted lambs, relative T RI protein
is decreased by 221% at 4 wk (P < 0.05). Values are means ±
SE. *P < 0.05 control vs. shunt.
|
|

View larger version (67K):
[in this window]
[in a new window]
|
Fig. 8. T RI protein expression in the lung in vivo from tissue from 1-day-old
and 1-, 4-, and 8-wk-old lambs (control and, after insertion of an
aorta-to-pulmonary artery vascular graft in utero, shunt). Immunohistochemical
localization of T RI expression in the lung in vivo from 1-day-old
(A and B), 1-wk-old (C and D), 4-wk-old
(E and F), and 8-wk-old (G and H) lambs
(control: A, C, E, and G; shunted: B, D, F, and
H). Polyclonal goat anti-T RI antiserum and monoclonal mouse
anti-smooth muscle cell-actin antibody were used to localize expression of
T RI. T RI expression is shown in red and smooth muscle cell-actin
expression is shown in green. Colocalization is shown in yellow.
Magnification, x200. Results are representative of at least 2 different
sets of twin matches (control and shunt). Arrows, pulmonary arteries. Note
that 1- and 4-wk-old controls, but not shunts, show intense T RI staining
in the endothelium of pulmonary arteries.
|
|
Finally, we analyzed the expression of ALK-1, which has been shown to
mediate TGF-
1 signaling in the endothelium. Immunoblotting experiments
showed that ALK-1 protein expression in the control lambs decreases with age
(Fig. 9). Compared with control
values, an increase in ALK-1 protein expression was evident at 1 day (150%
increase) and was significant at 1 and 4 wk of age (400% and 86% increase,
respectively, P < 0.05, Fig.
10). Therefore, ALK-1 and T
RI protein expression appear to
be regulated in opposing directions in control compared with shunted lambs.
Immunohistochemistry localized ALK-1 to the endothelium layer of pulmonary
microvessels in both control and shunt samples
(Fig. 10, AH).
However, at 1 and 4 wk of age, control lambs exhibited little expression of
ALK-1 (43.7 ± 10.2% positively stained arteries at 1 wk and 19.2
± 7.4% at 4 wk of age) in the endothelium of small-to-large pulmonary
vessels (Fig. 10, C
and E). In shunted lambs, ALK-1 was highly expressed in small
pulmonary arteries with 76.3 ± 15.3% immunoreactive arteries at 1 wk
and 58.4 ± 6.5% at 4 wk of age (P < 0.05 compared with
controls, Fig. 10, D
and F). There were no detectable differences in expression of ALK-1
in endothelium of small pulmonary arteries between control and shunted lambs
at 8 wk of age (Fig. 10,
D and G).

View larger version (29K):
[in this window]
[in a new window]
|
Fig. 9. Western blot analysis for activin receptor-like kinase 1 (ALK-1) in lung
tissue protein from 1-day-old and 1-, 4-, and 8-wk-old lambs (control and,
after insertion of an aorta-to-pulmonary artery vascular graft in utero,
shunt). A: representative Western blots are shown from protein
extracts (100 µg) prepared from lung tissue from 1-day-old and 1-, 4-, and
8-wk-old lambs (1 control and 1 shunt from each age), separated on a 12%
SDS-polyacrylamide gradient gel, electrophoretically transferred to Hybond
membranes, and analyzed using a specific antiserum raised against ALK-1. ALK-1
protein expression was increased in shunted lambs only a 1 and 4 wk.
B: densitometric values for relative ALK-1 protein from 5 control and
5 shunted lambs at each age. In shunted lambs, relative ALK-1 protein is
increased by 407% at 1 wk and by 86% at 4 wk (P < 0.05). Values
are means ± SE. *P < 0.05 control vs. shunt.
|
|

View larger version (63K):
[in this window]
[in a new window]
|
Fig. 10. ALK-1 protein expression in the lung in vivo from tissue from 1-day-old and
1-, 4-, and 8-wk-old lambs (control and, after insertion of an
aorta-to-pulmonary artery vascular graft in utero, shunt). Immunohistochemical
localization of ALK-1 expression in the lung in vivo from 1-day-old
(A and B), 1-wk-old (C and D), 4-wk-old
(E and F), and 8-wk-old (G and H) lambs
(control: A, C, E, and G; shunted: B, D, F, and
H). Polyclonal goat anti-ALK-1 antiserum and monoclonal mouse
anti-smooth muscle cell-actin antibody were used to localize expression of
ALK-1. ALK-1 expression is shown in red and smooth muscle cell-actin
expression is shown in green. Colocalization is shown in yellow.
Magnification, x200. Results are representative of at least 2 different
sets of twin matches (control and shunt). Arrows, pulmonary arteries. Note
that 1-day-old and 1- and 4-wk-old shunts, but not controls, have intense
ALK-1 (red) staining in the endothelium of pulmonary arteries.
|
|
 |
DISCUSSION
|
---|
The progressive structural abnormalities of the pulmonary vascular bed
associated with congenital heart disease and increased pulmonary blood flow
are well characterized. In 1958, Heath and Edwards
(9) described a progressive
structural classification that ranged from reversible medial hypertrophy to
terminal changes such as angiomatoid formation and fibrinoid necrosis. In
1978, Rabinovitch et al. (30)
described a modification based on lung biopsy specimens that featured
alterations in normal remodeling and growth. The three progressive stages
(AC) that have been correlated with perioperative hemodynamics are
characterized by progressive medial hypertrophy, abnormal muscle extension,
and reduced artery size and concentration. The vascular remodeling at 4 and 8
wk of age described in the present study would be characterized as grade B
alterations, with medial hypertrophy
2 times normal and abnormal
appearance of muscle in the walls of intra-acinar arteries. Between 4 and 8 wk
of life, the pulmonary-to-systemic blood flow ratio, mean pulmonary arterial
pressure, and left pulmonary vascular resistance all tended to increase in
shunted lambs, suggesting a persistent remodeling stimulus. However, since
there is a normal thinning of the medial layer with age, the less dramatic
medial thickness at 8 wk most likely represents changes relative to normal
development. The increase in pulmonary vessel number per unit area observed in
4-wk-old shunted lambs has not been reported in children with pulmonary
hypertension and increased pulmonary blood flow
(22,
31) and may represent an early
adaptive angiogenic and/or recruitment response to incorporate the increase in
pulmonary blood flow. During the second month of life, when alveolar growth is
significant, arterial vessel growth and/or recruitment was not maintained at
the same degree, such that 8-wk-old shunted lambs had similar vessel number
per unit area as age-matched control lambs.
Hemodynamic insult, as in increased blood flow and/or pressure, has been
known to play a critical role in the increase in smooth muscle hypertrophy and
hyperplasia and phenotypic changes of vascular cells
(23,
39); however, little is known
about the mechanisms by which biomechanical forces transduce intracellular
signals leading to gene regulation. TGF-
1 increases have been observed
in response to laminar shear stress in endothelial cells and conduit vessels
(4,
15) and to cyclic stretch in
cardiomyocytes (42). In
support of these findings, we have observed in our model of increased
pulmonary blood flow an increase in TGF-
1 expression in the pulmonary
vessels of shunted lambs compared with age-matched controls. In our studies, a
relatively normal arterial morphology was found in the 1-wk-old shunted lambs,
a time when TGF-
1 expression starts to increase. Four-week-old shunted
lambs have increased vessel number per unit area, a significant increase in
muscularity of the intraacinar arteries, and a significant increase in medial
thickness of small pulmonary arteries, a time when TGF-
1 expression
peaks. At 8 wk, the shunted lambs have normal vessel number per unit area but
maintain medial thickening and increased muscularity of the intra-acinar
arteries. However, at this age the levels of TGF-
1 expression are
similar in shunted and control lambs. These data suggest that dysregulation of
TGF-
1 signaling pathways is an early event that precedes the development
of vascular remodeling induced by increased pulmonary blood flow secondary to
congenital heart disease. Furthermore, studies will be required to determine
whether TGF-
1 is a stimulus or a marker of remodeling.
TGF-
1 plays a pivotal role in vascular homeostasis by regulating the
synthesis of extracellular matrix proteins that stabilize interactions between
endothelial, mesenchymal, and smooth muscle cells of the vessel wall
(25). TGF-
1 has been
ascribed both antiangiogenic and proangiogenic effects in vivo and in vitro
(6,
8,
1921,
33). However, the role of
TGF-
1 in vascular remodeling is not well understood. Previous
observations have shown a biphasic in vitro effect of TGF-
1 on
endothelial cell proliferation, where low doses of TGF-
1 stimulate
proliferation and migration and high doses inhibit these processes
(8,
26). Recently, it has been
shown that the biphasic effect of TGF-
1 on angiogenesis is due to
differences in receptor signaling
(8). Active TGF-
1 binds
to T
RII, which then recruits a type I receptor. In the endothelial cell,
two type I receptors have been described: T
RI and ALK-1
(8,
1920).
Both type I receptors have been shown to heterodimerize with T
RII and
endoglin (a type III receptor) and to bind TGF-
1 and TGF-
3
(8,
1920,
24). However, ALK-1 and
T
RI have opposing signaling events and biological functions
(8). T
RI phosphorylates
Smad 2 and Smad 3, leading to transcriptional activation of extracellular
matrix proteins (collagen and fibrin) and plasminogen activator inhibitor-1
(PAI-1) (8). ALK-1 induces the
phosphorylation of Smad 1 and Smad 5, leading to enhanced gene expression of
Id-1, a cell differentiation inhibitor
(8). T
RI activation leads
to inhibitory functions in endothelial cell migration and proliferation and,
therefore, signals the antiangiogenic effects of TGF-
1
(8,
38). Conversely, ALK-1
activation and Id-1 upregulation leads to the opposite effects of T
RI
signaling, thereby showing proangiogenic effects
(8). In agreement with these
studies, we have observed in our shunt model of increased pulmonary blood flow
a profound imbalance between the expression of T
RI and ALK-I in
endothelial cells of small pulmonary arteries in shunted lambs in which
structural differences were indicative of an active angiogenic process. In
addition, we have observed that plasminogen activator inhibitor-1 is decreased
in the shunt model as early as 1 wk of age, in conjunction with T
RI
downregulation (data not shown). Together, our data suggest that the decrease
in T
RI and increase in ALK-1 signaling would lead to endothelial
activation and increased production of extracellular matrix protein
degradation necessary for new blood vessel formation.
Other studies have suggested that TGF-
1 induces proangiogenic effects
indirectly by upregulating VEGF expression
(3,
12,
27). Moreover, TGF-
1 has
been shown to activate VEGF transcription in vascular smooth muscle cells
through activation of Smads 2 and 3, in conjunction with other transcriptional
factors, including hypoxia-inducible factor 1 (HIF-1) and activator protein-1
(34). These studies suggest
that increased VEGF transcription occurs through T
RI signaling. Our
immunohistochemical analysis demonstrated that, while T
RI is
downregulated in endothelial cells, the opposite is true in vascular smooth
muscle cells of
15% of small pulmonary arteries in shunted lambs of
14 wk of age. In addition, we have observed an increase in VEGF
expression in similar vessels. In support of our observations, various reports
show parallel increased expression of TGF-
1 and VEGF in models of atrial
fibrillation and adult pulmonary hypertension
(35). In addition, in vitro
studies have shown that cyclic stretch-induced VEGF expression is dependent on
previous activation of TGF-
1 since a TGF-
1 neutralizing antibody
abolished stretch-induced increases in VEGF mRNA expression and protein
secretion (42). This suggests
that TGF-
1 might be inducing proangiogenic effects by upregulating VEGF
expression in vascular smooth muscle cells of small pulmonary arteries in
models of increased blood flow, similar to HIF-1
factors in
hypoxia-induced pulmonary hypertension
(36). Increased TGF-
1
signaling in the smooth muscle cell layer, through TBRI, could also account
for hyperplasia and increased extracellular matrix protein production
(14,
23).
Considering that TGF-
1 signaling is fundamental for the homeostasis
of intimal, medial, and adventitial layers of vessels, we propose that the
dysregulation of TGF-
1 and its receptors due to increased blood flow is
likely to play an important role in the development of the pulmonary vascular
remodeling in our model of pulmonary hypertension secondary to increased
pulmonary blood flow. Further understanding of these mechanisms could lead to
potential new therapies for the management of secondary pulmonary hypertension
due to congenital heart disease.
 |
ACKNOWLEDGMENTS
|
---|
The authors thank Michael J. Johengen for expert technical assistance.
This research was supported in part by National Institutes of Health Grants
HL-60190 (S. M. Black), HL-67841 (S. M. Black), HD-398110 (S. M. Black), and
HL-61284 (J. R. Fineman), March of Dimes Grant FY00-98 (S. M. Black), and
American Heart Association, Midwest Affiliate Grant 0051409Z (S. M.
Black).
S. M. Black is a member of the Feinberg Cardiovascular Research
Institute.
 |
FOOTNOTES
|
---|
Address for reprint requests and other correspondence: S. M. Black, Division
of Neonatology, Northwestern Univ. Medical School, Ward 12-191, 303 E. Chicago
Ave., Chicago, IL 60611-3008 (E-mail:
steveblack{at}northwestern.edu).
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
 |
REFERENCES
|
---|
- Black SM,
Fineman JR, Steinhorn RH, Bristow J, and Soifer SJ. Increased endothelial
NOS in lambs with increased pulmonary blood flow and pulmonary hypertension.
Am J Physiol Heart Circ Physiol
275: H1643-H1651,
1998.[Abstract/Free Full Text]
- Black SM,
Johengen MJ, Ma ZD, Bristow J, and Soifer SJ. Ventilation and oxygenation
induce endothelial nitric oxide synthase gene expression in the lungs of fetal
lambs. J Clin Invest 100:
1448-1458, 1997.[Abstract/Free Full Text]
- Brogi E, Wu T,
Namiki A, and Isner JM. Indirect angiogenic cytokines upregulate VEGF and
bFGF gene expression in vascular smooth muscle cells, whereas hypoxia
upregulates VEGF expression only. Circulation
90: 649-652,
1994.[Abstract]
- Cucina A,
Sterpetti AV, Borrelli V, Pagliei S, Cavallaro A, and D'Angelo LS. Shear
stress induces transforming growth factor-
1 release by arterial
endothelial cells. Surgery 123:
212-217, 1998.[ISI][Medline]
- De Caestecker M and Meyrick B. Bone morphogenetic proteins, genetics and the
pathophysiology of primary pulmonary hypertension. Respir
Res 2: 193-197,
2001.[ISI][Medline]
- Fajardo LF,
Prionas SD, Kwan HH, Kowalski J, and Allison AC. Transforming growth
factor
1 induces angiogenesis in vivo with a threshold pattern.
Lab Invest 74:
600-608, 1996.[ISI][Medline]
- Folkman J and
D'Amore PA. Blood vessel formation: what is its molecular basis?
Cell 87:
1153-1155, 1996.[ISI][Medline]
- Goumans MJ,
Vladimarsdottir G, Itoh S, Rosendahl A, Sideras P, and ten Dijke P.
Balancing the activation state of the endothelium via two distinct TGF-
type I receptors. EMBO J 21:
1743-1753, 2002.[Abstract/Free Full Text]
- Heath D and
Edwards JE. The pathology of hypertensive pulmonary vascular disease.
Circulation 18:
533-547, 1958.[ISI]
- Hoffman JI,
Rudolph AM, and Heymann MA. Pulmonary vascular disease with congenital
heart lesions: pathologic features and causes.
Circulation 64:
873-877, 1981.[Abstract]
- Johnson JE,
Perkett EA, and Meyrick B. Pulmonary veins and bronchial vessels undergo
remodeling in sustained pulmonary hypertension induced by continuous air
embolization into sheep. Exp Lung Res
23: 459-473,
1997.[ISI][Medline]
- Koh GY, Kim SJ,
Klug MG, Park K, Soonpaa MH, and Field LJ. Targeted expression of
transforming growth factor-
1 in intracardiac grafts promotes vascular
endothelial cell DNA synthesis. J Clin Invest
95: 114-121,
1995.[ISI][Medline]
- Larsson J,
Goumans MJ, Sjoostrand LJ, van Rooijen MA, Ward D, Leveen P, Xu X, ten Dijke
P, Mummery CL, and Karlsson S. Abnormal angiogenesis but intact
hematopoietic potential in TGF-
type I receptor-deficient mice.
EMBO J 20:
1663-1673, 2001.[Abstract/Free Full Text]
- Li G, Li RK,
Mickle DAG, Weisel RD, Merante F, Ball WT, Christakis GT, Cusimano RJ, and
Williams WG. Elevated insulin-like growth factor-I and transforming growth
factor-
1 and their receptors in patients with idiopathic hypertrophic
obstructive cardiomyopathy: a possible mechanism.
Circulation 98, Suppl
II: 144-149, 1998.
- Lum RM, Wiley
LM, and Barakat AI. Influence of different forms of fluid shear stress on
vascular endothelial TGF-
1 mRNA expression. Int J Mol
Med 5: 653-641,
2000.
- Lux A, Attisano
L, and Marchuk DA. Assignment of transforming growth factor
1 and
3 and a third new ligand to the type I receptor ALK-1. J Biol
Chem 274:
9984-9992, 1999.[Abstract/Free Full Text]
- Machado RD,
Pauciulo MW, Thomson JR, Lane KB, Morgan NV, Wheeler L, Phillips JA, Newman J,
Williams D, Galie N, Manes A, McNeil K, Yacoub M, Mikhail G, Rogers P, Corris
P, Humbert M, Donnai D, Martensson G, Tranebjaerg L, Loyd JE, Trembath RC, and
Nichols WC. BMPR2 haploinsufficiency as the inherited molecular mechanism
for primary pulmonary hypertension. Am J Hum Genet
68: 92-102,
2001.[ISI][Medline]
- Madri JA, Bell
L, and Merwin JR. Modulation of vascular cell behaviour by transforming
growth factor
. Mol Reprod Dev
32: 121-126,
1992.[ISI][Medline]
- Massague J.
TGF-
signal transduction. Annu Rev Biochem
67: 753-791,
1998.[ISI][Medline]
- Massague J.
The transforming growth factor-
family. Annu Rev Cell
Biol 6: 597-641,
1990.[ISI][Medline]
- Meyrick B and
Reid L. Pulmonary hypertension: anatomic and physiologic correlates.
Clin Chest Med 4:
199-217, 1983.[ISI][Medline]
- Muller G,
Behrens J, Nussbaumer U, Bohlen P, and Birchmeier W. Inhibitory action of
transforming growth factor
on endothelial cells. Proc Natl
Acad Sci USA 84:
5600-5604, 1987.[Abstract]
- Nagumo K,
Yamaki S, and Takahashi T. Extremely thickened media of small pulmonary
arteries in fatal pulmonary hypertension with congenital heart disease: a
morphometric and clinicopathological study. Jpn Circ J
64: 909-914,
2000.[ISI][Medline]
- O'Callaghan CJ and Williams B. Mechanical strain-induced extracellular matrix production
by human vascular smooth muscle cells. Role of TGF-
1.
Hypertension 36:
319-324, 2000.[Abstract/Free Full Text]
- Oh SP, Seki T,
Goss KA, Imamura T, Yi Y, Donahoe PK, Li L, Miyazono K, ten Dijke P, Kim S,
and Li E. Activin receptor-like kinase 1 modulates transforming growth
factor-
1 signaling in the regulation of angiogenesis. Proc
Natl Acad Sci USA 97:
2626-2631, 2000.[Abstract/Free Full Text]
- Pepper MS.
Transforming growth factor-
: vasculogenesis, angiogenesis and vessel
wall integrity. Cytokine Growth Factor Rev
8: 21-43,
1997.[Medline]
- Pepper MS,
Vasalli JD, Orci L, and Montesano R. Biphasic effect of transforming
growth factor-
1 on in vitro angiogenesis. Exp Cell
Res 204: 356-363,
1993.[ISI][Medline]
- Pertovaara L,
Kaipainen A, Mustonen T, Orpana A, Ferrara N, Saksela O, and Alitalo K.
Vascular endothelial growth factor is induced in response to transforming
growth factor-
in fibroblastic and epithelial cells. J Biol
Chem 269:
6271-6274, 1994.[Abstract/Free Full Text]
- Pittet JF,
Griffiths MJ, Geiser T, Kaminski N, Dalton SL, Huang X, Brown LA, Gotwals PJ,
Koteliansky VE, Matthay MA, and Sheppard D. TGF-
is a critical
mediator of acute lung injury. J Clin Invest
107: 1537-1544,
2001.[Abstract/Free Full Text]
- Rabinovitch M,
Bothwell T, Hayakawa BN, Williams WG, Trusler GA, Rowe RD, Olley PM, and Cutz
E. Pulmonary artery endothelial abnormalities in patients with congenital
heart defects and pulmonary hypertension. A correlation of light with scanning
electron microscopy and transmission electron microscopy. Lab
Invest 55:
632-653, 1986.[ISI][Medline]
- Rabinovitch M,
Haworth SG, Castaneda AR, Nadas AS, and Reid LM. Lung biopsy in congenital
heart disease: a morphometric approach to pulmonary vascular disease.
Circulation 58:
1107-1122, 1978.[Abstract]
- Reddy VM,
Meyrick B, Wong J, Khoor A, Liddicoat JR, Hanley FL, and Fineman JR. In
utero placement of aortopulmonary shunts. A model of postnatal pulmonary
hypertension with increased pulmonary blood flow in lambs.
Circulation 92:
606-613, 1995.[Abstract/Free Full Text]
- Roberts AB.
Molecular and cell biology of TGF-
. Miner Electrolyte
Metab 24:
111-119, 1998.[ISI][Medline]
- Roberts AB,
Sporn MB, Assoian RK, Smith JM, Roche NS, Wakefield LM, Heine UI, Liotta LA,
Falanga V, Kehrl JH, and Fauci AS. Transforming growth factor type
:
rapid induction of fibrosis and angiogenesis in vivo and stimulation of
collagen formation in vitro. Proc Natl Acad Sci USA
83: 4167-4171,
1986.[Abstract]
- Sanchez-Elsner T, Botella LM, Velasco B, Corbi A, Attisano L, and
Bernabe C. Synergistic cooperation between hypoxia and transforming growth
factor-
pathways on human vascular endothelial growth factor gene
expression. J Biol Chem 276:
38527-38535, 2001.[Abstract/Free Full Text]
- Seko Y,
Nishimura H, Takahashi N, Ashida T, and Nagai R. Serum levels of vascular
endothelial growth factor and transforming growth factor-
1 in patients
with atrial fibrillation undergoing defibrillation therapy. Jpn
Heart J 41:
27-32, 2000.[ISI][Medline]
- Semenza GL,
Agani F, Feldser D, Iyer N, Kotch L, Laughner E, and Yu A. Hypoxia, HIF-1
and the pathophysiology of common human diseases. Adv Exp Med
Biol 475:
123-130, 2000.[ISI][Medline]
- Sime PJ and
O'Reilly KM. Fibrosis of the lung and other tissues: new concepts in
pathogenesis and treatment. Clin Immunol Immunopathol
99: 308-319,
2001.
- Stefansson S,
Petitclerc E, Wong MK, McMahon GA, Brooks PC, and Lawrence DA. Inhibition
of angiogenesis in vivo by plasminogen activator inhibitor-1. J
Biol Chem 276:
8135-8141, 1996.[Abstract/Free Full Text]
- Topper JN.
Transforming growth factor-
and vascular disease. CARP as a putative
TGF-
target gene in the vessel wall. Circ Res
88: 5-6,
2001.[Free Full Text]
- Trembath RC,
Thomson JR, Machado RD, Morgan NV, Atkinson C, Winship I, Simonneau G, Galie
N, Loyd JE, Humbert M, Nichols WC, and Morrell NW. Clinical and molecular
genetic features of pulmonary hypertension in patients with hereditary
hemorrhagic telangiectasia. N Engl J Med
345: 325-334,
2001.[Abstract/Free Full Text]
- Tuder RM,
Chacon M, Alger L, Wang J, Tarseviciene-Stewart L, Kasahara Y, Cool CD, Bishop
AE, Geraci M, Semenza GL, Yacoub M, Polak JM, and Voelkel NF. Expression
of angiogenesis-related molecules in plexiform lesions in severe pulmonary
hypertension: evidence for a process of disordered angiogenesis. J
Pathol 195:
367-374, 2001.[ISI][Medline]
- Zheng W, Seftor
EA, Meininger CJ, Hendrix MJ, and Tomanek RJ. Mechanisms of coronary
angiogenesis in response to stretch: role of VEGF and TGF-
.
Am J Physiol Heart Circ Physiol
280: H909-H917,
2001.[Abstract/Free Full Text]