Main

Pulmonary vascular tone in the perinatal period is regulated, in part, by substances produced by vascular endothelial cells (1–3). The most potent of these substances are NO and ET-1 (4,5). NO is synthesized from L-arginine by the enzyme ENOS (4). NO diffuses into vascular smooth muscle cells and activates SGC, increasing the production of cGMP. cGMP initiates a cascade that results in vascular smooth muscle relaxation and vasodilation. These effects are limited when cGMP is degraded by specific cGMP PDE, one of which, PDEV, is expressed in the lung (6).

ET-1 is produced as PreproET-1, a 203-amino acid peptide, which is cleaved into big ET-1, a 38-amino acid peptide. Big ET-1 is cleaved into its functional form (ET-1) by ECE-1 (7). Although ET-1 produces systemic vasoconstriction, its effects on the pulmonary circulation vary with age and vascular tone (8–10). These hemodynamic effects are mediated through two receptor subtypes: ETA receptors and ETB receptors (11,12). ETA receptors and a very small subpopulation of ETB receptors are located on vascular smooth muscle cells and are responsible for the vasoconstricting effects of ET-1 (11,12). A second very large subpopulation of ETB receptors are located on vascular endothelial cells and are responsible for the vasodilating effects of ET-1, that are mediated by NO production (13).

With the initiation of ventilation and oxygenation of birth, pulmonary vascular resistance decreases and pulmonary blood flow increases (2). There is evidence that increased ENOS gene expression, ENOS activity, and NO production contribute to these changes (1,3,14–16). However, in a number of clinical conditions, there is failure of the pulmonary circulation to undergo this normal transition to postnatal life, resulting in PPHN (2,17). The role of the NO-cGMP and ET-1 pathways in preventing the normal decrease in pulmonary vascular resistance in these newborns is not well understood.

Prolonged compression or ligation of the ductus arteriosus in utero in the lamb produces fetal and neonatal pulmonary hypertension (18–20). Similar to newborns who die with PPHN, these lambs have an increase in the thickness of the smooth muscle of the small pulmonary arteries, complete muscularization of normally partially muscularized pulmonary arteries, and extension of muscle to nonmuscularized arteries (17). These lambs demonstrate altered hemodynamic responses to and changes in the production and/or concentration of NO and cGMP. They have impaired endothelium-dependent, but not endothelium-independent pulmonary vasodilation (21–23). Although inhaled NO produces pulmonary vasodilation in these lambs, there is impaired relaxation to substances that stimulate NO production and SGC activity (including NO) in isolated fetal pulmonary artery rings (24). There is also a decrease in the expression of ENOS mRNA and protein in lung tissue (15,16). These lambs also demonstrate loss of ETB receptor-mediated pulmonary vasodilation, increased lung ET-1 content, and increased ETA receptor-mediated pulmonary vasoconstriction (18). We hypothesize that fetal pulmonary hypertension after ligation of the ductus arteriosus in utero alters the expression of genes of the NO-cGMP and ET-1 pathways in the lung which could help explain these changes.

cDNA fragments for ovine ENOS, PDEV, PreproET-1, ECE-1, and the ETA and ETB receptors were cloned and used in RNase protection assays. Antisera were raised against ENOS and the ETA receptor. Western blot analyses were performed, using these antisera and antisera raised against either the α1 or the β1 subunits of SGC. Our results confirm that fetal pulmonary hypertension induced by ligation of the ductus arteriosus in utero in lambs is associated with decreased expression of ENOS mRNA and protein in lung tissue (15,16). We report that there is also decreased expression of the α1 and the β1 subunits of SGC protein and increased expression of PDEV mRNA. These changes would limit NO and cGMP production and/or concentration, thereby decreasing pulmonary vasodilator activity. Simultaneously, there is increased expression of PreproET-1 mRNA and decreased expression of ETB receptor mRNA. These changes would increase ET-1 concentration and limit ETB receptor activation, thereby increasing pulmonary vasoconstrictor activity. This coordinated regulation in gene expression would increase fetal pulmonary vascular resistance, which could then contribute to the development of pulmonary hypertension after birth.

METHODS Surgical preparation. All procedures and protocols were approved by the Committee on Animal Research of the University of California, San Francisco. Seven mixed-breed Western pregnant ewes (123-126-d gestation, term = 145 d) were operated on under sterile conditions using local anesthesia (lidocaine hydrochloride, 2%), intramuscular sedation (ketamine hydrochloride, 15 mg/kg), and atropine, 2 mg), and i.v. anesthesia (ketamine hydrochloride, 0.3 mg kg-1 min-1, and diazepam, 0.002 mg kg-1 min-1). A venous catheter was placed in the maternal hind limb for the administration of intraoperative anesthesia and antibiotics.

A laparotomy was performed. Through a small uterine incision, a fetal hind limb was exposed. Lidocaine hydrochloride, 1%, was used for local anesthesia. Polyvinyl catheters were inserted into the pedal artery and vein and advanced to the descending aorta and inferior vena cava, respectively. The fetus was then anesthesized with an i.v. injection of ketamine hydrochloride (25 mg). To prevent spontaneous movement and to facilitate manipulation of the fetus, an i.v. injection of succinylcholine chloride (5 mg) was given. The fetal skin and uterine incisions were closed in layers.

Through a separate uterine incision, the fetal left hemithorax was exposed. A left lateral thoracotomy was performed in the fourth intercostal space. The heart and great vessels were exposed. The ductus arteriosus was visualized and ligated with umbilical tape (umbilical tape #10-A, Ethicon, Inc., Somerville, NJ) (19,20). The fetal skin incision was closed in layers. Amniotic fluid losses were replaced with warm (39°C) 0.9% saline. Penicillin G potassium (1 000 000 IU) and gentamicin sulfate (100 mg) were injected i.v. into the ewe and into the amniotic fluid. Daily, thereafter, these antibiotics were given by intramuscular injection to the ewe.

At 138-139-d gestation, 12-16 d (mean ± SEM = 13.6 ± 0.5 d) after ligation of the ductus arteriosus in utero, the ewe and fetuses were killed with an i.v. injection of pentobarbital sodium (Euthanasia CII, Central City Medical, Union City, CA) followed by bilateral thoracotomy. An autopsy was performed; the fetal lungs were removed and prepared for RNase protection assays and Western blot analysis. The unoperated twin fetal lamb was used as the control. A sham operation was not performed on control fetal lambs as this would increase mortality and require additional ewes and fetuses to complete the study. To document that there was rapid recovery of cardiovascular and hormonal function, and hemodynamic stability in this preparation, eight other fetal lambs (123-126-d gestation) were similarly operated on as described above. In addition, these fetal lambs were instrumented to measure vascular pressures and left pulmonary blood flow, and studied sequentially for 12-16 d (to 138-139-d gestation). After ligation of the ductus arteriosus in utero, mean pulmonary arterial pressure increased from 60.3 ± 5.3 to 77.1 ± 9.1 mm Hg (p < 0.05, mean ± SD). There were no changes in left pulmonary blood flow (40.0 ± 24.7 versus 67.9 ± 51.2 mL/min), mean descending aortic pressure (53.5 ± 8.0 versus 56.5 ± 5.6 mm Hg), descending aortic PO2 (21.9 ± 4.3 versus 21.0 ± 2.1 mm Hg), descending aortic PCO2 (55.3 ± 6.1 versus 56.5 ± 7.0 mm Hg), descending aortic pH (7.34 ± 0.32 versus 7.32 ± 0.04), or in plasma norepinephrine concentration (592.2 ± 347.8 versus 325.0 ± 226.5 pg/mL). These data confirm the stability of the preparation (25) and constancy of the physiologic function after ligation of the ductus arteriosus in utero, as we have seen (26). Preliminary studies also showed no change in expression of genes of the NO-cGMP or ET-1 pathways in the ovine fetal lung after thoracotomy.

Tissue preparation. To prepare total RNA, fresh lung tissue was snap-frozen in liquid nitrogen and stored at -70°C until used. The snap-frozen lung tissue was pulverized, then briefly homogenized in 4 M guanidium isothiocyanate. Total RNA was extracted with acid phenol and precipitated in isopropanol (14). To prepare protein, fresh lung tissue was rinsed in cold (4°C) 0.9% saline to remove blood, minced, and then homogenized, using a Tissuemizer (twice, 15 s each, at 80% power), in 4 volumes/wet weight of Triton lysis buffer [20 mM Tris-HCl (pH 7.6), 0.5% Triton X-100, and 20% glycerol] supplemented with protease inhibitors. Extracts were then centrifuged at 15 000 × g for 15 min. The supernatant was then removed for protein determination and Western blot analysis (14).

Generation of ovine cDNAs. Total ovine fetal lung RNA was used in reverse transcription-PCR reactions (kit from Perkin-Elmer, Foster City, CA). The generated cDNA fragment of interest (see below) was cloned directly into the pCR II vector (Invitrogen, San Diego, CA), sequenced (Sequenase kit from U. S. Biochemical Corp., Cleveland, OH), and then subcloned into pBluescript KS+ (Stratagene, La Jolla, CA).

Endothelial nitric oxide synthase. A region within the hemed binding domain was identified as region of minimal homology between the three isoforms of nitric oxide synthase (14). Oligonucleotides were synthesized (using the bovine ENOS as a template) to allow amplification of this region within the ovine ENOS sequence. The sequences of the oligonucleotides were 5′-CCTCCGGAGGGGCCCAAGTTCCCTCGC-3′ for oligonucleotide 1 and 5′-CACGTCGAAGCGCCGTTTCCGGGGGT-3′ for oligonucleotide 2. The region amplified (681 bp) corresponds to amino acids 62-288 of the ENOS protein. When sequenced, the cDNA fragment was found to be 96.6% identical to the bovine ENOS cDNA. The ovine ENOS cDNA fragment was digested with StuI. When used in RNase protection assays, the ENOS-protected fragment was 353 bp.

PDEV. A region within the amino-terminal end of bovine PDEV was identified as a region of minimal homology with other phosphodiesterases (6). Oligonucleotides were synthesized (using the bovine PDEV as a template) to allow amplification of this region within the ovine PDEV sequence. The sequences of the oligonucleotides were 5′-GACGATCACTGGGACTTTAC-3′ for oligonucleotide 1 and 5′-CCCCTTCATCATTATAAAC-3′ for oligonucleotide 2. The region amplified (337 bp) corresponds to amino acids 25-137 of the PDEV protein. When sequenced, the cDNA fragment was found to be 99.7% identical to the bovine PDEV cDNA. When used in RNase protection assays, the PDEV-protected fragment was 337 bp.

PreproET-1. Oligonucleotides were synthesized using the 522-bp cDNA fragment of ovine PreproET-1 as a template (27). The sequences of the oligonucleotides were 5′-CCTGAATTCCTCTGCTGTTTGTGGCTT-3′ for oligonucleotide 1 and 5′-GAAGAATTCGCTGTTGCTGATGGCCTC-3′ for oligonucleotide 2. The ovine PreproET-1 cDNA fragment was digested with Eco47III. When used in RNase protection assays, the PreproET-1 protected fragment was 242 bp.

ECE-1. Oligonucleotides were synthesized using the bovine ECE-1 cDNA as a template (28). The sequences of the oligonucleotides were 5′-AGCTCCATCTTGAGTTGAGTTCCATG-3′ for oligonucleotide 1 and 5′-CTGGAAGTTGTCCTTGTCCCA-3′ for oligonucleotide 2. The region amplified (338 bp) corresponds to amino acids 97-209 of the ECE-1 protein. When sequenced, the cDNA fragment was found to be 93.5% identical to the bovine ECE-1 cDNA. When used in RNase protection assays, the ECE-1-protected fragment was 338 bp.

ET receptors. Regions of minimal homology between the rat ETA and ETB receptors were identified (11,12). Oligonucleotides were synthesized (using the rat ET receptors as a template) to allow amplification of these regions within the ovine ETA and ETB receptor sequences. The sequences of the oligonucleotides for the ETA receptor were 5′-TATCTACGTGGTCATTGATCTCCCCAT-3′ for oligonucleotide 1 and 5′-CTTGTATTCGAAGGGTACCATGACGAA-3′ for nucleotide 2. The region amplified (309 bp) corresponds to amino acids 127-232 of the ETA receptor protein. When sequenced, the cDNA fragment was found to be 96.1% identical to the rat ETA receptor cDNA. The ovine ETA receptor cDNA fragment was digested with BamHI. When used in RNase protection assays, the ETA receptor protected fragment was 222 bp. The sequences of the oligonucleotides for ETB receptor were 5′-CTACACATCATCATCGACATTCCCATT-3′ for oligonucleotide 1 and 5′-TTTGATGTCCGACGTAATCACATCAAA-3′ for oligonucleotide 2. The region amplified (291 bp) corresponds to amino acids 148-247 of the ETB receptor protein. When sequenced, the cDNA fragment was found to be 99.3% identical to the rat ETB receptor cDNA. When used in RNase protection assays, the ETB receptor protected fragment was 291 bp.

Generation of ovine antisera. The ENOS cDNA fragment was subcloned in frame into the pET23a expression vector to overexpress the corresponding ENOS protein fragment (14) (Novagen, Madison, WI). After confirming the reading frame at both the 5′- and 3′-ends, the pET23a clone was transformed into the bacterial strain BL21(DE3)plys S, which contains a lysogen of T7 bacteriophage and a plasmid-encoding lysozyme to reduce constitutive expression of the T7 RNA polymerase. Cultures (1 L) were grown from a single colony under ampicillin selection until the OD600 reached ∼0.6; isopropylthiogalactoside was then added (final concentration 0.4 mM). After 3 h, the cells were pelleted, resuspended in 0.1 volume of imidazole buffer, and sonicated to disrupt the cell membranes and shear chromosomal DNA. The lysate was cleared by centrifugation, passed over a Ni2+ column, washed, and eluted with 1 M imidazole. The eluted fraction was concentrated by passage through a concentrator with the addition of sterile distilled water to reduce the imidazole concentration. The resultant partially purified ENOS protein fragment was then injected into New Zealand White rabbits (Animal Pharm. Services, Healdsberg, CA) to produce a polyclonal ENOS antiserum. Using the same procedures, a polyclonal ETA receptor antiserum was also generated.

RNA probe synthesis and RNase protection Assay. The plasmid containing the cDNA fragment of interest was linearized with the appropriate restriction enzyme (GIBCO-BRL, Grand Island, NY). Antisense [32P]UTP radiolabeled cRNA probes (New England Nuclear, Boston, MA) were synthesized by in vitro transcription using either T3 or T7 RNA polymerases (Boehringer Mannheim, Indianapolis, IN) in the presence of cold rCTP, rGTP, and rATP (14).

RNase protection assays were performed as previously described (14). Antisense radiolabeled cRNA probes were hybridized overnight at 42°C with total ovine fetal lung RNA isolated from ovine fetal lung (50 µg) in 80% formamide, 50 mM 1,4-piperazinediethanesulfonic acid (pH 6.4), 0.4 M NaCl, and 1 mM EDTA. Single-stranded RNA was digested for 1 h at 37°C with an RNase A/T1 mixture (Ambion, Austin, TX). After phenol/CHCl3 extraction and ethanol precipitation, the protected fragments were analyzed by electrophoresis on a 6% denaturing polyacrylamide gel. Also included was a probe for 18 S to serve as a control for the amount of input total RNA and the recovery of protected fragments.

Western blot analysis. Western blot analysis was performed as previously described (14). Protein extracts (100 µg) were separated on a 6% denaturing polyacrylamide gel and electrophoretically transferred to Hybond-polyvinylidene difluoride membranes (Amersham, Arlington Heights, IL). The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline containing 0.1% Tween. After blocking, the membranes were incubated at room temperature with the appropriate dilution of the antiserum of interest [1:1,000 for ENOS, 1:1,000 for the α1 subunit of SGC, 1:10,000 for the β1 subunit of SGC (antisera for the α1 subunit and for the β1 subunit of SGC were gifts from Peter Yuen, Ph.D.), 1:100 for PECAM (a gift from Steven M. Albelda, M.D.), or 1:2,000 for the ETA receptor], washed with Tris-buffered saline containing 0.1% Tween, and then incubated with a mouse anti-rabbit IgG-horseradish peroxidase conjugate. After washing, chemiluminescence was used to detect the protein bands. The specificity of each antisera was assessed by Western blot analysis on protein extracts prepared from a variety of tissues (31–33) (data not shown).

Data analysis. Quantitation of autoradiographic results were performed by scanning (SCA Jet IICX, Hewlett Packard Inc., Palo Alto, CA) the bands of interest into an image editing software program (Adobe Photoshop, Adobe Systems, Mt. View, CA). Band intensities from RNase protection assays and Western blot analysis were analyzed densitometrically on a Macintosh computer (model 9500, Apple Computer, Inc., Cupertino, CA) using the public domain National Institutes of Health Image Program (developed at the National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-image). For RNase protection assays, to control for the amount of input RNA and the recovery of protected fragments, the mRNA signal of interest was normalized to the corresponding 18 S signal for each lane. Results from control fetal lamb lungs were assigned the value of 1 (relative mRNA of interest). For Western blot analysis, to assure equal protein loading, duplicate polyacrylamide gels were run. One was stained with Coomassie Blue. Results from control fetal lamb lungs were assigned the value of 1 (relative protein of interest). The means ± SEM were calculated for the relative mRNA and the relative protein of interest from control and ductal ligation fetal lamb lungs, and were compared by the unpaired t test. A p < 0.05 was considered statistically significant.

RESULTS

There were changes in expression of genes of the No-cGMP pathway after ligation of the ductus arteriosus in utero. Compared with the normal near-term ovine fetal lung, the expression of ENOS mRNA and protein decreased by 30% after ligation of the ductus arteriosus in utero. (p < 0.05) (Fig. 1). This decrease in ENOS gene expression was not due to a decrease in pulmonary vessel number because there was no change in the protein expression of PECAM, an endothelial cell-specific marker, after ductal ligation (Fig. 2). The expression of the α1 and the β1 subunits of soluble guanylate cyclase protein decreased by 60 and 70%, respectively (p < 0.05) (Fig. 3). The expression of PDEV mRNA increased by 70% (p < 0.05) (Fig. 4).

Figure 1
figure 1

RNase protection assay (panels A and B) and Western blot analysis (panels C and D) for endothelial nitric oxide synthase (ENOS) in lung tissue from near-term fetal lambs: control and after ligation of the ductus arteriosus in utero (ductal ligation). (A) A cRNA probe for ovine ENOS was hybridized overnight to 50 µg of total lung RNA prepared from four near-term fetal lambs (two control and two ductal ligation). This cRNA probe was also hybridized without RNA (PA) and in the presence of tRNA, ENOS is an undigested probe. A cRNA for ovine 18 S was hybridized to serve as a control for RNA loading. (B) The densitometric values for relative ENOS mRNA (normalized to 18 S mRNA and to control) from seven control and seven ductal ligation near-term fetal lambs. Values are mean ± SEM. *p < 0.05, control vs ductal ligation). (C) Protein extracts (100 µg), prepared from lung tissue from four near-term fetal lambs (two control and two ductal ligation), were separated on a 6% denaturing polyacrylamide gel, electrophoretically transferred to Hybond membranes, and analyzed using a specific antiserum raised against ENOS. (D) The densitometric values for relative ENOS protein (normalized to control) from seven control and seven ductal ligation near-term fetal lambs. Values are mean ± SEM. *p < 0.05 control vs ductal ligation.

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Figure 2
figure 2

Western blot analysis for the PECAM (panels A and B) in lung tissue from near-term fetal lambs: control and after ligation of the ductus arteriosus in utero (ductal ligation). (A) Protein extracts (100 µg), prepared from lung tissue from two near-term fetal lambs (one control and one ductal ligation), were separated on a 6% denaturing polyacrylamide gel, electrophoretically transferred to Hybond membranes, and analyzed using a specific antiserum raised against PECAM protein. (B) The densitometric values for relative PECAM protein (normalized to control) from five control and five ductal ligation near-term fetal lambs. Values are mean ± SEM.

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Figure 3
figure 3

Western blot analyses for the α1 subunit (panels A and B) and the β1 subunit (panels C and D) of SGC protein in lung tissue from near-term fetal lambs: control and after ligation of the ductus arteriosus in utero (ductal ligation). (A) Protein extracts (100 µg), prepared from lung tissue from six near-term fetal lambs (three control and three ductal ligation), were separated on a 6% denaturing polyacrylamide gel, electrophoretically transferred to Hybond membranes, and analyzed using a specific antiserum raised against the α1 SGC protein. (B) The densitometric values for relative α1 SGC protein (normalized to control) from six control and six ductal ligation near-term fetal lambs. Values are mean ± SEM. *p < 0.05 control vs ductal ligation. (C) Protein extracts (100 µg), prepared from lung tissue from six near-term fetal lambs (three control and three ductal ligation), were separated on a 6% denaturing polyacrylamide gel, electrophoretically transferred to Hybond membranes, and analyzed using a specific antiserum raised against the β1 SGC protein. (D) The densitometric values for relative β1 SGC protein (normalized to control) from four control and four ductal ligation near-term fetal lambs. Values are mean ± SEM. *p < 0.05, control vs ductal ligation.

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Figure 4
figure 4

RNase protection assay for PDEV in lung tissue from near-term fetal lambs: control and after ligation of the ductus arteriosus in utero (ductal ligation). (A) A cRNA probe for ovine PDEV was hybridized overnight to 50 µg of total lung RNA prepared from two near-term fetal lambs (one control and one ductal ligation). This cRNA probe was also hybridized without RNA (PA) and in the presence of tRNA. PDEV is an undigested probe. A cRNA for ovine 18 S was hybridized to serve as a control for RNA loading. (B) The densitometric values for relative PDEV mRNA (normalized to 18 S mRNA and to control) from seven control and six ductal ligation near-term fetal lambs. Values are mean ± SEM. *p < 0.05 control vs ductal ligation.

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There were also changes in expression of genes of the ET-1 pathway after ligation of the ductus arteriosus in utero. Compared with the normal near-tern ovine fetal lung, the expression of PreproET-1 increased by 50% after ligation of the ductus arteriosus in utero (p < 0.05) (Fig. 5) without a change in the expression of ECE-1 mRNA (Fig. 6). The expression of ETB receptor mRNA decreased by 50% (p < 0.05) (Fig. 7) without changes in the expression of ETA receptor mRNA or protein (Fig. 8).

Figure 5
figure 5

RNase protection assay for PreproET-1 in lung tissue from near-term fetal lambs: control and after ligation of the ductus arteriosus in utero (ductal ligation). (A) A cRNA probe for ovine PreproET-1 was hybridized overnight to 50 µg of total lung RNA prepared from two near-term fetal lambs (one control and one ductal ligation). This cRNA probe was also hybridized without RNA (PA) and in the presence of tRNA. PreproET-1 is an undigested probe. A cRNA for ovine 18 S was hybridized to serve as a control for RNA loading. (B) The densitometric values for relative PreproET-1 mRNA (normalized to 18 S mRNA and to control) from seven control and seven ductal ligation near-term fetal lambs. Values are mean ± SEM. *p < 0.05, control vs ductal ligation.

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Figure 6
figure 6

RNase protection assay for ECE-1 in lung tissue from near-term fetal lambs: control and after ligation of the ductus arteriosus in utero (ductal ligation). (A) A cRNA probe for ovine ECE-1 was hybridized overnight to 50 µg of total lung RNA prepared from two near-term fetal lambs (one control and one ductal ligation). This cRNA probe was also hybridized without RNA (PA) and in the presence of tRNA. ECE-1 is an undigested probe. A cRNA for ovine 18 S was hybridized to serve as a control for RNA loading. (B) The densitometric values for relative ECE-1 mRNA (normalized to 18 S mRNA and to control) from five control and five ductal ligation near-term fetal lambs. Values are mean ± SEM.

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Figure 7
figure 7

RNase protection assay for the ETB receptor in lung tissue from near-term fetal lambs: control and after ligation of the ductus arteriosus in utero (ductal ligation). (A) A cRNA probe for the ovine ETB receptor was hybridized overnight to 50 µg of total lung RNA prepared from six near-term fetal lambs (three control and three ductal ligation). This cRNA probe was also hybridized without RNA (PA) and in the presence of tRNA. ETB receptor is an undigested probe. A cRNA for ovine 18 S was hybridized to serve as a control for RNA loading. (B) The densitometric values for relative ETB receptor mRNA (normalized to 18 S mRNA and to control) from seven control and seven ductal ligation near-term fetal lambs. Values are mean ± SEM. *p < 0.05 control vs ductal ligation.

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Figure 8
figure 8

RNase protection assay (panels A and B) and Western blot analysis (panels C and D) for the ETA receptor in lung tissue from near-term fetal lambs: control and after ligation of the ductus arteriosus in utero (ductal ligation). (A) A cRNA probe for the ovine ETA receptor was hybridized overnight to 50 µg of total lung RNA prepared from six near-term fetal lambs (three control and three ductal ligation). This cRNA probe was also hybridized without RNA (PA) and in the presence of tRNA. ETA receptor is an undigested probe. A cRNA for ovine 18 S was hybridized to serve as a control for RNA loading. (B) The densitometric values for relative ETA receptor mRNA (normalized to 18 S mRNA and to control) from seven control and five ductal ligation near-term fetal lambs. Values are mean ± SEM. (C) Protein extracts (100 µg), prepared from lung tissue from eight near-term fetal lambs (four control and four ductal ligation). were separated on a 6% denaturing polyacrylamide gel, electrophoretically transferred to Hybond membranes, and analyzed using a specific antiserum raised against the ETA receptor protein. (D) The densitometric values for relative ETA receptor protein (normalized to control) from four control and four ductal ligation near-term fetal lambs. Values are mean ± SEM.

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DISCUSSION

The purpose of this study was to evaluate whether fetal pulmonary hypertension alters the expression of genes of the NO-cGMP and ET-1 pathways in the lung, which could help explain the hemodynamic changes and the alterations in the production or concentration of NO, cGMP, and ET-1 observed after ligation of the ductus arteriosus in utero in lambs (19–21). Our results confirm that ligation of the ductus arteriosus in utero in lambs is associated with decreased lung expression of ENOS mRNA and protein. We report that there is also decreased expression of the α1 and the β1 subunits of soluble guanylate cyclase protein and increased expression of PDEV mRNA. Together, these changes would result in decreased production and/or concentration of NO and cGMP. Simultaneously, ligation of the ductus arteriosus in utero is associated with increased expression of PreproET-1 mRNA and decreased expression of ETB receptor mRNA. Together, these changes would result in increased production of ET-1 and decreased ETB receptor-mediated pulmonary vasodilation. Thus, ligation of the ductus arteriosus in utero induces coordinated regulation of genes of the NO-cGMP and ET-1 pathways which could result in decreased pulmonary vasodilator activity and increased pulmonary vasoconstrictor activity. These changes in gene expression could increase fetal pulmonary vascular resistance and lead to the failure of the fetal pulmonary circulation to undergo the normal transition to postnatal life with the initiation of ventilation and oxygenation at birth.

Impairment of NO and cGMP generation has been demonstrated in isolated pulmonary arteries, as well as in intact animals and humans with pulmonary hypertension (22,24,29–32). Rats exposed to either monocrotaline or chronic hypoxia have impairment of endothelium-dependent pulmonary vasodilation (29,30). Similarly, after ligation or constriction of the ductus arteriosus in utero, newborn lambs demonstrate impairment of endothelium-dependent pulmonary vasodilation, reduced pulmonary arterial sensitivity to NO, and decreased pulmonary arterial cGMP production (22,24). ENOS gene expression in these lungs is also decreased (15,16). In addition, there is impairment of endothelium-dependent pulmonary vasodilation in children with congenital heart defects and pulmonary hypertension (31). Finally, adults with severe pulmonary hypertension have impairment of endothelium-dependent pulmonary vasodilation and decreased ENOS gene expression in the lung (32,33). The results of our study show that fetal pulmonary hypertension not only decreases ENOS gene expression in the lung, but also alters the expression of other genes of the NO-cGMP pathway. Our results suggest that there is coordinated regulation of the genes of the NO-cGMP pathway in the lung, decreasing NO production and SGC activity, while increasing cGMP degradation, and leading to decreased cGMP content in the lung. These changes in gene expression would increase fetal pulmonary vascular resistance and lead to failure of pulmonary vasodilation at birth.

Alterations in ET-1 generation and ET-1-induced vasoactive responses have also been demonstrated in isolated pulmonary arteries, as well as in intact animals and humans with pulmonary hypertension (18,34–37). Rats exposed to either monocrotaline or chronic hypoxia have increased lung ET-1 content, and increased lung and pulmonary arterial expression of ET-1, ETA, and ETB mRNAs (35,36). Fetal lambs after ligation or compression of the ductus arteriosus in utero, and 4-wk-old lambs with chronic pulmonary hypertension have increased ET-1 content, loss of ETB receptor-mediated pulmonary vasodilation, and increased ETA receptor-mediated pulmonary vasoconstriction (18). In addition, plasma ET-1 concentrations are also increased in newborns with PPHN (38). Finally, adults with severe pulmonary hypertension secondary to a variety of conditions have increased pulmonary arterial endothelial cell ET-1 immunoreactivity and increased ET-1 mRNA (37). The results of our study show that fetal pulmonary hypertension increases the expression of ET-1 mRNA and decreases the expression of ETB receptor mRNA, without changing the expression of ECE-1 mRNA or ETA receptor mRNA or protein in the lung. These results suggest that there is coordinated regulation of the genes of the ET-1 pathway in the lung, increasing ET-1 content and decreasing ETB receptor-mediated pulmonary vasodilation. These changes would lead to increased activation of the ETA receptor, resulting in pulmonary vasoconstriction and increased fetal pulmonary vascular resistance. The latter would contribute to the development of pulmonary hypertension after birth.

As descriptions of the NO-cGMP and ET-1 pathways have evolved, it has been suggested that NO and ET-1 may each participate in the regulation of the other through an autocrine negative feedback loop (39). The ETB receptor may play a critical role in this feedback loop because it is involved in both pathways. The ETB receptor has a high affinity for ET-1, and ET-1/ETB receptor complexes are not easily dissociated (40). Therefore, ETB receptor signaling is dependent upon the number of ETB receptors. When the ETB receptor is highly expressed (as in the lung of the normal fetal lamb), ET-1 activates the ETB receptor, which stimulates ENOS activity and NO production, producing vascular smooth muscle relaxation and pulmonary vasodilation (5,41). In addition, the NO that is produced further inhibits ET-1 production and gene expression, enhancing pulmonary vasodilation (42). When the ETB receptor is down-regulated (as in the lung of the fetal lamb after ligation of the ductus arteriosus in utero), ET-1 activates the ETA receptor, producing pulmonary vasoconstriction. In addition, the ET-1 that is produced further decreases the expression of ETB receptor mRNA, enhancing pulmonary vasoconstriction (43).

Our study shows that in fetal pulmonary hypertension, produced by ligation of the ductus arteriosus in utero in the lamb, there is a coordinated regulation of genes of the NO-cGMP and ET-1 pathways. Fetal pulmonary hypertension decreased the expression of ENOS mRNA and protein, and the α1 and the β1 subunits of SGC protein. Because the expression of PDEV mRNA was increased, these changes would lead to decreased production and/or concentration of NO and cGMP. Simultaneously, fetal pulmonary hypertension increased the expression of ET-1 mRNA and decreased the expression of ETB receptor mRNA. Together, these changes would shift the balance toward substances or stimuli producing pulmonary vasoconstriction rather than pulmonary vasodilation and would help explain the failure of the pulmonary circulation to undergo vasodilation with the initiation of ventilation and oxygenation at birth. A better understanding of the mechanisms responsible for these changes in gene expression and their effect on the pulmonary circulation may lead to new prevention and improved treatment strategies for pulmonary hypertension after birth.