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1 expression in lambs with increased pulmonary blood flow and pulmonary hypertension
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
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONREFERENCES |
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1 in this vascular
remodeling, we investigated vascular changes in expression and localization of
TGF-1 and its receptors TRI, ALK-1, and TRII 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 TRI was decreased at 4
wk of age (P < 0.05). Immunohistochemistry demonstrated shunts had
increased TGF-1 and TRI 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, TRI 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
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
(18–20,
32,
33). However, the actions of
TGF- in vivo are complex and largely dependent on the environment of
individual target cells
(18–20).
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 (TRII) is the primary receptor target for
TGF-. On binding to TGF-1, TRII recruits type I TGF-
receptor (TRI) (19,
20). TRI, 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 TRII
(26). TRI 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 TRII, TRI, and ALK-1 from 1 day to 8 wk in lambs with
increased pulmonary blood flow.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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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 (2–3 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 4–20% (TGF-1) and 12% (TRI, TRII, 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 TRI
and TRII (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 TRI, 62 kDa for ALK-1, and 70 kDa
for TRII.
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, TRI, TRII, 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-TRI, ALK-1, and
TRII. 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, TRI,
TRII, 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 1–103 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.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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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.
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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, A–F).
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.
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TGF receptor expression. Active TGF-1 binds to TRII,
inducing its dimerization with either TRI 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. TRII
expression was unchanged between shunted and control lambs over all
developmental periods (Fig. 5).
Immunohistochemical analysis localized TRII expression mainly in the
endothelial layers of medium-sized (500–200 µm), small-sized (<200
µm), and microvessels (capillaries, <10 µm), in both control and
shunt samples (Fig. 6).
Differences in TRII localization were observed between shunt and
controls. Specifically, compared with age-matched controls, decreased
expression of TRII was observed in the capillaries of 1-wk-old shunts
(Fig. 6, C and
D), whereas increased staining of TRII was found in the small
pulmonary arterial endothelium of 4-wk-old shunts. Also, we observed an
increased localization of TRII in the smooth muscle layer of vessels and
airways in 8-wk-old shunts (Fig.
6, G and H).
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Although TRI protein expression showed little change in control
animals over time, shunted lambs showed a significant decrease in expression
of TRI 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 TRI 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 TRI 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 TRI in their
smooth muscle layer compared with only 1.5 ± 0.1% in agematched
controls (P < 0.05, Fig.
8, C and D). TRI 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,
A–H).
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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 TRI 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, A–H).
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).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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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,
19–21,
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 TRII, which then recruits a type I receptor. In the endothelial cell,
two type I receptors have been described: TRI and ALK-1
(8,
19–20).
Both type I receptors have been shown to heterodimerize with TRII and
endoglin (a type III receptor) and to bind TGF-1 and TGF-3
(8,
19–20,
24). However, ALK-1 and
TRI have opposing signaling events and biological functions
(8). TRI 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). TRI 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 TRI
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 TRI 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 TRI
downregulation (data not shown). Together, our data suggest that the decrease
in TRI 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 TRI signaling. Our
immunohistochemical analysis demonstrated that, while TRI is
downregulated in endothelial cells, the opposite is true in vascular smooth
muscle cells of 15% of small pulmonary arteries in shunted lambs of
1–4 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.
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ACKNOWLEDGMENTS |
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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.
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FOOTNOTES |
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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.
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