Fetal Pulmonary Veins Research Articles

Effect of long-term high-altitude hypoxia on fetal pulmonary vascular contractility

Hypoxia in the fetus and/or newborn is associated with an increased risk of pulmonary hypertension. The present study tested the hypothesis that long-term high-altitude hypoxemia differentially regulates contractility of fetal pulmonary arteries (PA) and veins (PV) mediated by differences in endothelial NO synthase (eNOS). PA and PV were isolated from near-term fetuses of pregnant ewes maintained at sea level (300 m) or high altitude of 3,801 m for 110 days (arterial Po(2) of 60 Torr). Hypoxia had no effect on the medial wall thickness of pulmonary vessels and did not alter KCl-induced contractions. In PA, hypoxia significantly increased norepinephrine (NE)-induced contractions, which were not affected by eNOS inhibitor N(G)-nitro-l-arginine (l-NNA). In PV, hypoxia had no effect on NE-induced contractions in the absence of l-NNA. l-NNA significantly increased NE-induced contractions in both control and hypoxic PV. In the presence of l-NNA, NE-induced contractions of PV were significantly decreased in hypoxic lambs compared with normoxic animals. Acetylcholine caused relaxations of PV but not PA, and hypoxia significantly decreased both pD(2) and the maximal response of acetylcholine-induced relaxation in PV. Additionally, hypoxia significantly decreased the maximal response of sodium nitroprusside-induced relaxations of both PA and PV. eNOS was detected in the endothelium of both PA and PV, and eNOS protein levels were significantly higher in PV than in PA in normoxic lambs. Hypoxia had no significant effect on eNOS levels in either PA or PV. The results demonstrate heterogeneity of fetal pulmonary arteries and veins in response to long-term high-altitude hypoxia and suggest a likely common mechanism downstream of NO in fetal pulmonary vessel response to chronic hypoxia in utero.

P45.13: Imaging of fetal coronary arteries and pulmonary veins with gray and color flow Doppler sonography

P45.13: Imaging of fetal coronary arteries and pulmonary veins with gray and color flow Doppler sonography

Regulation of cGMP-dependent protein kinase-mediated vasodilation by hypoxia-induced reactive species in ovine fetal pulmonary veins

We previously reported that hypoxia attenuates cGMP-dependent protein kinase (PKG)-mediated relaxation in pulmonary vessels (Am J Physiol Lung Cell Mol Physiol 279: L611-L618, 2003). To determine whether hypoxia-induced reactive oxygen and nitrogen species (ROS and RNS, respectively) may be involved in the downregulation of PKG-mediated relaxation, ovine fetal intrapulmonary veins were exposed to 4 h of normoxia or hypoxia, with or without scavengers of ROS [N-acetylcysteine (NAC)] or peroxynitrite (quercetin and Trolox) and preconstricted with endothelin-1. Hypoxia decreased the relaxation response to 8-bromo-cGMP, PKG protein expression, and kinase activity and increased tyrosine nitration in PKG. However, ROS and RNS scavengers prevented these changes. To determine whether increased PKG nitration diminishes PKG activity, pulmonary vein smooth muscle cells (PVSMC) were exposed to shorter-term (30 min) hypoxia, which increased PKG nitration and decreased PKG activity but did not alter PKG protein expression. Increased dihydro-2,7-dichlorofluorescein diacetate (DCFH(2)-DA) fluorescence in PVSMC after 4 h or 30 min of hypoxia was not observed in the presence of NAC, quercetin, or Trolox, suggesting increased ROS and RNS production. Increased PKG nitration and the associated decrease in PKG activity in PVSMC after 30 min of hypoxia were also reversed on reoxygenation. The consequences of PKG nitration were assessed by exposure of purified PKG-Ialpha to peroxynitrite, which caused increased 3-nitrotyrosine immunoreactivity and inhibition of kinase activity. Our data suggest that, after 30 min of hypoxia, reversible covalent modification of PKG by hypoxia-induced reactive species may be an important mechanism by which the relaxation response to cGMP is regulated. However, after 4 h of hypoxia, PKG nitration and decreased PKG expression are involved.

Effect of chronic hypoxia on fetal pulmonary vascular contractility

The present study tested the hypothesis that chronic hypoxia differentially regulates contractility of fetal pulmonary arteries and veins. Fourth generation pulmonary arteries and veins were isolated from near-term fetuses of normoxic control and chronically hypoxic (exposure to high altitude 3,801m for 110 days, PaO2: 60 Torr) pregnant ewes. Chronic hypoxia did not significantly change KCl-induced contractions. In pulmonary arteries, chronic hypoxia significantly enhanced NE-induced contractions, increasing the maximal response from 104.6±2.6 to 136.4±4.0 %Kmax (P<0.05). Inhibition of eNOS with L-NNA showed no effect on NE-induced contractions in either control or hypoxic arteries. In pulmonary veins, chronic hypoxia had no effect on NE-induced contractions in the absence of L-NNA. L-NNA significantly increased NE-induced contractions in control vessels, and this effect was significantly reduced in hypoxic vessels. In the presence of L-NNA, chronic hypoxia significantly decreased NE-induced contractions of pulmonary veins (217.1±10.1 vs 149.7±10.1 %Kmax, P<0.05). In addition, chronic hypoxia significantly decreased SNP-induced relaxations of both arteries and veins. The results suggest that chronic hypoxia increases NE-induced contractions in fetal pulmonary arteries that are not regulated by NO. In contrast, chronic hypoxia decreases both NE-induced contractions and NO-mediated relaxations in fetal pulmonary veins. Furthermore, chronic hypoxia decreases smooth muscle sensitivity to NO-induced relaxations in both pulmonary arteries and veins. (Supported in part by NIH grant HD31226)

Hypoxia Down-Regulates Cyclic Guanidine Monophosphate-Dependent Protein Kinase in Fetal Pulmonary Vascular Smooth Muscle Cell Through Generation of Reactive Oxygen Species and Promotes Development of Pulmonary Hypertension

We have previously reported that cyclic guanidine monophosphate (cGMP) mediates vasodilation primarily through protein kinase G (PKG) in fetal pulmonary vessels 1 Dhanakoti SN Gao Y Nguyen MQ et al. Involvement of cGMP-dependent protein kinase in the relaxation of ovine pulmonary arteries to cGMP and cAMP. J Appl Physiol. 2000; 88: 1637-1642 PubMed Google Scholar and that acute hypoxia down-regulates cGMP-PKG-mediated relaxation in these vessels. 2 Gao Y Dhanakoti SN Trevino EM et al. Effect of oxygen on cyclic GMP-dependent protein kinase-mediated relaxation in ovine fetal pulmonary arteries and veins. Am J Physiol Lung Cell Mol Physiol. 2003; 285: L611-L618 Crossref Scopus (34) Google Scholar The mechanism by which this occurs is not known. Using a microarray, we found that in human pulmonary vascular smooth muscle cells (PVSMCs) 4 h of hypoxia altered the expression of > 900 genes fourfold or more. This was reduced to 121 genes by the reactive oxygen species (ROS) scavenger ascorbate, suggesting a role for ROS in hypoxic gene expression. Hence, first we used dichlorodihydrofluorescein (DCF) to measure ROS production in the pulmonary artery and pulmonary vein SMC during hypoxia. We determined identity of ROS by using scavengers for superoxide, hydrogen peroxide, and peroxynitrite during DCF fluorescence measurement. Ovine fetal pulmonary artery smooth muscle cells and PVSMCs were grown in Dulbecco minimum essential medium, preloaded with DCF and incubated under conditions of hypoxia (0% O2-5% CO2) or normoxia (20% O2-5% CO2). After 30 min, hypoxia ROS production increased fourfold to sixfold compared to normoxia but returned to normoxic levels after 4 h. Pretreatment of PVSMCs with ROS scavengers (Tiron for superoxide radical and Trolox for peroxynitrite radical) or with mitochondria complex III inhibitor myxothiazol significantly decreased ROS generated by hypoxia. After 4 h, hypoxia reduced PKG protein and PKG1 messenger RNA expression in PVSMCs. The scavenging of superoxide and peroxynitrite generated during hypoxia prevented the hypoxic decrease in PKG protein and PKG1 messenger RNA expression. Our findings indicate that the hypoxic generation of ROS in PVSMCs leads to the down-regulation of cGMP-mediated vasodilation by the inhibition of PKG and predisposes the patient to the development of pulmonary hypertension.

Dynamics of the pulmonary venous flow in the fetus and its association with vascular diameter.

The usual positioning of the Doppler sample volume to assess fetal pulmonary vein flow is in the distal portion of the vein, where the vessel diameter is maximal. This study was performed to test the association of the pulmonary vein pulsatility index (PVPI) with the vessel diameter. Twenty-three normal fetuses (mean gestational age, 28.6+/-5.3 weeks) were studied by Doppler echocardiography. Pulmonary right upper vein flow was assessed adjacent to the venoatrial junction ("distal" position) and in the middle of the vein ("proximal" position). The vessel diameter was measured by 2D echocardiography with power Doppler, and the PVPI was obtained by the ratio (maximal velocity [systolic or diastolic peak]-minimal velocity [presystolic peak])/mean velocity. The statistical analysis used t test and exponential correlation studies. Mean distal diameter was 0.33+/-0.10 cm (0.11 to 0.57 cm), and mean proximal diameter was 0.16+/-0.08 cm (0.11 to 0.25 cm) (P<0.0001). Mean distal PVPI was 0.84+/-0.21 (0.59 to 1.38), and mean proximal PVPI was 2.09+/-0.59 (1.23 to 3.11) (P<0.0001). Exponential inverse correlation between pulmonary vein diameter and pulsatility index was highly significant (P<0.0001), with a determination coefficient of 0.439. In the normal fetus, the pulmonary venous flow pulsatility decreases from the lung to the heart, and this parameter is inversely correlated to the diameter of the pulmonary vein, which increases from its proximal to its distal portion. This study emphasizes the importance of the correct positioning of the Doppler sample volume, adjacent to the venoatrial junction, to assess pulmonary venous flow dynamics.

Effect of oxygen on cyclic GMP-dependent protein kinase-mediated relaxation in ovine fetal pulmonary arteries and veins.

Cyclic GMP-dependent protein kinase (PKG) plays an important role in regulating pulmonary vasomotor tone in the perinatal period. In this study, we tested the hypothesis that a change in oxygen tension affects PKG-mediated pulmonary vasodilation. Isolated intrapulmonary arteries and veins of near-term fetal lambs were first incubated for 4 h under hypoxic and normoxic conditions (Po2 of 30 and 140 mmHg, respectively) and then contracted with endothelin-1. 8-Bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP), a cell membrane-permeable analog of cGMP, induced a greater relaxation in vessels incubated in normoxia than in hypoxia. beta-Phenyl-1,N2-etheno-8-bromoguanosine-3',5'-cyclic monophosphorothioate, Rp isomer (Rp-8-Br-PET-cGMPS), a selective inhibitor of PKG, attenuated relaxation induced by 8-BrcGMP (10-4 and 3 x 10-4 M). In the presence of Rp-8-Br-PET-cGMPS, the differential responses to 8-BrcGMP between hypoxia and normoxia treatment were abolished in veins but not in arteries. cGMP-stimulated PKG activity was present in arteries but not in veins after 4 h of hypoxia. Both vessel types showed significant increase in cGMP-stimulated PKG activity after 4 h of normoxia. PKG protein (Western blot analysis) and PKG mRNA levels (quantitative RT-PCR) were greater in veins but not in arteries after 4-h exposure to normoxia vs. hypoxia. These results demonstrate that oxygen augments cGMP-mediated vasodilation of fetal pulmonary arteries and veins. Furthermore, the effect of oxygen on response of the veins to cGMP is due to an increase in the activity, protein level, and mRNA of PKG.

The influence of a restrictive atrial septal defect on pulmonary vascular morphology in patients with hypoplastic left heart syndrome.

Hypoplastic left heart syndrome (HLHS) with a restrictive atrial septal defect (ASD) is a form of congenital heart disease with considerable morbidity and mortality. This morphologic analysis assesses the pulmonary vasculature in this patient population. Pulmonary arteries, the persistence of high-resistance fetal arterioles, pulmonary veins, and lymphatics from multiple lung sections from each of five patients with HLHS and a restrictive ASD were compared to those of five patients with HLHS and nonrestrictive ASD. Lung sections from each patient were qualitatively graded in severity of pathology from 0 to 3 for each of the structures described previously, with the pathologist blinded to the status of the ASD. Patients with a restrictive ASD exhibited more significant pulmonary venous thickening and lymphatic dilatation (p = 0.02), with a tendency toward persistence of high-resistance fetal vessels (p = 0.2), compared to patients with a nonrestrictive ASD. These findings imply that patients with HLHS and a restrictive ASD possess pulmonary vascular abnormalities that place them at higher risk for the current surgical interventions available compared to patients with a nonrestrictive ASD.

Content, in vivo release, and bioactivity of fetal pulmonary immunoreactive adrenocorticotropin

We hypothesized that fetal lung contains and releases immunoreactive (ir) adrenocorticotropin (ACTH). Fetal sheep lung (89-145 days gestation, n = 13) irACTH content was 4,100 +/- 672 (SE) pg/g lung wet wt compared with 5,425 +/- 1,403 (SE) pg/g in adults (n = 5) when measured by radioimmunoassay (RIA). irACTH values normalized to protein content were 158 +/- 18 and 95 +/- 22 (SE) ng/g protein for fetuses and adults, respectively. Grouped by age, < 90 days (n = 4), 131-139 days (n = 5), and 143-145 days (n = 4), the values for irACTH were 184 +/- 28, 173 +/- 33, and 114 +/- 33 (SE) ng/g protein, respectively, and declined significantly from < 90 days to adulthood. RIA serial dilution curve slopes were different from standard in 7 of 13 fetuses and in all adults, suggesting that irACTH structure differs from ACTH-(1-39). Acute in vivo fetal pulmonary artery and vein sampling (n = 5, 120-128 days gestation) revealed a mean difference of -609 +/- 476 (SE) pg/ml, a statistically significant release of irACTH across the lung. Western blotting revealed that pulmonary irACTH migrates in multiple immunostaining bands between molecular weight 14,000 and 46,000 and does not colocalize with ACTH-(1-39). Adrenal cell bioassay revealed that pulmonary irACTH is not corticotropic. We concluded that ovine fetal lung contains large-molecular-weight irACTH, that content declines from 90 days gestation to adulthood, and that, under acute conditions, the fetal lung releases irACTH substances in sufficient quantity to contribute to circulating hormone concentrations of irACTH.