Department of Respiratory Medicine, Juntendo University School of Medicine, Bunkyo-Ku, Tokyo 113-8421, Japan
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Pulmonary veins
show greater sensitivity to endothelin (ET)-1-induced vasoconstriction
than pulmonary arteries, and remodeling was observed in pulmonary veins
under hypoxic conditions. We examined, using an immunohistochemical
method, the expression of Big ET-1, ET-converting enzyme (ECE), and
ETA and ETB receptors in rat pulmonary veins
under normoxic and hypoxic conditions. In control rats, Big ET-1 and
ECE were coexpressed in the intima and media of the pulmonary veins,
with an even distribution along the axial pathway. ETA and
ETB receptors were expressed in the pulmonary veins, with a
predominant distribution in the proximal segments. The expression of
Big ET-1 was more abundant in the pulmonary veins than in the pulmonary
arteries. After exposure to hypoxia for 7 or 14 days, the expression of
Big ET-1, ECE, and ET receptors increased in small pulmonary veins.
Increases in the medial thickness, wall thickness, and immunoreactivity
for -smooth muscle actin were also observed in the small pulmonary
veins under hypoxic conditions. The upregulation of ET-1 and ET
receptors in the small pulmonary veins is associated with vascular
remodeling, which may lead to the development of hypoxic pulmonary hypertension.
endothelin-1; endothelin-converting enzyme; hypoxia; pulmonary hypertension; immunohistochemistry
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
ENDOTHELINS (ETs) are potent vasoconstrictors and smooth muscle mitogens consisting of 21-residue vasoactive peptides synthesized from Big ETs by a specific cleavage at Trp21-Val22/Ile22 by ET-converting enzymes (ECEs) (15, 16). The pulmonary vascular ETs are represented by ET-1, which is synthesized in various cells including endothelial cells, smooth muscle cells, and tissue macrophages in the pulmonary vessels (16). The actions of ET-1 are mediated by two different receptors, ETA and ETB receptors (15, 16). ETA receptors are found in pulmonary vascular smooth muscle cells and mediate smooth muscle contraction and the proliferation of smooth muscle cells and fibroblasts, whereas ETB receptors are expressed in endothelial cells and smooth muscle cells and cause either relaxation or contraction (11, 16, 22).
ET-1 and its receptors play important roles in the regulation of the pulmonary circulation under normal and hypoxic conditions (11, 15, 16). Blocking ETA receptors or both ETA and ETB receptors with selective antagonists attenuates the increase in pulmonary arterial pressure and the development of vascular remodeling associated with chronic hypoxia, suggesting significant roles of the ET system in vascular remodeling (4, 5, 7, 28). The primary site of generation of pulmonary vascular resistance under normal and hypoxic conditions is postulated to be the pulmonary arteries; therefore, extensive studies utilizing pharmacological and physiological methods on the effects of ET-1 and ET receptors on pulmonary arteries have been conducted (reviewed in Ref. 16). Interestingly, several investigators (19, 25) have reported that pulmonary veins show a greater sensitivity to ET-1-induced vasoconstriction than pulmonary arteries and that the responses of pulmonary arteries and veins to ET-1 are different under chronic hypoxia. However, few studies have described the expression of ET-1 and its receptors in pulmonary veins under normoxic condition and the influence of hypoxia on them (24). Furthermore, vascular remodeling similar to that seen in the pulmonary arteries has been observed in pulmonary veins in some clinical cases and in experimental models of pulmonary hypertension due to hypoxia and other causes (3, 6, 10, 12, 17, 20, 26). But the molecular mechanism involving vascular remodeling in the pulmonary veins has not been elucidated. We hypothesize that the ET system is present in the pulmonary veins as well as in the pulmonary arteries and plays a certain role in the development of pulmonary venous remodeling associated with chronic hypoxia.
In this study, we investigated the locality and distribution of Big ET-1, ECE, and ETA and ETB receptors in the rat pulmonary vasculature under normoxic condition, with an emphasis on the pulmonary veins. We also studied the temporal changes in the expression of the ET systems in pulmonary veins after exposure to hypoxia to explore the roles of the ET system in the remodeling of the pulmonary veins.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Animals.
The methods utilized to isolate rat lungs are almost identical to those
previously reported except that strict precautions were exercised to
keep the animals under hypoxic conditions before lung preparation
because lung ET-1 mRNA expression has been reported to decrease rapidly
in rats exposed to chronic hypoxic pulmonary hypertension after a brief
exposure to normoxia (14, 22). Briefly, adult male
Sprague-Dawley rats (8 wk old, 260-280 g) were placed for 0.5, 3, 7, or 14 days in a specially designed hypobaric chamber. The chamber
was depressurized to 380 mmHg (oxygen concentration reduced to ~10%)
in a room with a 12:12-h light-dark cycle. To minimize exposure to
normoxia, the rats raised in the hypobaric chamber were transferred to
and kept in a chamber filled with 10% oxygen-90% nitrogen just before
lung preparation. Age-matched control rats were maintained in room air.
The lungs were isolated from the rats after the intraperitoneal
administration of 60 mg of pentobarbital sodium and an intracardiac
injection of 100 U of heparin. Cannulas were inserted into the
pulmonary artery and left atrium, and the lungs were perfused at 36 cmH2O through the pulmonary arterial cannula with PBS. The
lungs were perfused with 4% paraformaldehyde (PFA), inflated by
infusion of 4% PFA through the cannula inserted in the trachea, and
fixed in 4% PFA overnight at 4°C. After removal of PFA by repeated
instillation and withdrawal of PBS through the tracheal cannula, the
lung tissue was filled with optimum cutting temperature compound
through the tracheal cannula, embedded in optimum cutting temperature
compound, and stored at 80°C until used for immunohistochemical
studies. The development of hypoxic pulmonary hypertension was
determined by right ventricle-to-left ventricle plus septum weight
ratio as previously described (8). Each group consisted of
four to six experimental animals.
Immunohistochemistry.
To detect ET-1 expression on the pulmonary vasculature, we utilized
anti-Big ET-1 antibody and anti-ECE antibody instead of the anti-ET-1
antibody because immunostaining with three types of anti-ET-1
antibodies purchased from different companies showed a certain degree
of nonspecific staining (data not shown). Frozen sections (4 µm) cut
in a cryostat at 20°C were mounted on slides and incubated with
10% normal goat serum to reduce nonspecific binding of secondary
antibodies. The tissue sections utilized for ECE staining were
pretreated by placing the slides in 0.01 M citrate buffer in an
autoclave at 120°C for 10 min before incubation. The serum was
removed, and the sections were incubated with anti-Big ET-1 antibody
(IBL, Gumma, Japan) at 10 µg/ml, anti-ECE (AEC27-121; a gift from Dr.
K. Tanzawa, Sankyo, Tokyo, Japan) (23) at 60 µg/ml, anti-ETA receptor antibody (IBL) at 5 µg/ml, or
anti-ETB receptor antibody (IBL) at 5 µg/ml for 12 h
at 4°C. In addition, monoclonal antibodies against vascular cell
adhesion molecule-1 (VCAM-1; 5 µg/ml; Santa Cruz Biotechnology, Santa
Cruz, CA) and
-smooth muscle (SM) actin (0.2 µg/ml; DAKO,
Glostrup, Denmark) were used as an endothelial marker and smooth muscle
marker, respectively. The sections were further incubated with a
biotinylated goat anti-rabbit IgG antibody (diluted 1:100) for 30 min
and then with an avidin-biotin-peroxidase complex (diluted 1:100;
Vector Laboratories, Burlingame, CA) for 30 min. Subsequently, the
immunoperoxidase color reaction was performed by incubation for 15 min
with Tris · HCl containing 0.05% 3,3'-diaminobenzidine
tetrahydrochloride and 0.01% hydrogen peroxide. Negative controls
were prepared with rabbit nonimmune serum instead of the primary
antibody or by omitting steps in the avidin-biotin-peroxidase complex
procedure and showed little nonspecific staining (data not shown). The
sections were counterstained with methyl green (for Big ET-1,
ETA and ETB receptors, VCAM-1, and
-SM
actin) or hematoxylin (for ECE). The pulmonary veins were distinguished
from the pulmonary arteries on the basis of anatomic location and
structure. The pulmonary veins either were located at the margin of
acini or ran midway between two airways, whereas the pulmonary arteries
could be identified by the accompanying airways. In addition, serial
sections were stained with elastic van Gieson stain for accurate
identification of arteries and veins by the presence or absence of
internal elastic lamina (18). Only those veins that were
cut in true cross section or in an oblique section in which the length
of profile was less than twice its diameter were included in the
semiquantitative and morphometric analyses. The external diameter (ED)
was measured as the shortest luminal distance from the external edge of
the single elastic lamina on one side of the vein to the other. For
each rat, pulmonary veins were grouped according to ED: 60-200,
200-500, and 500-1,000 µm. In this study, we omitted the
intra-acinar vessels with an ED of <60 µm because it was difficult
to distinguish veins from arteries by the criterion of the presence or
absence of internal elastic lamina. The sections were examined by light
microscopy without knowledge of the treatment group, and the intensity
of immunostaining was graded semiquantitatively from 0 to 3: grade 0, no staining; grade 1, focal staining or weak staining; grade 2, diffuse
moderate staining; and grade 3, strong staining (22). To
assess the changes in the expression of Big ET-1, ECE, ETA and ETB receptors, and
-SM actin after exposure to
hypoxia, the immunostaining grades of the pulmonary veins were
estimated in lung sections from each animal, and the data were
calculated for each group. For the assessment of remodeling of the
pulmonary veins, the wall thickness and medial thickness were measured
in pulmonary veins with a 60- to 200-µm ED from the alveolar region with an eyepiece reticle in each tissue section. The wall of each vein
profile was noted as muscular, partially muscular, or nonmuscular. The
wall thickness was measured along the same plane used to measure the
ED, from the luminal surface to the external edge of the single elastic
lamina, including the collagenous perivascular sheath when present. The
medial thickness of muscular and partially muscular veins was assessed
as the distance between the internal edge of the single elastic lamina
and the adluminal edge of the intimal muscle layer. The relationship
between vessel size and wall thickness is expressed as the percent wall
thickness (%WT), calculated as [(WT1 + WT2)/ED] × 100, where WT1 and WT2
are the wall thickness of each side. Similarly, the percent medial
thickness (%MT) was calculated as [(MT1 + MT2)/ED] × 100 for muscular veins, where MT1
and MT2 are the medial thickness of each side, and
(MT/ED) × 100 for partially muscular veins (3,
10).
Northern blot analysis.
Northern blot analysis was performed as previously described
(22). Briefly, 10 µg of denatured total RNA isolated
from control and hypoxia-exposed rat lungs was electrophoresed in 1%
agarose-formamide gels and transferred to nylon membranes (Hybond-N+,
Amersham Japan, Tokyo, Japan). After prehybridization, the membranes
were hybridized for 24 h at 42°C with random-primed
32P-labeled ET-1, ECE-1, ETA receptor, or
ETB receptor cDNA probes. The cDNA probes for rat ET-1,
ECE-1, and ETA receptor were synthesized by reverse
transcriptase (RT)polymerase chain reaction (PCR). The primers
used for PCR were sense, 5'-GCGATCCTTGAAAGACTTAC-3', and antisense,
5'-CTCGGTTGTGTATCAACTTC-3', for preproET-1; sense, 5'-TTCCAGCTGGAATCCTGCAG-3', and antisense, 5'-GTTCTGAGAACTCCTTGGAG-3', for ECE-1; sense, 5'-TGTTGCTGTTGTCACCAGTCC-3', and antisense, 5'-GAGCGCAGCTGCTGCGTGACCG-3', for the ETA receptor;
and sense, 5'-CTGCTGGTCCAAACGTTTGAG-3', and antisense,
5'-CCATGGCTTTCTTAGGTTGTA-3', for the ETB receptor (2,
21, 27). After hybridization, the membrane was washed at a final
stringency in 0.1× sodium-saline citrate (0.15 M NaCl and 0.015 M
sodium citrate, pH 7.4)-0.1% sodium dodecyl sulfate at 65°C.
Autoradiography was performed at 80°C, and the bands were
quantitated by densitometry with a computer-assisted image analyzer
(BAStation, Scanalytics, Billerica, MA). After being probed with ET-1,
ECE-1, ETA receptor, or ETB receptor cDNAs, the
membranes were stripped and reprobed with a 32P-labeled 28S
rRNA cDNA as a positive control. The amount of ET-1, ECE-1, and
ETA and ETB receptor mRNAs is expressed as the
ratio of ET-1, ECE-1, ETA receptor, or ETB
receptor mRNA to 28S rRNA.
Statistical methods. Numerical data are means ± SE. Statistical analyses of immunohistochemical grading and autoradiographic densitometry were conducted with one-way analysis of variance with a proprietary software (Statview, Abacus Concepts, Berkeley, CA). P < 0.05 was considered significant.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The measurement of ventricular weight revealed the right ventricle-to-left ventricle plus septum weight ratios to be 0.31 ± 0.03 in control rats and 0.33 ± 0.01, 0.36 ± 0.01, 0.38 ± 0.01, and 0.61 ± 0.03 in rats exposed to hypoxia for 0.5, 3, 7, and 14 days, respectively (control vs. 7 and 14 day, P < 0.05; n = 4-6). Histological findings of elastic van Gieson staining in rats exposed to hypoxia for 7 and 14 days were also consistent with a previous report (9) of a hypoxic pulmonary hypertension model, e.g., a gradual increase in arterial wall thickness and progressive extension of muscle into smaller and more peripheral arteries up to 200-µm ED. The results indicate that a significant degree of pulmonary hypertension developed after 7 and 14 days of exposure to hypoxia.
In addition to the well-known vascular remodeling of pulmonary
arteries, a significant but smaller degree of medial thickening was
observed in small pulmonary veins of <200-µm ED after
exposure to hypoxia for 14 days (Fig. 1,
a-f). Although the degree of medial thickening was variable in the pulmonary veins, smaller vessels exhibited a more pronounced thickening. In accordance with the increase
in venous wall thickness, there was a progressive increase in the
expression of -SM actin in the media of pulmonary veins after
exposure of the rats to hypoxia (Fig. 1,
a-f). Semiquantitative evaluation
showed that immunoreactivity for
-SM actin in the media of pulmonary
veins of 60- to 200-µm ED significantly increased after exposure to
hypoxia for 7 and 14 days (Fig. 1g). However, the expression
of
-SM actin in pulmonary veins of >500-µm ED appeared unchanged
after exposure to hypoxia (data not shown). Morphometric analysis
revealed that %WT and %MT increased by 1.5- to 1.9-fold and 1.8- to
2.1-fold after exposure to hypoxia for 7 and 14 days, respectively
(P < 0.001). Furthermore, the ratio of muscular and
partially muscular veins to nonmuscular veins tended to increase under
hypoxic conditions (Table 1).
|
|
In normoxic rat lungs, specific expression of Big ET-1 was observed in
the media of large and small pulmonary veins with smooth muscle layers
and, to a lesser degree, in the intima. Predominant localization of Big
ET-1 in the media of pulmonary veins was confirmed with the different
pattern of immunostaining for VCAM-1 that is specific for the
endothelium (Fig. 2,
a-f). The distribution of Big ET-1
seemed to be consistent in the pulmonary veins along the axial pathway.
After exposure of the rats to hypoxia for 7 and 14 days, there was an
increase in immunoreactivity for Big ET-1 in the media of the large and
small pulmonary veins (Fig. 2, a-f).
Semiquantitative analysis revealed that the expression of Big ET-1 was
increased in the media of pulmonary veins of both 500- to 1,000-µm
and 60- to 200-µm ED after 7 and 14 days under hypoxic conditions
(Fig. 2g). It is noteworthy that the intensity of
immunoreactivity was higher in pulmonary veins than in pulmonary arteries of similar diameter under normoxic and hypoxic conditions (Fig. 3).
|
|
The expression of ECE in the pulmonary veins is similar to that of Big
ET-1, although its localization was dominant in the intima rather than
in the media. Immunoreactivity for ECE was observed in the intima and
media of pulmonary veins having smooth muscular layers in control rats.
The distribution of ECE immunoreactivity along the axial pathway of the
pulmonary veins seemed to be relatively consistent. After exposure to
hypoxia for 7 and 14 days, the expression of ECE increased
significantly in the vascular walls of large and small pulmonary veins
(Fig. 4, a-d).
Semiquantitative analysis revealed that the immunoreactivity for ECE
increased in the vascular wall of large and small pulmonary veins after
exposure to hypoxia for 7 and 14 days (Fig. 4e).
|
Weak immunoreactivity for ETA receptors was observed in the
media of pulmonary veins having smooth muscle layers in normoxic rats.
The distribution of ETA receptors in the pulmonary veins appeared to be dominant in the proximal segments (Fig.
5,
a-f). Although it was difficult to
clearly separate and identify the endothelium in the pulmonary veins,
the pattern of immunostaining was different from that of VCAM-1,
suggesting that expression of ETA receptors was localized
mainly in the media. Semiquantitative evaluations revealed that the
immunoreactivity for ETA receptors tended to increase after
exposure to hypoxia in pulmonary veins of >500-µm ED. The expression
of ETA receptors in pulmonary veins of 60- to 200-µm ED
significantly increased after exposure to hypoxia for 7 and 14 days
(Fig. 5g). In general, immunoreactivity for ETA
receptors is greater in pulmonary arteries than in pulmonary veins of
similar diameter (data not shown).
|
A small number of ETB receptors were expressed in the
intima and media of the proximal segments of the pulmonary veins,
whereas faint staining was observed in the vascular wall of the distal segments in normoxic rats. There was a slight increase in
immunoreactivity for ETB receptors in the media of large
pulmonary veins and a significant increase in the small pulmonary veins
after exposure to hypoxia for 14 days (Fig.
6, a-d).
Semiquantitative analysis revealed that the immunoreactivity for
ETB receptors was significantly increased in the media of
the small pulmonary veins after 7 and 14 days, whereas the increase was
relatively small in the large pulmonary veins (Fig. 6e).
Expression of ETB receptors in the intima and media was
more intense in pulmonary arteries than in pulmonary veins of similar
ED (data not shown).
|
Northern blot analysis was utilized to detect changes in mRNA
expression of ET-1, ECE-1, ETA receptors, and
ETB receptors in rat lung tissues after exposure to hypoxia
(Fig. 7A). A 2.3-kb preproET-1
mRNA transcript was expressed in normoxia- and hypoxia-exposed rat
lungs. Compared with that in control rats, the ratio of ET-1 mRNA to
28S rRNA was ~1.7-fold greater after 0.5 day of exposure to hypoxia
and then decreased after 3 days of exposure, and there was a 2.0- and
2.2-fold increase in the ratio after 7 and 14 days of exposure,
respectively (Fig. 7B). A faint band for ECE-1 at 4.4 kb was
detected in the lung tissues from control and hypoxia-exposed rats
(Fig. 7A). The ratio of ECE-1 mRNA to 28S rRNA tended to increase at 0.5 and 3 days and then to decrease slightly after 7 and 14 days of exposure, but the changes were not significant (Fig.
7B). The ETA receptor-specific probe hybridized
with 5.2- and 4.3-kb bands (Fig. 7A). The results revealed
an 80-100% increase in the ratio of ETA receptor mRNA
to 28S rRNA after exposure to hypoxia, although a transient decrease in
the ratio at 3 days was observed (Fig. 7B). The
ETB receptor-specific probe hybridized with a single 5.0-kb
band (Fig. 7A). Hypoxic exposure for 7 and 14 days was associated with
an increase in the ratio of ETB receptor to 28S rRNA
(Fig. 7B).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this study, we examined the locality and distribution of Big ET-1, ECE, and ETA and ETB receptors in the pulmonary veins of control rats and the changes in their expression after exposure to hypoxia. Several interesting findings were observed. First, we demonstrated that Big ET-1 and ECE are coexpressed in the endothelium and smooth muscle cells of pulmonary veins of normoxia- and hypoxia-exposed rats. These findings suggest that ET-1 synthesis in the pulmonary veins is regulated by synchronous expression and corresponds to the localization of ECE and its substrate substance Big ET-1. Interestingly, localization of Big ET-1 was more prominent in the media rather than in the intima. The data indicate that smooth muscle cells as well as endothelial cells appear to be an important source of ET-1 in the pulmonary veins. In contrast to Big ET-1, the expression pattern for ECE appears to be more dominant in the endothelium. The reason for the discrepant localization of Big ET-1 and ECE is not clear. Because expression for ECE is more abundant in the endothelium, conversion of Big ET-1 to mature ET-1 should be more rapid in the endothelium than in the smooth muscle cells. This may result in a more abundant expression of Big ET-1 in the smooth muscle cells than in the endothelium even if Big ET-1 production is equal or dominant in the endothelium. However, further studies are necessary to elucidate the precise mechanism.
We also demonstrated that a certain number of ETA receptors were expressed in the media, whereas ETB receptors were observed in the intima and media of the large pulmonary veins. These observations suggested that ET-1 synthesized in the pulmonary veins exerts its actions through ETA and ETB receptors. They are compatible with recent pharmacological and physiological experiments on the study of responses of pulmonary veins to ET-1 utilizing an ETA receptor antagonist and an ETB receptor agonist, which suggested the existence of both ETA and ETB receptors in pulmonary veins (13). Several investigators (19, 25) utilizing vascular rings prepared from large pulmonary arteries and veins have revealed that pulmonary veins exhibit greater sensitivity to ET-1-induced vasoconstriction than pulmonary arteries. The mechanism of the different responses of pulmonary arteries and veins to ET-1 is uncertain. We observed that ETB receptors in the intima are rather abundant in the pulmonary arteries compared with those in the pulmonary veins (22). If we postulate that ETB receptors expressed in the endothelium exert a vasodilating action in the pulmonary veins as seen in the pulmonary arteries, a possible mechanism of the different responses to ET-1-induced vasoconstriction may be due to more potent dilatory actions mediated by ETB receptors expressed in the intima of large pulmonary arteries than of large pulmonary veins. Although this may partially explain the different sensitivity of ET-1-induced vasoconstriction between large pulmonary arteries and veins, further studies are necessary to elucidate the mechanisms of the different responses to ET-1.
Second, the immunoreactivity for Big ET-1 was more intense in pulmonary veins than in pulmonary arteries in both normoxia- and hypoxia-exposed rats. Rodman et al. (19) reported that 27% of the increase in pulmonary arterial pressure caused by ET-1 in blood-perfused rat lungs was due to an increase in microvascular pressure, presumably as a result of postcapillary vasoconstriction. In their experiments, pulmonary edema was not observed despite the increase in microvascular pressure. Their study showed that an increase in postcapillary resistance by ET-1-induced venoconstriction could certainly affect pulmonary arterial pressure without causing pulmonary edema. Furthermore, Aharinejad et al. (1) demonstrated with scanning electron microscopy that the mean pulmonary arterial pressure change caused by ET-1 correlates with the extent of focal pulmonary venous contraction in normal rats. These findings support the hypothesis that ET-1 and ETA and ETB receptors expressed in the pulmonary veins are associated with the regulation of pulmonary venous tone under normoxic conditions.
Third, we confirmed the vascular remodeling of pulmonary veins after
exposure to hypoxia. Vascular remodeling of small pulmonary veins
associated with chronic hypoxia has been found in some clinical cases
and experimental models, although there are few changes in large
pulmonary veins. For instance, thickening or muscularization of small
pulmonary veins was reported in patients with hypoxic pulmonary
hypertension associated with chronic bronchitis, emphysema, and cystic
fibrosis (20, 26). Experimentally, muscularization of the
small pulmonary veins was observed in rats exposed to hypoxia for 28 days (17). We also revealed increases in the wall
thickness, medial thickness, and immunoreactivity for -SM actin in
the distal segments of pulmonary veins after 7 and 14 days of exposure
to hypoxia. These observations indicate that vascular remodeling readily occurs in small pulmonary veins as well as in pulmonary arteries in hypoxic pulmonary hypertension. As previously described (19), an increase in postcapillary resistance could
certainly affect pulmonary arterial pressure without causing pulmonary
edema. Although the principal site of vascular remodeling that affects the development of hypoxic pulmonary hypertension is most likely the
precapillary segment of pulmonary arteries, it is possible that
remodeling of small pulmonary veins could also contribute to the
pathophysiology of hypoxic pulmonary hypertension by increasing postcapillary vascular resistance.
The most striking finding in this study is that chronic hypoxia is also
associated with an increase in the coexpression of Big ET-1 and
ETA and ETB receptors in the distal segment of
the pulmonary veins. These findings were supported by Northern blot analyses demonstrating a persistent increase in the expression of the
mRNA expression of ET-1 and ETA and ETB
receptors after 7 and 14 days of exposure to hypoxia, although the
change in whole lung mRNA cannot distinguish between changes in
arteries and veins. The pathophysiological significance of the
upregulation is uncertain, but there are several possibilities. The
increase in the coexpression of ET-1 and ETA and
ETB receptors in the media of small pulmonary veins may
contribute to vascular remodeling for the following reasons. First,
ET-1 is known to have mitogenic activity through ET receptors to
stimulate DNA synthesis and cell proliferation of smooth muscle cells
and fibroblasts (11, 15, 16). Second, as shown in the
immunohistochemical study of -SM actin, the localization of the
increase in both big ET-1 and ET receptors expression induced by
hypoxia corresponds closely to the site where medial thickening occurs
after exposure to hypoxia. Third, temporal changes in ET-1 and
ETA and ETB receptor expression seem to occur
with or precede those of remodeling; e.g., both an upregulation of
ET-1, ET receptors, and
-SM actin and an increase in the wall
thickness and medial thickness are evident after 7 and 14 days of
exposure to hypoxia. Another hypothesis is that the increased ET-1 in
the pulmonary vein may cause enhancement of postcapillary
vasoconstriction. Dingemans and Wagenvoort (6)
utilizing an electron-microscopic technique demonstrated morphological
evidence of vasoconstriction of small pulmonary veins in rats exposed
to hypoxia for 28 days. This study suggests not only medial thickening
in pulmonary veins under hypoxic conditions but also the actual
occurrence of venoconstriction, and this venoconstriction may
be a contributing factor to the establishment of hypoxic pulmonary
hypertension. In addition, several investigators (19, 25)
have revealed that pulmonary veins are more sensitive to ET-1-induced
vasoconstriction than pulmonary arteries. These findings suggest that
the increased expression of ET-1 and ET receptors in the media of small
pulmonary veins may enhance venoconstriction, thereby resulting in the
increase in microvascular pressure. It is uncertain how much the
pressure actually increases due to remodeling of postcapillary vessels and/or venoconstriction associated with the upregulation of ET-1 and ET
receptors; however, the ET system in small pulmonary veins may
contribute to the pathophysiology of hypoxic pulmonary hypertension. In
contrast, there were no significant changes in ECE-1 mRNA expression, and the increases in ECE expression under hypoxic conditions were less
significant than those of Big ET-1 and the ET receptors. These findings
suggest that the physiological role of the change in ECE expression in
the development of remodeling may be less than that of ET-1 and its receptors.
In conclusion, ET-1 and its receptors are coexpressed in pulmonary veins in normoxic rats. In addition, the expression of Big ET-1, ECE, and ETA and ETB receptors increases in small pulmonary veins after exposure to hypoxia, which may be associated with pulmonary venous remodeling.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Kazuhiko Tanzawa (Biological Research Laboratories, Sankyo) for providing the monoclonal anti-endothelin-converting enzyme antibodies and Dr. Kuniaki Fukuda (Drug Metabolism and Pharmacokinetics Research Laboratories, Sankyo) for advice on immunostaining.
![]() |
FOOTNOTES |
---|
Address for reprint requests and other correspondence and present address of H. Takahashi: Clinic, Personnel Division, Mitsui & Co., Ltd., 1-2-1 Ote-machi, Chiyoda-Ku, Tokyo 100-0004, Japan (E-mail: Hide.Takahashi{at}xm.mitsui.co.jp).
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.
Received 7 August 2000; accepted in final form 19 December 2000.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Aharinejad, S,
Schraufnagel DE,
Miksovsky A,
Larson EK,
and
Marks SC, Jr.
Endothelin-1 focally constricts pulmonary veins in rats.
J Thorac Cardiovasc Surg
110:
148-156,
1995
2.
Arai, H,
Hori S,
Aramori I,
Ohkubo H,
and
Nakanishi S.
Cloning and expression of cDNA encoding an endothelin receptor.
Nature
348:
730-732,
1990[ISI][Medline].
3.
Chazova, I,
Loyd JE,
Zhdanov VS,
Newman JH,
Belenkov Y,
and
Meyrick B.
Pulmonary artery adventitial changes and venous involvement in primary pulmonary hypertension.
Am J Pathol
146:
389-397,
1995[Abstract].
4.
Chen, SJ,
Chen YF,
Meng CQ,
Durand J,
DiCarlo VS,
and
Oparil S.
Endothelin-receptor antagonist bosentan prevents and reverses hypoxic pulmonary hypertension in rats.
J Appl Physiol
79:
2122-2131,
1995
5.
DiCarlo, VS,
Chen SJ,
Meng QC,
Durand J,
Yano M,
Chen YF,
and
Oparil S.
ETA-receptor antagonist prevents and reverses chronic hypoxia-induced pulmonary hypertension in rat.
Am J Physiol Lung Cell Mol Physiol
269:
L690-L697,
1995
6.
Dingemans, KP,
and
Wagenvoort CA.
Pulmonary arteries and veins in experimental hypoxia.
Am J Pathol
93:
353-368,
1978[Abstract].
7.
Eddahibi, S,
Raffestin B,
Clozel M,
Levame M,
and
Adnot S.
Protection from pulmonary hypertension with an orally active endothelin receptor antagonist in hypoxic rats.
Am J Physiol Heart Circ Physiol
268:
H828-H835,
1995
8.
Fulton, RM,
Hutchinson EC,
and
Jones AM.
Ventricular weight in cardiac hypertrophy.
Br Heart J
14:
413-420,
1952.
9.
Hislop, A,
and
Reid L.
New findings in pulmonary arteries of rats with hypoxia-induced pulmonary hypertension.
Br J Exp Pathol
57:
542-554,
1976[ISI][Medline].
10.
Hu, L-M,
and
Jones R.
Injury and remodeling of pulmonary veins by high oxygen. A morphometric study.
Am J Pathol
134:
253-262,
1989[Abstract].
11.
Janakidevi, K,
Fisher MA,
Del Vecchio PJ,
Tirupathi C,
Figge J,
and
Malik AB.
Endothelin-1 stimulates DNA synthesis and proliferation of pulmonary artery smooth muscle cells.
Am J Physiol Cell Physiol
263:
C1295-C1301,
1992
12.
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].
13.
Lal, H,
Williams KI,
and
Woodward B.
Chronic hypoxia differentially alters the responses of pulmonary arteries and veins to endothelin-1 and other agents.
Eur J Pharmacol
371:
11-21,
1999[ISI][Medline].
14.
Le Cras, TD,
Richter G,
Sato K,
Abman SH,
and
McMurtry IF.
Rapid decrease in lung preproendothelin-1 mRNA in rats with chronic hypoxic pulmonary hypertension after brief exposure to normoxia (Abstract).
Am J Respir Cell Mol Biol
157:
A725,
1998.
15.
MacLean, MR.
Endothelin-1: a mediator of pulmonary hypertension?
Pulm Pharmacol Ther
11:
125-132,
1998[ISI][Medline].
16.
Michael, JR,
and
Markewitz BA.
Endothelins and the lungs.
Am J Respir Crit Care Med
154:
555-581,
1996[ISI][Medline].
17.
Naeye, RL.
Pulmonary vascular changes with chronic unilateral hypoxia.
Circ Res
17:
160-167,
1965[ISI].
18.
Resta, TC,
Gonzales RJ,
Dail WG,
Sanders TC,
and
Walker BR.
Selective upregulation of arterial endothelial nitric oxide synthase in pulmonary hypertension.
Am J Physiol Heart Circ Physiol
272:
H888-H896,
1997.
19.
Rodman, DM,
Stelzner TJ,
Zamora MR,
Bonvallet ST,
Oka M,
Sato K,
O'Brien RF,
and
McMurtry IF.
Endothelin-1 increases the pulmonary microvascular pressure and causes pulmonary edema in salt solution but not blood-perfused rat lungs.
J Cardiovasc Pharmacol
20:
658-663,
1992[ISI][Medline].
20.
Ryland, D,
and
Reid L.
The pulmonary circulation in cystic fibrosis.
Thorax
30:
285-292,
1975[Abstract].
21.
Shimada, K,
Takahashi M,
and
Tanzawa K.
Cloning and functional expression of endothelin-converting enzyme from rat endothelial cells.
J Biol Chem
269:
18275-18278,
1994
22.
Soma, S,
Takahashi H,
Muramatsu M,
Oka M,
and
Fukuchi Y.
Localization and distribution of endothelin receptor subtypes in pulmonary vasculature of normal and hypoxia-exposed rats.
Am J Respir Cell Mol Biol
20:
620-630,
1999
23.
Takahashi, M,
Fukuda K,
Shimada K,
Barnes K,
Turner AJ,
Ikeda M,
Koike H,
Yamamoto Y,
and
Tanzawa K.
Localization of rat endothelin-converting enzyme to vascular endothelial cells and some secretory cells.
Biochem J
311:
657-665,
1995[ISI][Medline].
24.
Tchekneva, E,
Quertermous T,
Christman BW,
Lawrence ML,
and
Meyrick B.
Regional variability in prepro-endothelin-1 gene expression in sheep pulmonary artery and lung during the onset of air-induced chronic pulmonary hypertension.
J Clin Invest
101:
1389-1397,
1998
25.
Toga, H,
Ibe BO,
and
Raj JU.
In vitro responses of ovine intrapulmonary arteries and veins to endothelin-1.
Am J Physiol Lung Cell Mol Physiol
263:
L15-L21,
1992
26.
Wagenvoort, CA,
and
Wagenvoort N.
Pulmonary venous changes in chronic hypoxia.
Virchows Arch A Pathol Anat Histol
372:
51-56,
1976[Medline].
27.
Yanagisawa, M,
Kurihara H,
Kimura S,
Tomobe Y,
Kabayashi M,
Mitsui Y,
Yazaki Y,
Goto K,
and
Masaki T.
A novel potent vasoconstrictor peptide produced by vascular endothelial cells.
Nature
332:
411-415,
1988[ISI][Medline].
28.
Zamora, MA,
Dempsey EC,
Walchak SJ,
and
Stelzner TJ.
BQ123, an ETA receptor antagonist, inhibits endothelin-1-mediated proliferation of human pulmonary artery smooth muscle cells.
Am J Respir Cell Mol Biol
9:
429-433,
1993[ISI][Medline].