Endothelin receptor blockade is an emerging therapy for pulmonary hypertension. However, hemodynamic and structural effects and potential changes in endogenous nitric oxide (NO)-cGMP and endothelin-1 signaling of chronic endothelin A receptor blockade in pulmonary hypertension secondary to congenital heart disease are unknown. Therefore, the objectives of this study were to determine hemodynamic and structural effects and potential changes in endogenous NO-cGMP and endothelin-1 signaling of chronic endothelin A receptor blockade in a lamb model of increased pulmonary blood flow following in utero placement of an aortopulmonary shunt. Immediately after spontaneous birth, shunt lambs were treated lifelong with either an endothelin A receptor antagonist (PD-156707) or placebo. At 4 wk of age, PD-156707-treated shunt lambs (n = 6) had lower pulmonary vascular resistance and right atrial pressure than placebo-treated shunt lambs (n = 8, P < 0.05). Smooth muscle thickness or arterial number per unit area was not different between the two groups. However, the number of alveolar profiles per unit area was increased in the PD-156707-treated shunt lambs (190.7 ± 5.6 vs. 132.9 ± 10.0, P < 0.05). Plasma endothelin-1 and cGMP levels and lung NOS activity, cGMP, eNOS, preproendothelin-1, endothelin-converting enzyme-1, endothelin A, and endothelin B receptor protein levels were similar in both groups. We conclude that chronic endothelin A receptor blockade attenuates the progression of pulmonary hypertension and augments alveolar growth in lambs with increased pulmonary blood flow.
- pulmonary heart disease
- congenital heart defect
altered pulmonary vascular reactivity and pulmonary hypertension are a common accompaniment of congenital heart disease with increased pulmonary blood flow (16). Normal pulmonary vascular tone and vascular smooth muscle cell proliferation is regulated by a complex interaction of vasoactive substances that are locally produced by the vascular endothelium, such as nitric oxide (NO) and endothelin-1 (ET-1) (7, 9, 28). Endothelial injury secondary to increased pulmonary blood flow and/or pressure disrupts these regulatory mechanisms and is a potential factor in the development of pulmonary hypertension secondary to congenital heart disease (10, 11).
ET-1 is a 21-amino acid polypeptide produced by vascular endothelial cells that has potent vasoactive properties and is comitogenic for vascular smooth muscle cells (14, 28). Recent data suggest a role for ET-1 in the pathophysiology of pulmonary hypertension. For example, ET-1 concentrations are increased in children with increased pulmonary blood flow and pulmonary hypertension, and prepro-ET-1 gene expression is increased in adults with advanced pulmonary vascular disease (11, 25, 29). In addition, chronic endothelin receptor blockade has demonstrated clinical improvement in adults with advanced pulmonary vascular disease (24). However, the early role of ET-1 in the pathophysiology of pulmonary hypertension secondary to congenital heart disease with increased pulmonary blood flow remains unclear (8). In addition, the potential effects of chronic ET receptor blockade on endogenous endothelial signaling are unknown.
To better define the role of ET-1 in the pathogenesis of pulmonary hypertension, we established a model of pulmonary hypertension with increased pulmonary blood flow in the lamb following in utero placement of an aorta-to-pulmonary vascular graft. At 1 mo of age, these lambs (shunt lambs) have a pulmonary-to-systemic blood flow ratio of ∼2:1, a mean pulmonary arterial pressure that is 50% of mean systemic arterial pressure, and pulmonary vascular remodeling characteristic of children with pulmonary hypertension and increased pulmonary blood flow (20). Previously, we demonstrated that shunt lambs display alterations in the ET-1 cascade at 4 wk of age. These include increased plasma ET-1 levels, decreased endothelin B (ETB) receptor protein with loss of ETB receptor-mediated vasodilation, and increased endothelin A (ETA) receptor protein with augmentation of ETA receptor-mediated vasoconstriction, suggesting a role for ET-1 in the pathophysiology of pulmonary hypertension secondary to increased pulmonary blood flow (1, 27).
The objectives of this study were to determine 1) the role of ETA receptor activity in the early progression of pulmonary hypertension secondary to congenital heart disease with increased pulmonary blood flow and 2) the potential effects of ET receptor blockade on endothelial signaling. Therefore, we studied the effects of chronically administered PD-156707 (an ETA receptor antagonist) or placebo in our lamb model of congenital heart disease. Four weeks after spontaneous delivery of the lambs, hemodynamic variables, ET-1-induced vasoactive responses, and pulmonary artery morphology were determined and compared. In addition, we determined potential alterations in the endogenous NO-cGMP and ET-1 pathways induced by chronic ETA receptor blockade by comparing plasma ET-1, plasma and lung tissue NO/nitrate/nitrite (NOx) and cGMP levels, tissue nitric oxide synthase (NOS) activity, and tissue endothelial NOS, prepro-ET-1, endothelin-converting enzyme-1 (ECE-1), and ETA and ETB receptor protein levels in lambs with and without ET receptor blockade.
Surgical Preparation and Care
All procedures were approved by the Committee on Animal Research of the University of California, San Francisco.
Before spontaneous delivery, the lambs were randomly divided into a treatment and a placebo group. The treatment group (n = 12) received PD-156707, an ETA receptor antagonist diluted in water (150 mg·kg−1·day−1). The placebo group (n = 8) received daily water. All treatments were started within 12 h of spontaneous delivery and continued for 4 wk. The lambs were weighed daily, and the respiratory rate and heart rates were obtained weekly by an animal care nurse who was blinded to the therapy. Furosemide (1 mg/kg im) was administered twice daily. At 4 wk of age, vascular pressures and flow was measured as previously described (1, 27).
After baseline measurements of the hemodynamic variables were obtained, ET-1 (250 μg/kg) was injected into the pulmonary artery over 1 min. The hemodynamic variables were measured continuously.
Blocks of tissue of the left lung were removed and stored for later Western blot analysis. Additionally, in five of the PD-156707-treated shunt lambs and five of the placebo-treated shunt lambs, the pulmonary arterial bed of the right lung was distended with a barium gelatin suspension, and the lungs were inflated by way of the trachea with formalin and placed in a bath of formalin for fixation.
Six random blocks for routine light microscopic examination were taken from each left lung. The sections were then examined for the characteristic structural changes of chronic pulmonary hypertension by the use of well-established quantitative techniques (19, 21). Briefly, the 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 by the calculation: percent medial thickness = 2 × medial thickness/external diameter × 100. The structure of each artery was also noted (muscular, partially muscular, 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 intraacinar arteries was also assessed. Using a ×25 objective and an eyepiece reticule, we counted the number of barium-filled arteries of <200 μm external diameter and related that to the number of alveolar profiles in these same fields. Lastly, the number of alveolar per unit area was counted. At least 25 consecutive microscopic fields were counted for each animal.
Plasma and Tissue Determinations
Blood samples were assayed for immunoreactive ET-1 as previously described (27).
Blood samples and tissue were assayed with a cGMP [125I] assay kit (Amersham International) according to the manufacturer's instructions (4).
Utilizing a vanadium(III) and hydrochloric acid reduction, we detected total NO, nitrite, and NOx by chemiluminescence (NOA 280; Sievers Instruments, Boulder, CO) in blood samples and lung tissue (2).
NOS activity in the lung tissue was determined by the conversion of [3H]arginine to [3H]citrulline as previously described (5).
Western Blot Analysis
Western blot analysis (ETA and ETB receptor, prepro-ET-1, ECE-1, endothelial NOS) was performed as previously described (1).
The means ± SD was calculated for the hemodynamic variables, systemic arterial blood gases, pH and ET-1 levels, structural changes, cGMP, NOx, and NOS activity. Comparisons were made by the paired t-test using the Bonferroni correction, the unpaired t-test, or ANOVA for repeated measures with multiple-comparison testing.
We performed quantitation of autoradiographic results by scanning the bands of interest into an image editing software program (Adobe Photoshop; Adobe Systems, Mountain View, CA). The means ± SD was calculated for the relative protein. Protein and structural data were compared by the unpaired t-test. A P < 0.05 was considered statistically significant.
Spontaneous delivery occurred 2–19 days after fetal surgery. Six of the PD-156707-treated shunt lambs died between days 1 and 21 of life. Two lambs died from presumed sepsis, one from necrotic enterocolitis, and one suddenly from unknown causes. Two additional lambs were killed because of respiratory distress following an aspiration event. All of the placebo-treated shunt lambs survived the 4-wk study period. In general, we observed that the lambs in the treatment group were clinically sicker, with increased work of breathing and decreased activity level. However, this could not be quantified, and there were no differences in body weight, respiratory rate, or heart rate between the two groups (data not shown). A total of 14 lambs (n = 6 treated and n = 8 placebo) completed the 4-wk study period and underwent the hemodynamic study and biochemical and morphometric analysis.
All lambs had an audible continuous murmur and an increase in oxygen saturation between the right ventricle and distal pulmonary artery. The baseline systemic arterial Pco2, Po2, and hemoglobin were similar between the two groups and within the normal limits of the laboratory. There were no differences in systemic arterial pH between the two groups (7.46 ± 0.04 vs. 7.52 ± 0.06); however, both were greater than historical controls for the laboratory (1, 27). Left pulmonary vascular resistance, mean right atrial pressure, and systemic pulse pressure were decreased in PD-156707-treated shunt lambs compared with placebo-treated shunt lambs (P < 0.05, Table 1). All other hemodynamic variables measured were not different between the two groups. However, mean pulmonary arterial pressure in PD-156707-treated shunt lambs was similar to values previously noted in age-matched control lambs (1, 27).
In placebo-treated shunt lambs, the intrapulmonary injection of ET-1 increased left pulmonary vascular resistance (18.5 ± 26.0%) and mean systemic arterial pressure (9.4 ± 5.0%, P < 0.05). However, in all PD-156707-treated shunt lambs both the pulmonary and systemic vasoconstriction to ET-1 was blocked.
Biochemical and Protein Determinations
Lung tissue NOx levels, plasma and lung tissue cGMP levels, plasma ET-1 (P = 0.06), and tissue NOS activity were all similar between the two groups. Plasma NOx levels were decreased in PD-156707-treated shunt lambs compared with placebo-treated shunt lambs (P < 0.05, Table 2). Protein levels of eNOS, prepro-ET-1, ECE-1, and ETA and ETB receptors were are similar between the two groups (Table 3).
Morphometric analysis revealed no differences in the percent medial thickness of pulmonary arteries between the two groups (Fig. 1). However, the presence of muscular arteries in the intraacinar region at the alveolar duct and alveolar wall levels was significantly increased in the PD-156707-treated shunt lambs compared with the placebo-treated shunt lambs (Table 4). In addition, although there was no difference in the number of barium-filled small arteries per unit area between the two groups (3.8 ± 0.2, n = 5 vs. 4.0 ± 0.3, n = 5), the number of alveolar profiles per unit area was increased by 43.5% in PD-156707-treated shunt lambs (P < 0.05, Fig. 2). This led to a significant reduction in the number of small barium-filled arteries per 100 alveolar profiles in the PD-156707-treated shunt lambs (2.1 ± 0.3 vs. 2.9 ± 0.3 arteries/100 alveoli, P = 0.002).
Activation of the ET-1 cascade has been demonstrated in a variety of pulmonary hypertensive disorders, including children and animals with congenital heart disease and increased pulmonary blood flow. The present study is the first to demonstrate that chronic ET receptor blockade lowers pulmonary vascular resistance in a model of increased pulmonary blood flow secondary to congenital heart disease, suggesting a role for ET-1 in the early pathophysiology of this disorder. After in utero aortopulmonary graft placement in the lamb, we found that chronic postnatal ETA receptor blockade lowered pulmonary vascular resistance and right atrial pressure without affecting basal endogenous NO-cGMP or ET-1 signaling. We also demonstrate the novel finding that ETA receptor blockade amplified alveolar growth, suggesting a role for ET-1 in the regulation of postnatal alveolar development in this setting.
Although chronic ET receptor blockade has demonstrated symptomatic improvement in adults with advanced primary pulmonary hypertension, this would not be expected in the setting of increased pulmonary blood flow (24). Because it is well established that pulmonary vasodilator therapy in the setting of a left-to-right shunt worsens congestive heart failure and mortality, the objective of this study was to utilize PD-156707 as a pharmacological tool to further delineate the role of ET-1 in the pathophysiology of this disorder, rather than a potential therapy in this setting (26). As expected, PD-156707-treated shunt lambs had increased work of breathing, decreased activity, and a greater mortality (50% vs. 0%, P < 0.05) than placebo-treated shunt lambs. Postmortem investigation of the deaths suggested that two occurred from aspiration events, one from an intrauterine infection, one from a postnatal urachus infection, and one sudden death of unclear etiology. In an attempt to prevent the increased symptomatology of increased pulmonary blood flow, both groups of lambs were treated with furosemide twice a day, beginning on the first day of life. It is noteworthy that both PD-156707- and placebo-treated shunt lambs had lower pulmonary arterial pressure, higher systemic arterial pH, and less vascular remodeling than historical shunt controls, which received furosemide only on an intermittent basis in response to increased symptomatology (20). The effects of the aggressive diuretic regimen, in addition to the high mortality of the treatment group were unavoidable, but significant, limitations of the current study. We cannot exclude the possibility that the high mortality selected out lambs with differential hemodynamics and vascular morphology and that a shorter treatment course with a less aggressive diuretic regimen may have demonstrated greater differences between the PD-156707- and placebo-treated shunt lambs. Additionally, we also cannot exclude the possibility that the 50% mortality has introduced other unknown serious biases to the study. Further studies are needed to better delineate these potential factors.
In previous animal models of pulmonary hypertension, chronic ET receptor blockade attenuated the progression of vascular smooth muscle remodeling. For example in rats exposed to either hypoxia or monocrotaline, ET receptor blockade resulted in decreased medial thickness of the small pulmonary arteries compared with controls (6, 15). Similarly, in postnatal piglets with increased pulmonary blood flow, ET receptor blockade prevented the increase in medial thickness of the small pulmonary arteries (23). However, ETA receptor blockade did not prevent pulmonary vascular remodeling in a rat model of myocardial infarction (18). In the present study, PD-156707-treated shunt lambs displayed no differences in percent medial thickness of the pulmonary arteries compared with controls. In fact, the abnormal presence of muscularized pulmonary arteries in the region of the alveolar duct and wall was greater in PD-156707-treated shunt lambs (Table 4). These surprising findings are not completely understood, but a few potential explanations are noteworthy. First, as previously mentioned, the degree of vascular remodeling was not generally significant in either the PD-156707-treated shunt lambs or the placebo-treated shunt lambs, and much less than historical shunt controls, which is likely due to the aggressive diuretic regimen utilized in this study. Second, the high mortality in the PD-156707-treated shunt lambs may have selected for a group of survivors with more vascular remodeling, which could limit pulmonary blood flow and clinical symptomatology. Lastly and most importantly, this study is unique in that the abnormal hemodynamic state was present from birth, and treatment was started immediately following birth. Because the early morphological abnormalities in children with increased pulmonary blood flow are similar to the normal fetal vascular morphology, it is likely that the early vascular changes represent a failure of the fetal morphological state to regress rather that remodeling. Therefore, the abnormal extension of muscle present in the PD-156707-treated shunt lambs may represent a failure of the normal regression of fetal morphology and may suggest a role for ET-1 in this process.
Previously, we demonstrated that increased pulmonary blood flow induces significant alterations in the endogenous ET-1 and NO-cGMP cascades (1, 3, 27). In the present study, we demonstrate that chronic ETA receptor blockade does not significantly alter these changes. For example, there were no differences in plasma ET-1 levels, plasma and tissue cGMP levels, NOS activity, and protein levels of eNOS, prepro-ET-1, ECE-1, and ETA and ETB receptors. Plasma NOx levels were increased in PD-156707-treated shunt lambs. However, plasma NOx levels may be altered by differences in extracellular volume and renal clearance (30). Because lung tissue NOx levels were unchanged, differences in plasma NOx are unlikely to represent differences in basal pulmonary NO production.
To achieve ETA receptor blockade, treated lambs received 150 mg·kg−1·day−1 of PD-156707, an orally active, selective ETA receptor antagonist (22). The dose of PD-156707 was chosen after several previous studies showed that this dose completely blocked the vasoconstricting effects of exogenous ET-1 and resulted in steady-state plasma concentrations that blocked ETA receptors in vivo (17). To insure adequate ETA receptor blockade with PD-156707 in this study, we studied the effect of exogenous ET-1 in both groups of lambs. Similar to our previous studies, ET-1 induced both pulmonary and systemic vasoconstriction in vehicle-treated lambs (27). However, in all PD-156707-treated lambs, the vasoconstricting effect of ET-1 was blocked, suggesting adequate ETA receptor blockade. Interestingly, ET-1 did not elicit pulmonary vasodilation. These physiological data in conjunction with the protein data demonstrating no change in ETB receptor protein suggest that ETB receptors were not upregulated during ETA receptor blockade.
Lastly, this study demonstrates the novel finding that chronic ETA receptor blockade increases alveolar and, presumably, capillary growth in lambs with increased pulmonary blood flow. As seen in Fig. 2, the number of alveolar-capillary profiles per unit area was increased by 44% in PD-156707-treated shunt lambs (P < 0.05). Although ET-1 is known to have mitogenic actions on cardiovascular tissues and is comitogenic with a variety of growth factors and vasoactive substances, there is very limited data on the role of ET-1 in the developing lung (12). However, ET-1 is expressed in the airway epithelium of the perinatal mouse, suggesting a role for ET-1 in lung development (13). In the current study, we have uncovered a previously unknown role for ET-1 in postnatal lung development during the stimulus of increased pulmonary blood flow. These novel observations suggest that ET-1 may play an important role in regulating lung growth in both normal and pathological situations. Although age-matched control lambs with lower pulmonary arterial pressures have similar alveolar growth as vehicle-treated shunt lambs (20), our results cannot rule out the possibility that the effect on alveolarization in the ET antagonist group was not secondary to some indirect, hemodynamic effect. Therefore, further investigations will be required to elucidate the role of ET-1 in regulating lung development. It is likely that such studies will yield important biological and clinically relevant information.
This research was supported by National Heart, Lung, and Blood Institute Grants HL-61284 (J. R. Fineman), HL-48536 (B. Meyrick), HL-60190, and HL-67841 (S. M. Black) and Deutsche Herzstiftung Grant S/05/01 (S. Fratz).
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- Copyright © 2004 the American Physiological Society