Endothelin (ET)-1, a potent vasoconstrictor and smooth muscle mitogen, is produced from its precursor, preproET-1, by endothelin-converting enzyme (ECE)-1 activity. ET-1 may bind to two receptors, ETA and ETB, that mediate vasoconstriction and vasodilation in the ovine fetal lung, respectively. ET-1 contributes to high pulmonary vascular resistance in experimental perinatal pulmonary hypertension induced by ligation of the ductus arteriosus in the fetal lamb. Physiological studies in this model have demonstrated enhanced ETA- and diminished ETB-receptor activities and a threefold increase in lung immunoreactive ET-1 protein content. We hypothesized that increased ET production and an imbalance in receptor expression would favor vasoconstriction and smooth muscle cell hypertrophy in pulmonary hypertension and may be partially due to alterations in gene expression. To test this hypothesis, we studied lung mRNA expression of preproET-1, ECE-1, and the ETA and ETB receptors in normal and hypertensive fetal lambs. Total RNA was isolated from whole lung tissue in normal late-gestation fetuses (135 ± 3 days; 147 days = term) and from animals with pulmonary hypertension after ductus arteriosus ligation for 8 days (134 ± 4 days). Ductus arteriosus ligation increased right ventricular hypertrophy [control 0.56 ± 0.02 vs. hypertension 0.85 ± 0.05; right ventricle/(left ventricle + septum); P < 0.05]. Northern blot analysis was performed using cDNA probes and was normalized to the signal for 18S rRNA. We found a 71 ± 24% increase in steady-state preproET-1 mRNA (P < 0.05) and a 62 ± 5% decrease in ETB mRNA (P < 0.05) expression in ductus arteriosus ligation. ECE-1 and ETA-receptor mRNA expression did not change. We conclude that chronic intrauterine pulmonary hypertension after ductus arteriosus ligation increases steady-state preproET-1 mRNA and decreases ETB-receptor mRNA without changing ECE-1 mRNA or ETA-receptor mRNA expression. These findings suggest that increased ET-1 production and decreased ETB-receptor expression may contribute to increased vasoconstrictor tone in this experimental model of neonatal pulmonary hypertension.
- endothelin receptors
- endothelin-converting enzyme
- persistent pulmonary hypertension of the newborn
- pulmonary circulation
persistent pulmonary hypertension of the newborn (PPHN) is a clinical syndrome characterized by elevated pulmonary vascular resistance (PVR), resulting in right-to-left shunting across the foramen ovale and ductus arteriosus with severe hypoxemia (26). Clinical and experimental studies suggest that chronic pulmonary hypertension in utero leads to failure of the normal transition at birth (1, 26, 33, 34). Chronic intrauterine pulmonary hypertension due to ligation of the ductus arteriosus in fetal lambs causes marked elevation of intrauterine pulmonary artery pressure, right ventricular hypertrophy, and hypertensive lung structural changes, as well as abnormal pulmonary vasoreactivity and failure to achieve the normal decline in pulmonary resistance at birth (1, 3, 33, 34). Ligation of the ductus arteriosus in late-gestation fetal lambs has provided an experimental model for studying mechanisms contributing to structural and functional changes associated with perinatal pulmonary hypertension (1, 3, 31, 33).
Past studies of this experimental model of PPHN suggest that high PVR is partly due to structural changes and an imbalance in production or responsiveness to vasodilator and vasoconstrictor stimuli (1, 3, 31,33, 34). Vasodilation in response to agents that act directly on the smooth muscle cell is preserved, as opposed to endothelium-dependent vasodilation and endogenous nitric oxide (NO) activity (31). Endothelial NO synthase activity, protein content, and mRNA content are decreased in this model of pulmonary hypertension (38, 43). Endothelin (ET)-1 is a potent vasoactive peptide with mitogenic effects on vascular smooth muscle and is produced primarily by the vascular endothelium in the normal lung circulation (10, 50). In the normal fetal lung, ET-1 is present and contributes to high PVR (19, 30, 35,46). The effects of ET-1 in the ovine fetal lung are dependent on stimulation of ETA and ETB receptors, which mediate vasoconstriction and vasodilation, respectively (19). We have previously shown that chronic intrauterine pulmonary hypertension causes the loss of ETB-mediated vasodilation, progressive ETA-mediated vasoconstriction, and increased lung ET-1 protein content (21). We have also shown that ETA-receptor stimulation contributes to the pathophysiology of experimental perinatal pulmonary hypertension because chronic intrauterine ETA-receptor blockade with BQ-123 decreases pulmonary artery pressure in utero, decreases right ventricular hypertrophy and distal muscularization of small pulmonary arteries in the lung, and increases the fall in PVR at delivery (20). Endothelin-converting enzyme (ECE) catalyzes the final step in the biosynthesis of the mature ET peptide. However, little is known about the role of ECE-1 in pulmonary hypertension or the exact mechanisms by which ET contributes to high PVR in chronic intrauterine pulmonary hypertension.
We hypothesized that increased ET production and an imbalance in receptor expression would favor vasoconstriction and smooth muscle cell hypertrophy in pulmonary hypertension and may be partially due to alterations in gene expression. Therefore, we studied mRNA expression of preproET-1, ECE-1, and the ETAand ETB receptors in the normal ovine fetal lung and after ductus arteriosus ligation.
All procedures and protocols were previously reviewed and approved by the Animal Care and Use Committee at the University of Colorado Health Sciences Center. Eight mixed-breed (Columbia-Rambouillet) pregnant ewes between 125 and 129 days gestation (term = 147 days) were fasted 24 h before surgery. Ewes were sedated with intravenous pentobarbital sodium (2–4 g) and anesthetized with 1% tetracaine hydrochloride (3 mg) by lumbar puncture. Ewes remained sedated with pentobarbital sodium but breathed spontaneously throughout the surgery. Penicillin (500 mg) and streptomycin (1 g) were administered to the ewe at surgery. Under sterile conditions, the left forelimb of the fetal lamb was delivered through a uterine incision. A skin incision was made under the left forelimb after local infiltration with lidocaine (2–3 ml, 1% solution). A left axillary to sternal thoracotomy exposed the heart and great arteries. In four animals, the ductus arteriosus was partially ligated using umbilical tape (21), and the ductus arteriosus remained intact in the other four animals. The thoracotomy incision was closed in layers. The uteroplacental circulation was kept intact, and the fetus was gently placed in the uterus, with exposed surfaces bathed in warm towels. Ampicillin (500 mg) was added to the amniotic cavity before closure of the hysterotomy. Ampicillin (250 mg) was infused in the fetal vein and amniotic cavity during the first 3 days after surgery. The ewe was allowed to recover. After 8 days, the ewe and fetus were anesthetized with pentobarbital sodium. Pieces of distal whole lung were rapidly frozen in liquid nitrogen. The right ventricle and left ventricle + septum were weighed.
Northern blot analysis.
Total RNA was purified from hypertensive and control fetal lungs using Tri-Reagent (Molecular Research Center, Cincinnati, OH) and the method of Chomczynski and Sacchi (9). The RNA was quantified by measuring the absorbance at 260 nm. Twenty micrograms of total RNA per lung were analyzed using standard Northern blot and hybridization techniques using cDNA probes. Northern analysis was performed on mRNA from each of the four animals in the control and the four animals in the hypertensive group. Ovine preproET-1, human ETA, human ETB, and bovine ECE-1 cDNA probes were labeled with [α-32P]dCTP using random-primed labeling (RTS Random Primer DNA Labeling System; Gibco BRL, Gaithersburg, MD). The 522-bp sheep-specific preproET-1 cDNA probe was kindly provided by Dr. Thomas Quertermous (Vanderbilt Univ. School of Medicine, Nashville, TN) (12). The 2-kb bovine ECE-1 cDNA probe was kindly provided by Dr. Masashi Yanagisawa (Univ. of Texas Southwestern, Dallas, TX) (49). The 1,350-bp human ETA and human ETB cDNA probes were kindly provided by Dr. Scott Magnuson (Abbott Laboratories, Abbott Park, IL). An 18S rRNA oligonucleotide was labeled using terminal deoxytransferase and [α-32P]dCTP. After hybridization, blots were washed at room temperature in 1× SSC (1× SSC is 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0)-0.1% SDS (low stringency) and then at 65°C in 0.4× SSC-0.1% SDS (high stringency). Autoradiographs were obtained by exposure to Hyperfilm-MP (Amersham) at −70°C with intensifying screens, and densitometry was performed using National Institutes of Health (NIH) Image software. Normalization to 18S rRNA levels was used in quantification of mRNA signals.
Data are presented as means ± SE. Statistical analysis was performed with the Statview software package (Abacus Concepts, Berkeley, CA). Statistical comparisons were made using a Student’s unpaired t-test.P < 0.05 was considered significant.
Right ventricular hypertrophy.
Chronic ductus arteriosus ligation has been shown to increase fetal pulmonary artery pressure and PVR without affecting aortic pressure. Right ventricular hypertrophy increased in the present study (Fig. 1) as previously reported (20, 21).
Northern blot analysis.
Expression of mRNA for preproET-1 revealed a single 2.3-kb transcript as previously reported (27, 51). Expression of mRNA for ECE-1 revealed a 4.7- and 3.1-kb transcript as previously described (49). Expression of mRNA for the ETA receptor revealed a 5.2- and 4.2-kb transcript as previously described (27, 51). Expression of mRNA for the ETBreceptor revealed a single 5.0-kb transcript as previously described (27, 51). Intrauterine ductus arteriosus ligation increased preproET-1 mRNA expression by 71 ± 24% (P < 0.05; Fig.2). ECE-1 (Fig.3) and ETA-receptor (Fig.4) gene expression did not change. There was a 62 ± 5% decrease in ETBmRNA expression (P < 0.05; Fig.5) compared with control animals, as determined by Northern blot analysis.
On the basis of previous studies showing that chronic intrauterine pulmonary hypertension causes the loss of ETB-mediated vasodilation, progressive ETA-mediated vasoconstriction, and increased lung ET-1 protein content (21), we hypothesized that an imbalance in expression of preproET-1 and the ET receptors contributes to the hypertensive changes in the lungs after chronic intrauterine pulmonary hypertension. To test this hypothesis, we measured preproET-1, ECE-1, and ETA- and ETB-receptor mRNA in lungs from normal fetal lambs and in an experimental model of perinatal pulmonary hypertension caused by intrauterine ductus arteriosus ligation. Compared with age-matched fetal lambs, chronic pulmonary hypertension in utero increased preproET-1 mRNA expression by 71% and decreased ETB-receptor gene expression 62%, without changing ECE-1 and ETA-receptor gene expression. We conclude that the endothelin system favors increased vasoconstrictor tone and contributes to high PVR in experimental neonatal pulmonary hypertension.
These findings are interesting because little is known about the role of ET-1 and its receptors in development of perinatal pulmonary hypertension. Past studies of an experimental model of PPHN suggest that high PVR is partly due to structural changes and an imbalance in production of and responsiveness to vasodilator and vasoconstrictor stimuli (1, 3, 31, 33, 34). Endothelium-dependent vasodilation and endogenous NO activity are impaired in the hypertensive fetal lung (31, 38, 43). Several studies suggest that ET-1 may contribute to vasoconstriction and altered vasoreactivity in experimental PPHN (6, 7, 22). An imbalance in the NO-cGMP system and ET-1 system may also contribute to pulmonary hypertensive states. The decrease in NO production may lead to increased ET-1 production (25). Increased ET-1 activity also stimulates smooth muscle proliferation, which may further increase PVR (52).
Blockade of ETA-receptor activity attenuates and may reverse the development of pulmonary hypertension and right ventricular hypertrophy in adult rat models of pulmonary hypertension caused by chronic hypoxia or monocrotaline (4, 8, 11, 13,32). We have shown that ETA-receptor stimulation contributes to the pathophysiology of experimental perinatal pulmonary hypertension, as chronic intrauterine ETA-receptor blockade with BQ-123 decreases pulmonary artery pressure in utero, decreases right ventricular hypertrophy and distal muscularization of small pulmonary arteries in the lung, and increases the fall in PVR at delivery (20). Furthermore, adult animal models of pulmonary hypertension demonstrate increased expression of ETA-receptor mRNA (27) and decreased expression of ETB-receptor mRNA (51). Diminished ETB-receptor binding has also been found in hypoxia-induced pulmonary hypertension in the newborn piglet (16). Increased production of ET-1 has also been shown in adult rat pulmonary hypertension models (40, 51). We have previously shown that pulmonary hypertension due to ductus arteriosus ligation increases ET-1 production and leads to diminished ETB-mediated vasodilation and enhanced ETA-mediated vasoconstriction, suggesting that increased ET-1 and changes in its receptor activity at least in part mediate the altered reactivity in this model of pulmonary hypertension (21). The present study significantly extends these observations and demonstrates that an imbalance in gene expression of preproET-1 and the ETA and ETB receptors contributes to the hypertensive lung changes in this model of pulmonary hypertension.
ET-1 is present in the normal perinatal lung (30) and is vasoactive in the fetus (7, 18, 19, 22, 41). Brief infusion of ET-1 causes potent vasodilation acutely (6, 29, 47, 48); however, with prolonged infusion, hypertension prevails (7, 28). Evidence suggests that ET-1 acts as a local autocrine and paracrine factor rather than as a circulating hormone, since secretion of ET-1 by endothelial cells is polar and directed abluminally toward the interstitial region (45). Blockade of the ETA receptors causes vasodilation (19, 47), whereas selective blockade of the ETB1 (vasodilation) or ETB2 (vasoconstriction) receptors does not change basal pulmonary tone in the normal ovine fetus (21). However, brief and prolonged stimulation of the ETB receptors with sarafotoxin S6c causes only vasodilation, suggesting the presence of only ETB1 receptors in the normal ovine fetal lung (19). In contrast, studies in newborn piglets suggest the presence of both ETB1(vasodilation) and ETB2(vasoconstriction) receptors in the neonatal lung (36).
Little is known about the role of ECE-1 in pulmonary hypertension. Two isoenzymes of ECE have been described: ECE-1 and ECE-2 (14, 17, 49). ECE-1 is associated with the plasma membrane and has optimum activity at neutral pH, whereas ECE-2 is intracellular and active at an acidic pH (14). ECE-1 has two isoforms: ECE-1α and ECE-1β (39, 42). No functional differences between the ECE-1α and ECE-1β isoforms have been identified. The cDNA used in the present study encodes for both ECE-1α and ECE-1β (49). Although mRNA expression for ECE-1 does not change with chronic intrauterine pulmonary hypertension, ECE-1 activity and protein content have not been studied and may be increased. Furthermore, future studies should involve cell-specific localization of ET-1, ECE-1, and the ET receptors because pulmonary hypertension may cause local changes in components of the ET system that may not be revealed by studying total lung expression of mRNA or protein.
ET-1 levels are increased in many human disorders associated with pulmonary hypertension. Elevated immunoreactive ET-1 levels have been found in primary pulmonary hypertension, Eisenmenger syndrome (5), PPHN (37), children with pulmonary hypertension associated with congenital heart disease and bronchopulmonary dysplasia (2), and children with congenital heart disease and increased pulmonary blood flow (24, 44). Increased expression of ET-1 in vascular endothelial cells has been reported in adult patients with primary pulmonary hypertension, suggesting that the local production of endothelin-1 may contribute to the altered vascular reactivity and structural changes seen in pulmonary hypertension (15). Recently, acute infusion of the combined ETA- and ETB-receptor antagonist bosentan has been shown to lower PVR and increase cardiac output in congestive heart failure and pulmonary hypertension (23). However, the role of ET-1 in clinical disorders of pulmonary hypertension such as PPHN remains incompletely understood.
In summary, chronic intrauterine pulmonary hypertension caused by ligation of the ductus arteriosus increases steady-state preproET-1 mRNA expression while decreasing ETB-receptor gene expression. Expression of the mRNA for the ETAreceptor and ECE-1 does not change in this model of pulmonary hypertension. We speculate that an imbalance of ET-1 production and ET-receptor expression contributes to the pathophysiological changes in perinatal pulmonary hypertension by favoring increased vasoconstriction and smooth muscle proliferation.
We thank Dr. Thomas Quertermous (Vanderbilt Univ. School of Medicine, Nashville, TN), Dr. Scott Magnuson (Abbott Laboratories, Abbott Park, IL), and Dr. Masashi Yanagisawa (Univ. of Texas Southwestern, Dallas, TX) for kind gifts of cDNA probes.
Address for reprint requests: D. D. Ivy, Dept. of Cardiology, Box B100, The Children’s Hospital, 1056 E. 19th Ave., Denver, CO 80218.
This work was supported by The Children’s Hospital Research Institute Career Development Award (to D. D. Ivy); National Center for Research Resources General Clinical Research Centers Program Grant M01-RR-00069; National Heart, Lung, and Blood Institute Grants HL-41012 and HL-46481 (to S. H. Abman); the March of Dimes Birth Defects Foundation (D. D. Ivy); the Bugher Physician-Scientist Training Program (D. D. Ivy); and the American Heart Association Established Investigator Award (to S. H. Abman).
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