Upregulation of ET-1 and its receptors and remodeling in small pulmonary veins under hypoxic conditions

Hideki Takahashi, Sanae Soma, Masashi Muramatsu, Masahiko Oka, and Yoshinosuke Fukuchi

Department of Respiratory Medicine, Juntendo University School of Medicine, Bunkyo-Ku, Tokyo 113-8421, Japan


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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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 alpha -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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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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
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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 alpha -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 alpha -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 alpha -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.


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RESULTS
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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 alpha -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 alpha -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 alpha -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).


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Fig. 1.   a-f: representative photomicrographs showing distal segments of pulmonary vessels stained with the elastic van Gieson method (a-d) or immunostained with anti-alpha -smooth muscle (SM) actin antibody (e and f). a and c: pulmonary artery and pulmonary vein, respectively, from a control rat. b: marked vascular remodeling of a pulmonary artery after exposure to hypoxia for 14 days. d: medial hypertrophy of a pulmonary vein from a rat exposed to hypoxia for 14 days. The vein has smooth muscle layers over the entire circumference rather than isolated pads found in veins of similar caliber in control rats. e and f: immunostaining for alpha -SM actin in small pulmonary veins from a normal and a hypoxia-exposed rat, respectively. Counterstaining was with methyl green. Bars, 30 µm. g: time course of the effects of exposure to hypoxia on the grading of alpha -SM actin immunoreactivity in pulmonary veins (PV) with external diameter (ED) of 60-200 µm. Immunostaining for alpha -SM actin increased in the small pulmonary veins after exposure to hypoxia for 7 and 14 days. C, control. * Significant difference in the intensity of immunostaining compared with that of control as determined by semiquantitative analysis.


                              
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Table 1.   Changes in wall thickness and medial thickness of small pulmonary veins after exposure to hypoxia

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).


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Fig. 2.   a-f: representative immunostaining of pulmonary veins with anti-Big endothelin (ET)-1 antibody from a control rat (a and e) and a rat exposed to hypoxia for 14 days (b and f). Faint staining for Big ET-1 was detected in the intima and media of the proximal and distal segments of the pulmonary veins from the control rat (a and e, respectively). Increased immunoreactivity was observed in the vascular wall of the proximal and distal segments of the pulmonary veins from a hypoxia-exposed rat (b and f, respectively). Serial sections of each lung tissue (a and b) were stained with anti-vascular cell adhesion molecule (VCAM)-1 (c and d, respectively). Different patterns of immunostaining suggested that Big ET-1 expression in the pulmonary vein was dominant in the media than in the intima. Counterstaining was with methyl green. Bar, 30 µm. g: time course of the effects of exposure to hypoxia on the grading of Big ET-1 immunoreactivity in pulmonary veins. Grading of Big ET-1 immunoreactivity significantly increased in the media of large and small pulmonary veins after hypoxic conditions of 7 and 14 days. * Significant difference in the intensity of immunostaining compared with that of control as determined by semiquantitative analysis.



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Fig. 3.   Immunostaining with anti-Big ET-1 antibody of a pulmonary artery (PA) and a pulmonary vein from a rat exposed to hypoxia for 14 days. a: predominant expression of Big ET-1 in pulmonary vein compared with that in pulmonary artery. BR, bronchus. b: serial section of lung tissue stained by the elastic van Gieson method. Bar, 600 µm.

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).


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Fig. 4.   a-d: representative photomicrographs of lungs from control and hypoxia-exposed rats stained with anti-ET-converting enzyme (ECE) antibody. a and c: moderate staining in the intima and media of a large and small vein, respectively, in a control rat. b and d: increased immunoreactivity in the media and intima of the proximal and distal segments, respectively, of a pulmonary vein after exposure to hypoxia. Counterstaining was with hematoxylin. Bar, 30 µm. e: time course of the effects of exposure to hypoxia on the grading of ECE immunoreactivity in pulmonary veins. Increased expression of ECE-1 was observed in the media of proximal and distal segments of pulmonary veins after hypoxic conditions of 7 and 14 days. * Significant difference in the intensity of immunostaining compared with that of control as determined by semiquantitative analysis.

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).


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Fig. 5.   a-f: representative photomicrographs showing immunostaining with anti-ETA receptors of pulmonary veins from a control rat (a and c) and a rat exposed to hypoxia for 14 days (b and f). a and b: faint immunoreactivity for ETA receptors in the media of large pulmonary veins from a normal and a hypoxia-exposed rat, respectively. Faint staining found in the intima is considered to be nonspecific due to a technical problem with staining. e: slight immunostaining in a small pulmonary vein from a normal rat. f: increase in the immunoreactivity in a small vein from a rat exposed to hypoxia for 14 days. Serial sections of each lung tissue (a and b) were stained with anti-VCAM-1 (c and d, respectively). Different patterns of immunostaining suggested that ETA receptor expression in the pulmonary vein appears to be localized in the media. Counterstaining was with methyl green. Bar, 30 µm. g: time course of the effects of exposure to hypoxia on the grading of ETA receptor immunoreactivity in pulmonary veins. A significant increase in ETA receptor immunoreactivity was found in the media of pulmonary veins of 60- to 200-µm ED after hypoxic conditions of 7 and 14 days, but there was no significant change in the pulmonary arteries and veins of 500- to 1,000-µm ED. * Significant difference in the intensity of immunostaining compared with that of control as determined by semiquantitative analysis.

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).


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Fig. 6.   Representative photomicrographs showing immunostaining with anti-ETB receptors of pulmonary veins from a control rat (a and c) and a rat exposed to hypoxia for 14 days (b and d). a and b: fairly strong immunoreactivity for ETB receptors in the intima and media of large pulmonary veins from a normal and a hypoxia-exposed rat, respectively. c: slight immunostaining in a small pulmonary vein from a normal rat. d: significant increase in immunoreactivity in a small vein after exposure to hypoxia for 14 days. Counterstaining was with methyl green. Bar, 30 µm. e: time course of the effects of exposure to hypoxia on the grading of ETB receptor immunoreactivity in pulmonary veins. A significant increase in ETB receptor immunoreactivity was observed in the media of pulmonary veins of 60- to 200-µm ED after hypoxic conditions of 7 and 14 days. * Significant difference in the intensity of immunostaining compared with that of control as determined by semiquantitative analysis.

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).


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Fig. 7.   Effects of exposure to hypoxia on mRNAs of ET-1, ECE-1, and ETA and ETB receptors in rat lungs. A: representative Northern blots of ET-1, ECE-1, and ETA and ETB receptors in lung tissue from rats exposed to air and hypoxia for indicated days. A 2.3-kb band for ET-1, 4.4-kb band for ECE-1, 2 bands at 5.2 and 4.3 kb for ETA receptor, and a 5.0-kb band for ETB receptor were detected in control and hypoxia-exposed lungs. B: mRNA quantitation of ET-1, ECE-1, and ETA and ETB receptors by densitometry. Data were normalized to allow for variations in RNA loading with a 28 S rRNA probe; n = 4 animals/group. The mRNAs for ET-1, and ETA and ETB receptors increased 7 and 14 days after exposure to hypoxia. * P < 0.05 vs. control animals.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -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 alpha -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 alpha -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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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].


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