Improvement in fetal pulmonary hypertension and maturity after reversal of ductal constriction: prospective cohort study.

OBJECTIVES: To test the hypotheses that estimated mean pulmonary arterial pressure (MPAP) decreases and pulmonary vascular maturation, assessed by the ratio of pulmonary arterial flow acceleration time to ejection time (AT/ET ratio), increases after reversal of fetal ductus arteriosus constriction by reducing maternal intake of the causal agent (prostaglandin inhibitors, such as polyphenol-rich foods or non-steroidal anti-inflammatory drugs), and that these effects are independent of gestational age, which are inferences not yet demonstrated in the clinical setting. METHODS: This was a prospective cohort study comparing Doppler echocardiographic ductal flow dynamics, MPAP and pulmonary arterial flow AT/ET ratio in third-trimester fetuses (≥ 28 weeks' gestation) with ductus arteriosus constriction, at the time of diagnosis and after 2 weeks of reduced maternal intake of prostaglandin inhibitors either by suspending the use of pharmacological agents with potential for prostaglandin inhibition or by restricting the consumption of polyphenol-rich foods. MPAP was estimated using the Dabestani equation (MPAP = 90 - (0.62 × AT)), and pulmonary vascular maturity was assessed using the AT/ET ratio, according to reported validation studies. Student's t-test was used for comparison of variables at diagnosis with those after reversal of ductal constriction. Change in MPAP and pulmonary AT/ET ratio between the two assessments was compared with the expected change in the same gestational period in normal fetuses based on reference curves of MPAP and pulmonary AT/ET ratio constructed in normal fetuses from healthy pregnant women at 19-37 weeks' gestation, encompassing the same gestational age range as the study group (28-37 weeks). RESULTS: Seventy pregnancies with fetal ductus arteriosus constriction were included in the study. After 2 weeks of reduced maternal intake of prostaglandin inhibitors, normalization of mean systolic (change from 1.86 ± 0.34 m/s at diagnosis to 1.38 ± 0.41 m/s; P < 0.001) and diastolic (change from 0.41 ± 0.11 m/s to 0.21 ± 0.065 m/s; P < 0.001) ductal velocities and of mean pulsatility index (change from 1.99 ± 0.20 to 2.55 ± 0.42; P < 0.001) was demonstrated. MPAP decreased between the assessments (change from 66.7 ± 6.90 mmHg at diagnosis to 54.5 ± 6.70 mmHg after 2 weeks; P < 0.001) and mean pulmonary AT/ET ratio increased (change from 0.20 ± 0.06 to 0.33 ± 0.07; P < 0.001). Change in MPAP between diagnosis and after 2 weeks of reduced maternal intake of prostaglandin inhibitors was -12.2 ± 0.30 mmHg, which was 5.3-times higher than that in 305 normal fetuses over 2 weeks during the same gestational period (-2.3 ± 0.19 mmHg) (P < 0.001), and change in pulmonary AT/ET ratio between the two assessments was 0.13 ± 0.08, which was 8.7-times higher than that in normal fetuses in the same gestational period (0.015 ± 0.08) (P < 0.001). CONCLUSIONS: Resolution of fetal ductal constriction is followed by a fall in MPAP and by an increase in pulmonary vascular maturity, to a significantly greater degree than is observed in normal fetuses in the same gestational-age period. © 2021 International Society of Ultrasound in Obstetrics and Gynecology.

Persistent pulmonary hypertension alters the epigenetic characteristics of endothelial nitric oxide synthase gene in pulmonary artery endothelial cells in a fetal lamb model.

Decreased expression of endothelial nitric oxide synthase (eNOS), a key mediator of perinatal transition, characterizes persistent pulmonary hypertension of the newborn (PPHN) in neonates and a fetal lamb model; the mechanisms are unclear. We investigated whether increased DNA CpG methylation at the eNOS promoter in estrogen response elements (EREs) and altered histone code together contribute to decreased eNOS expression in PPHN. We isolated pulmonary artery endothelial cells (PAEC) from fetal lambs with PPHN induced by prenatal ductus arteriosus constriction from 128 to 136 days gestation or gestation-matched twin controls. We measured right ventricular systolic pressure (RVSP) and Fulton index and determined eNOS expression in PAEC in control and PPHN lambs. We determined DNA CpG methylation by pyrosequencing and activity of ten eleven translocase demethylases (TET) by colorimetric assay. We quantified the occupancy of transcription factors, specificity protein 1 (Sp1), and estrogen receptors and density of four histone marks around Sp1 binding sites by chromatin immunoprecipitation (ChIP) assays. Fetal lambs with PPHN developed increased RVSP and Fulton index. Levels of eNOS mRNA and protein were decreased in PAEC from PPHN lambs. PPHN significantly increased the DNA CpG methylation in eNOS promoter and decreased TET activity in PAEC. PPHN decreased Sp1 occupancy and density of the active mark, lysine 12 acetylation of histone 4, and increased density of the repression mark, lysine 9 trimethylation of histone 3 around Sp1 binding sites in eNOS promoter. These results suggest that epigenetic modifications are primed to decrease Sp1 binding at the eNOS gene promoter in PPHN.

Maternal omega-3 PUFA supplementation prevents hyperoxia-induced pulmonary hypertension in the offspring.

Pulmonary hypertension (PH) and right ventricular hypertrophy (RVH) affect 16-25% of premature infants with bronchopulmonary dysplasia (BPD), contributing significantly to perinatal morbidity and mortality. Omega-3 polyunsaturated fatty acids (PUFA ω-3) can improve vascular remodeling, angiogenesis, and inflammation under pathophysiological conditions. However, the effects of PUFA ω-3 supplementation in BPD-associated PH are unknown. The present study aimed to evaluate the effects of PUFA ω-3 on pulmonary vascular remodeling, angiogenesis, and inflammatory response in a hyperoxia-induced rat model of PH. From embryonic day 15, pregnant Sprague-Dawley rats were supplemented daily with PUFA ω-3, PUFA ω-6, or normal saline (0.2 ml/day). After birth, pups were pooled, assigned as 12 per litter, randomly assigned to either air or continuous oxygen exposure (fraction of inspired oxygen = 85%) for 20 days, and then euthanized for pulmonary hemodynamic and morphometric analysis. We found that PUFA ω-3 supplementation improved survival, decreased right ventricular systolic pressure and RVH caused by hyperoxia, and significantly improved alveolarization, vascular remodeling, and vascular density. PUFA ω-3 supplementation produced a higher level of total ω-3 in lung tissue and breast milk and was found to reverse the reduced levels of VEGFA, VEGF receptor 2, angiopoietin-1 (ANGPT1), endothelial TEK tyrosine kinase, endothelial nitric oxide synthase, and nitric oxide concentrations in lung tissue and the increased ANGPT2 levels in hyperoxia-exposed rats. The beneficial effects of PUFA ω-3 in improving lung injuries were also associated with an inhibition of leukocyte infiltration and reduced expression of the proinflammatory cytokines IL-1ß, IL-6, and TNF-α. These data indicate that maternal PUFA ω-3 supplementation strategies could effectively protect against infant PH induced by hyperoxia.

Downregulated Elastin Microfibril Interfacer 1 Expression in the Pulmonary Vasculature of Experimental Congenital Diaphragmatic Hernia.

AIM: Pulmonary hypertension (PH) is a severe complication of congenital diaphragmatic hernia (CDH). Transforming growth factor-ß (TGFß) signaling is suggested to be involved in PH development by regulating embryonic angiogenesis, cell proliferation, and cell differentiation. Altered TGFß signaling has been demonstrated in experimental CDH lungs. Elastin microfibril interfacer 1 (Emilin-1) is an extracellular matrix glycoprotein expressed in endothelial and vascular smooth muscle cells and known to regulate TGFß processing and arterial diameter. We designed this study to investigate the pulmonary vascular expression of Emilin-1 in nitrofen-induced CDH rats. MATERIALS AND METHODS: Following ethical approval (REC913b, REC1103), time-pregnant Sprague Dawley rats received nitrofen or vehicle on gestational day 9 (D9). Fetuses were sacrificed on D21 and divided into CDH group and control group. Quantitative real-time polymerase chain reaction (n = 11 each group), Western blot analysis, and confocal microscopy were used to determine the gene and protein expression of Emilin-1. MAIN RESULTS: Relative Emilin-1 messenger RNA (ribonucleic acid) levels were significantly downregulated in CDH lung tissue compared with controls (CDH: 0.043 ± 0.003; control: 0.067 ± 0.004; p < 0.001). Western blotting confirmed the decreased pulmonary Emilin-1 protein expression in CDH lungs. Confocal microscopy demonstrated a markedly diminished expression of Emilin-1 in the CDH pulmonary vasculature compared with controls. CONCLUSION: To our knowledge, this study demonstrates for the first time a decreased Emilin-1 gene and protein expression in the pulmonary vasculature of nitrofen-induced CDH. Emilin-1 deficiency through its interaction with TGFß may result in abnormal vascular remodeling resulting in PH in this model.

Increased c-kit and stem cell factor expression in the pulmonary vasculature of nitrofen-induced congenital diaphragmatic hernia.

PURPOSE: Persistent pulmonary hypertension(PPH) in congenital diaphragmatic hernia (CDH) is caused by increased vascular cell proliferation and endothelial cell (EC) dysfunction, thus leading to obstructive changes in the pulmonary vasculature. C-Kit and its ligand, stem cell factor(SCF), are expressed by ECs in the developing lung mesenchyme, suggesting an important role during lung vascular formation. Conversely, absence of c-Kit expression has been demonstrated in ECs of dysplastic alveolar capillaries. We hypothesized that c-Kit and SCF expression is increased in the pulmonary vasculature of nitrofen-induced CDH. METHODS: Timed-pregnant rats received nitrofen or vehicle on gestational day 9(D9). Fetuses were sacrificed on D15, D18, and D21, and divided into control and CDH group. Pulmonary gene expression levels of c-Kit and SCF were analyzed by qRT-PCR. Immunofluorescence double staining for c-Kit and SCF was combined with CD34 to evaluate protein expression in ECs of the pulmonary vasculature. RESULTS: Relative mRNA levels of c-Kit and SCF were significantly increased in lungs of CDH fetuses on D15, D18, and D21 compared to controls. Confocal laser scanning microscopy confirmed markedly increased vascular c-Kit and SCF expression in mesenchymal ECs of CDH lungs on D15, D18, and D21 compared to controls. CONCLUSION: Increased expression of c-Kit and SCF in the pulmonary vasculature of nitrofen-induced CDH lungs suggest that increased c-Kit signaling during lung vascular formation may contribute to vascular remodeling and thus to PPH.

Increased expression of activated pSTAT3 and PIM-1 in the pulmonary vasculature of experimental congenital diaphragmatic hernia.

PURPOSE: Signal transducer and activator of transcription (STAT) protein family (STAT1-6) regulates diverse cellular processes. Recently, the isoform STAT3 has been implicated to play a central role in the pathogenesis of pulmonary hypertension (PH). In human PH activated STAT3 (pSTAT3) was shown to directly trigger expression of the provirus integration site for Moloney murine leukemia virus (Pim-1), which promotes proliferation and resistance to apoptosis in SMCs. We designed this study to investigate the hypothesis that pSTAT3 and Pim-1 pulmonary vascular expression is increased in nitrofen-induced CDH. METHODS: Pregnant rats were exposed to nitrofen or vehicle on D9.5. Fetuses were sacrificed on D21 and divided into nitrofen (n=16) and control group (n=16). QRT-PCR, western blotting, and confocal-immunofluorescence were performed to determine pulmonary gene and protein expression levels of pSTAT3 and Pim-1. RESULTS: Pulmonary Pim-1 gene expression was significantly increased in the CDH group compared to controls. Western blotting and confocal-microscopy confirmed increased pulmonary protein expression of Pim-1 and increased activation of pSTAT3 in CDH lungs compared to controls. CONCLUSION: Markedly increased gene and protein expression of Pim-1 and activated pSTAT3 in the pulmonary vasculature of nitrofen-induced CDH lungs suggest that pSTAT3 and Pim-1 are important mediators of PH in nitrofen-induced CDH.

Antenatal maternally-administered phosphodiesterase type 5 inhibitors normalize eNOS expression in the fetal lamb model of congenital diaphragmatic hernia.

PURPOSE: Pulmonary hypertension (pHTN), a main determinant of survival in congenital diaphragmatic hernia (CDH), results from in utero vascular remodeling. Phosphodiesterase type 5 (PDE5) inhibitors have never been used antenatally to treat pHTN. The purpose of this study is to determine if antenatal PDE5 inhibitors can prevent pHTN in the fetal lamb model of CDH. METHODS: CDH was created in pregnant ewes. Postoperatively, pregnant ewes received oral placebo or tadalafil, a PDE5 inhibitor, until delivery. Near term gestation, lambs underwent resuscitations, and lung tissue was snap frozen for protein analysis. RESULTS: Mean cGMP levels were 0.53±0.11 in placebo-treated fetal lambs and 1.73±0.21 in tadalafil-treated fetal lambs (p=0.002). Normalized expression of eNOS was 82%±12% in Normal-Placebo, 61%±5% in CDH-Placebo, 116%±6% in Normal-Tadalafil, and 86%±8% in CDH-Tadalafil lambs. Normalized expression of ß-sGC was 105%±15% in Normal-Placebo, 82%±3% in CDH-Placebo, 158%±16% in Normal-Tadalafil, and 86%±8% in CDH-Tadalafil lambs. Endothelial NOS and ß-sGC were significantly decreased in CDH (p=0.0007 and 0.01 for eNOS and ß-sGC, respectively), and tadalafil significantly increased eNOS expression (p=0.0002). CONCLUSIONS: PDE5 inhibitors can cross the placental barrier. ß-sGC and eNOS are downregulated in fetal lambs with CDH. Antenatal PDE5 inhibitors normalize eNOS and may prevent in utero vascular remodeling in CDH.

Fetal stenting of the atrial septum: technique and initial results in cardiac lesions with left atrial hypertension.

BACKGROUND: Hypoplastic left heart syndrome with a highly restrictive or intact atrial septum (HLHS-RAS) has a very high mortality. Fetal left atrial (LA) hypertension results in abnormal lung development with lymphangiectasia and pulmonary vein muscularization. We report our initial experience with percutaneous ultrasound-guided stenting of the fetal atrial septum to decompress the LA. METHODS: Retrospective review of fetuses with HLHS-RAS or a variant that underwent active perinatal management from 2000 to 2012. RESULTS: Ten fetuses were identified. Two died in utero (33, 29 weeks). Four required the urgent creation of an atrial communication immediately after birth but died subsequently (5-54 days). Four fetuses (28-36 weeks) underwent percutaneous stenting of the atrial septum, with ultrasound guidance and intravenous maternal sedation. Elevated LA pressure, pulmonary vein dilation and MRI estimated pulmonary perfusion all improved after stenting. Three of four stented fetuses were delivered vaginally. Atrial septectomy was performed within 48 h of delivery to ensure complete LA decompression, rather than for hypoxemia. Intraoperative lung biopsy demonstrated muscularized pulmonary veins and lymphangiectasia in all four. Two fetuses developed stent stenosis in utero and died after birth, from pulmonary hypertension and sepsis respectively. Two are alive, representing an improved outcome over our previous experience (p=0.03). CONCLUSION: Fetal atrial septal stenting is feasible without maternal complications and allows vaginal delivery of a more stable neonate. Fetal LA decompression ameliorates rather than reverses lung injury, and is one component of an approach that may improve survival in HLHS-RAS.

Hipertensión y edema pulmonar de altura: Rol de la disfunción endotelial y de la programación fetal / Pulmonary hypertensión and lung edema at high altitude: Role of endothelial dysfunction and fetal programming

La altura constituye un fascinante laboratorio natural para la investigación médica. Si bien al principio el objetivo de la investigación en la altura fue la comprensión de los mecanismos de adaptación del organismo a la hipoxia y la búsqueda de tratamientos para las enfermedades relacionadas con la altura, durante la última década el alcance de esta investigación se ha ampliado considerablemente. Dos importantes observaciones han generado las bases para el crecimiento del alcance científico de la investigación en la altura. Primero, el hecho de que el edema pulmonar agudo de la altura constituye un modelo único para estudiar los mecanismos fundamentales de la hipertensión pulmonar y el edema pulmonar en humanos. Segundo, que la hipoxia ambiental asociada con la exposición a la altura facilita la detección de disfunción vascular pulmonar y sistémica en un estadio precoz. Aquí revisaremos los estudios que, capitalizando estas observaciones, han llevado a la descripción de nuevos mecanismos subyacentes del edema pulmonar y de la hipertensión pulmonar, y a la primera demostración directa de la existencia de una programación fetal sobre la disfunción vascular en humanos.

High altitude constitutes an exciting natural laboratory for medical research. While initially, the aim of high-altitude research was to understand the adaptation of the organism to hypoxia and find treatments for altitude-related diseases, over the past decade or so, the scope of this research has broadened considerably. Two important observations led to the foundation for the broadening of the scientific scope of high-altitude research. First, high-altitude pulmonary edema (HAPE) represents a unique model which allows studying fundamental mechanisms of pulmonary hypertension and lung edema in humans. Secondly, the ambient hypoxia associated with high-altitude exposure facilitates the detection of pulmonary and systemic vascular dysfunction at an early stage. Here, we review studies that, by capitalizing on these observations, have led to the description of novel mechanisms underpinning lung edema and pulmonary hypertension and to the first direct demonstration of fetal programming of vascular dysfunction in humans.

Nitric oxide activity through guanylate cyclase and phosphodiesterase modulation is impaired in fetal lambs with congenital diaphragmatic hernia.

BACKGROUND: Congenital diaphragmatic hernia (CDH) is associated with pulmonary hypertension and death. Administration of nitric oxide (NO) alone remains ineffective in CDH cases. We investigated in near full-term lambs with and without CDH the role of guanylate cyclase (GC), the enzyme activated by NO in increasing cyclic 3'-5'-guanylosine monophosphate, and the role of phosphodiesterase (PDE) 5, the enzyme-degrading cyclic 3'-5'-guanylosine monophosphate. METHODS: Congenital diaphragmatic hernia was surgically created in fetal lambs at 85 days of gestation. Pulmonary hemodynamics were assessed by means of pressure and blood flow catheters (135 days). In vitro, we tested drugs on rings of isolated pulmonary vessels. RESULTS: In vivo, sodium nitroprusside, a direct NO donor, and methyl-2(4-aminophenyl)-1,2-dihydro-1-oxo-7-(2-pyridinylmethoxy)-4-(3,4,5 trimethoxyphenyl)-3-isoquinoline carboxylate sulfate (T-1032) and Zaprinast, both PDE 5 blockers, reduced pulmonary vascular resistance in CDH and non-CDH animals. The activation of GC by sodium nitroprusside and the inhibition of PDE 5 by T-1032 were less effective in CDH animals. In vitro, the stimulation of GC by 3(5'hydroxymethyl-2'furyl)-1-benzyl indazole (YC-1) (a benzyl indazole derivative) and the inhibition of PDE 5 by T-1032 were less effective in pulmonary vascular rings from CDH animals. The YC-1-induced vasodilation in rings from CDH animals was higher when associated with the PDE 5 inhibitor T-1032. CONCLUSIONS: Guanylate cyclase and PDE 5 play a role in controlling pulmonary vascular tone in fetal lambs with or without CDH. Both enzymes seem to be impaired in fetal lambs with CDH.

Betamethasone attenuates oxidant stress in endothelial cells from fetal lambs with persistent pulmonary hypertension.

We investigated the effects of betamethasone on oxidative stress and impaired vasodilation in a lamb model of persistent pulmonary hypertension (PPHN). We treated pregnant ewes following fetal ductal ligation with betamethasone or saline for 48 h before delivery. Response of fetal pulmonary arteries to nitric oxide synthase (NOS) agonist adenosine triphosphate (ATP) and nitric oxide (NO) donor, s-nitroso-n-acetyl-penicillamine (SNAP) was determined in tissue bath. Pulmonary artery endothelial cells (PAEC) from fetal lambs with ductal ligation or sham ligation were treated with betamethasone or its vehicle for 48 h. Expression of endothelial NOS (eNOS), endothelin, endothelin-B (ET-B) receptor, and CuZn- and Mn-superoxide dismutase (SOD) in PAEC was studied. Intracellular cGMP and superoxide levels and interaction of eNOS with heat shock protein 90 (Hsp90) were determined in PAEC. Antenatal betamethasone improved the relaxation response of pulmonary arteries to ATP and SNAP in PPHN. PPHN was associated with decreases in eNOS and ET-B receptor and increase in prepro-endothelin mRNA levels. Betamethasone decreased prepro-endothelin mRNA and ET-1 pro-peptide levels and increased eNOS and MnSOD protein levels in PPHN. Betamethasone reversed the increased superoxide/decreased cGMP levels and restored Hsp90-eNOS interactions in PPHN. Betamethasone reduces oxidative stress and improves response of pulmonary arteries to vasodilators in lambs with PPHN.

Chronic intrauterine pulmonary hypertension increases capacitative calcium entry in fetal pulmonary artery smooth muscle cells.

Oxygen causes perinatal pulmonary dilatation. Although fetal pulmonary artery smooth muscle cells (PA SMC) normally respond to an acute increase in oxygen (O2) tension with a decrease in cytosolic calcium ([Ca2+]i), an acute increase in O2 tension has no net effect on [Ca(2+)](i) in PA SMC derived from lambs with chronic intrauterine pulmonary hypertension (PHTN). The present experimental series tests the hypothesis that an acute increase in O2 tension decreases capacitative calcium entry (CCE) in normal, but not hypertensive, fetal PA SMC. PA SMC were isolated from late-gestation fetal lambs after either ligation of the ductus arteriosus (PHTN) or sham (control) operation at 127 days gestation. PA SMC were isolated from the distal PA (>or=4th generation) and maintained under hypoxic conditions ( approximately 25 Torr) in primary culture. After fura 2 loading, apparent [Ca2+]i in PA SMC was determined as the ratio of 340- to 380-nm fluorescence intensity. Under both hypoxic and normoxic conditions, cyclopiazonic acid (CPA) increased [Ca2+]i more in PHTN than in control PA SMC. CCE was determined in PA SMC under hypoxic and normoxic conditions, after superfusion with zero extracellular Ca2+ and intracellular store depletion with CPA, followed by superfusion with Ca2+-containing solution, in the presence of the voltage-operated calcium channel blockade. CCE was increased in PHTN compared with control PA SMC under conditions of both acute and sustained normoxia. Transient receptor potential channel gene expression was greater in control compared with PHTN PA SMC. PHTN may compromise perinatal pulmonary vasodilation, in part, by modulating PA SMC CCE.

Intrauterine hypertension decreases lung VEGF expression and VEGF inhibition causes pulmonary hypertension in the ovine fetus.

Although vascular endothelial growth factor (VEGF) plays a vital role in lung vascular growth in the embryo, its role in maintaining endothelial function and modulating vascular structure during late fetal life has not been studied. We hypothesized that impaired lung VEGF signaling causes pulmonary hypertension, endothelial dysfunction, and structural remodeling before birth. To determine whether lung VEGF expression is decreased in an experimental model of persistent pulmonary hypertension of the newborn (PPHN), we measured lung VEGF and VEGF receptor protein content from fetal lambs 7-10 days after ductus arteriosus ligation (132-140 days gestation; term = 147 days). In contrast with the surge in lung VEGF expression during late gestation in controls, chronic intrauterine pulmonary hypertension reduced lung VEGF expression by 78%. To determine whether VEGF inhibition during late gestation causes pulmonary hypertension, we treated fetal lambs with EYE001, an aptamer that specifically inhibits VEGF(165). Compared with vehicle controls, EYE001 treatment elevated pulmonary artery pressure and pulmonary vascular resistance by 22 and 50%, respectively, caused right ventricular hypertrophy, and increased wall thickness of small pulmonary arteries. EYE001 treatment reduced lung endothelial nitric oxide synthase protein content by 50% and preferentially impaired the pulmonary vasodilator response to ACh, an endothelium-dependent agent. We conclude that chronic intrauterine pulmonary hypertension markedly decreases lung VEGF expression and that selective inhibition of VEGF(165) mimics the structural and physiological changes of experimental PPHN. We speculate that hypertension downregulates VEGF expression in the developing lung and that impaired VEGF signaling may contribute to the pathogenesis of PPHN.

Contribution of the K(Ca) channel to membrane potential and O2 sensitivity is decreased in an ovine PPHN model.

Ca2+-sensitive K+ (K(Ca)) channels play an important role in mediating perinatal pulmonary vasodilation. We hypothesized that lung K(Ca) channel function may be decreased in persistent pulmonary hypertension of the newborn (PPHN). To test this hypothesis, pulmonary artery smooth muscle cells (PASMC) were isolated from fetal lambs with severe pulmonary hypertension induced by ligation of the ductus arteriosus in fetal lambs at 125-128 days gestation. Fetal lambs were killed after pulmonary hypertension had been maintained for at least 7 days. Age-matched, sham-operated animals were used as controls. PASMC K+ currents and membrane potentials were recorded using amphotericin B-perforated patch-clamp techniques. The increase in whole cell current normally seen in response to normoxia was decreased (333.9 +/- 63.6% in control vs. 133.1 +/- 16.0% in hypertensive fetuses). The contribution of the K(Ca) channel to the whole cell current was diminished in hypertensive, compared with control, fetal PASMC. In PASMC from hypertensive fetuses, a change from hypoxia to normoxia caused no change in membrane potential compared with a -14.6 +/- 2.8 mV decrease in membrane potential in PASMC from control animals. In PASMC from animals with pulmonary hypertension, 4-aminopyridine (4-AP) caused a larger depolarization than iberiotoxin, whereas in PASMC from control animals, iberiotoxin caused a larger depolarization than 4-AP. These data confirm the hypothesis that the contribution of the K(Ca) channel to membrane potential and O2 sensitivity is decreased in an ovine model of PPHN, and this may contribute to the abnormal perinatal pulmonary vasoreactivity associated with PPHN.

Pulmonary expression of vascular endothelial growth factor and myosin isoforms in rats with congenital diaphragmatic hernia.

Abnormalities of the pulmonary vasculature are well documented in cases of congenital diaphragmatic hernia (CDH). Vascular endothelial growth factor (VEGF), an angiogenic factor, is a recently described endothelial cell-specific growth factor. Myosin heavy chain (MHC) isoforms such as SMemb, SM1 and SM2 are important molecular markers used to study vascular smooth muscle cell differentiation. SMemb is predominantly expressed in immature smooth muscle cells (SMC), and SM2 is expressed in mature SMCs. The authors investigated the expression of VEGF and SMC differentiation in pulmonary vessels in CDH rat lungs and in controls. The lungs of nitrofen-induced CDH rat fetuses (n = 16, gestational age 16, 18, 20, and 22 days) were stained immunohistochemically using antibodies against VEGF, SMemb and SM2, while alpha-actin was used as a general marker of vascular smooth muscle cells. In the CDH group VEGF expression was negative in pulmonary vessels before birth, and in the control group VEGF was positive in smooth muscle cells in vessel walls from 20 days both in vessels at the hilum and in pulmonary parenchyma. In both control and CDH groups, SMemb expression was positive from 16 days' gestation. SM2 expression was negative in vessel walls during the prenatal period in both groups. Alpha-actin was localized in both lungs obtained from control and CDH groups in the lung hilum from 16 days and around peripheral vessels from 18 days. Differences in vascular smooth muscle cell differentiation were not observed between control and CDH lung. These findings suggest that differences in pulmonary vascular development exist between control and CDH rats for VEGF expression, and maturational differences in smooth muscle cell differentiation are not present. This role of altered endothelial cell growth might be related to the different pulmonary vascular reactivity present in CDH lungs.