Timing of hyperoxic exposure during alveolarization influences damage mediated by leukotrienes

Hyperoxic exposure of rat pups during alveolarization (postnatal days 4-14) severely retards alveolar development. Some aspects of this inhibition are mediated by leukotrienes (LTs) and may be time sensitive. We determined 1) the effects of exposure to hyperoxia (O(2)) during discrete periods before and during alveolarization on developing alveoli and 2) whether a relationship exists between O(2) and LTs in these periods. Pups were exposed to >95% O(2) from days 1 to 4, 4 to 9, 9 to 14, or 4 to 14 in the absence and presence of the LT synthesis inhibitor MK-0591. Both the level of in vitro lung tissue LT output on days 4, 9, and 14 and the degree of alveolarization on day 14 were determined. Pups exposed to O(2) from days 4 to 9 had a more profound inhibition of alveolarization on day 14 compared with those exposed to O(2) from days 1 to 4 or 9 to 14. Peptido-LT levels were significantly higher in pups exposed to O(2) on days 9 and 14 compared with pups in air and returned to normal once normoxia was restored. LT inhibition from days 4 to 14, 4 to 9, or 9 to 14 in pups exposed to O(2) from days 4 to 14 prevented the O(2)-induced inhibition of alveolarization. These data suggest that developing alveoli are sensitive to LTs shortly before and after day 9, significantly retarding certain parameters of alveolarization on day 14. We conclude that some of the effects of O(2) are not uniform throughout different stages of alveolarization and that this is likely related to the timing of LT exposure.     

Cyclooxygenase-2 in newborn hyperoxic lung injury

Supraphysiological O₂ concentrations, mechanical ventilation, and inflammation significantly contribute to the development of bronchopulmonary dysplasia (BPD).Exposure of newborn mice to hyperoxia causes inflammation and impaired alveolarization similar to that seen in infants with BPD.Previously, we demonstrated that pulmonary cyclooxygenase-2 (COX-2) protein expression is increased in hyperoxia-exposed newborn mice.The present studies were designed to define the role of COX-2 in newborn hyperoxic lung injury.We tested the hypothesis that attenuation of COX-2 activity would reduce hyperoxia-induced inflammation and improve alveolarization.Newborn C3H/HeN micewere injected daily with vehicle, aspirin (nonselective COX-2 inhibitor), or celecoxib (selective COX-2 inhibitor) for the first 7 days of life.Additional studies utilized wild-type (C57Bl/6, COX-2^+/+), heterozygous (COX-2^+/-), and homozygous (COX-2^-/-) transgenic mice.Micewere exposed to room air (21% O₂) or hyperoxia (85% O₂) for 14 days.Aspirin-injected and COX-2^-/- pups had reduced levels of monocyte chemoattractant protein (MCP-1) in bronchoalveolar lavage fluid (BAL).Both aspirin and celecoxib treatment reduced macrophage numbers in the alveolar walls and air spaces.Aspirin and celecoxib treatment attenuated hyperoxia-induced COX activity, including altered levels of prostaglandin (PG)D₂ metabolites.Decreased COX activity, however, did not prevent hyperoxia-induced lung developmental deficits.Our data suggest thatincreased COX-2 activity may contribute to proinflammatory responses, including macrophage chemotaxis, during exposure to hyperoxia.Modulation of COX-2 activity may be a useful therapeutic target to limit hyperoxia-induced inflammation in preterm infants at risk of developing BPD.     

Sildenafil attenuates pulmonary inflammation and fibrin deposition, mortality and right ventricular hypertrophy in neonatal hyperoxic lung injury

Background

Phosphodiesterase-5 inhibition with sildenafil has been used to treat severe pulmonary hypertension and bronchopulmonary dysplasia (BPD), a chronic lung disease in very preterm infants who were mechanically ventilated for respiratory distress syndrome.

Methods

Sildenafil treatment was investigated in 2 models of experimental BPD: a lethal neonatal model, in which rat pups were continuously exposed to hyperoxia and treated daily with sildenafil (50–150 mg/kg body weight/day; injected subcutaneously) and a neonatal lung injury-recovery model in which rat pups were exposed to hyperoxia for 9 days, followed by 9 days of recovery in room air and started sildenafil treatment on day 6 of hyperoxia exposure. Parameters investigated include survival, histopathology, fibrin deposition, alveolar vascular leakage, right ventricular hypertrophy, and differential mRNA expression in lung and heart tissue.

Results

Prophylactic treatment with an optimal dose of sildenafil (2 × 50 mg/kg/day) significantly increased lung cGMP levels, prolonged median survival, reduced fibrin deposition, total protein content in bronchoalveolar lavage fluid, inflammation and septum thickness. Treatment with sildenafil partially corrected the differential mRNA expression of amphiregulin, plasminogen activator inhibitor-1, fibroblast growth factor receptor-4 and vascular endothelial growth factor receptor-2 in the lung and of brain and c-type natriuretic peptides and the natriuretic peptide receptors NPR-A, -B, and -C in the right ventricle. In the lethal and injury-recovery model we demonstrated improved alveolarization and angiogenesis by attenuating mean linear intercept and arteriolar wall thickness and increasing pulmonary blood vessel density, and right ventricular hypertrophy (RVH).

Conclusion

Sildenafil treatment, started simultaneously with exposure to hyperoxia after birth, prolongs survival, increases pulmonary cGMP levels, reduces the pulmonary inflammatory response, fibrin deposition and RVH, and stimulates alveolarization. Initiation of sildenafil treatment after hyperoxic lung injury and continued during room air recovery improves alveolarization and restores pulmonary angiogenesis and RVH in experimental BPD.     

Phosphodiesterase 4 inhibition attenuates persistent heart and lung injury by neonatal hyperoxia in rats

Phosphodiesterase (PDE) 4 inhibitors are potent anti-inflammatory drugs with antihypertensive properties, and their therapeutic role in bronchopulmonary dysplasia (BPD) is still controversial. We studied the role of PDE4 inhibition with piclamilast on normal lung development and its therapeutic value on pulmonary hypertension (PH) and right ventricular hypertrophy (RVH) in neonatal rats with hyperoxia-induced lung injury, a valuable model for premature infants with severe BPD. The cardiopulmonary effects of piclamilast treatment (5 mg·kg(-1)·day(-1)) were investigated in two models of experimental BPD: 1) daily treatment during continuous exposure to hyperoxia for 10 days; and 2) late treatment and injury-recovery in which pups were exposed to hyperoxia or room air for 9 days, followed by 9 or 42 days of recovery in room air combined with treatment started on day 6 of oxygen exposure until day 18. Prophylactic piclamilast treatment reduced pulmonary fibrin deposition, septum thickness, arteriolar wall thickness, arteriolar vascular smooth muscle cell proliferation and RVH, and prolonged survival. In the late treatment and injury-recovery model, hyperoxia caused persistent aberrant alveolar and vascular development, PH, and RVH. Treatment with piclamilast in both models reduced arteriolar wall thickness, attenuated RVH, and improved right ventricular function in the injury recovery model, but did not restore alveolarization or angiogenesis. Treatment with piclamilast did not show adverse cardiopulmonary effects in room air controls in both models. In conclusion, PDE4 inhibition attenuated and partially reversed PH and RVH, but did not advance alveolar development in neonatal rats with hyperoxic lung injury or affect normal lung and heart development.     

Antenatal inflammation induced TGF-beta1 but suppressed CTGF in preterm lungs

Chorioamnionitis is frequently associated with preterm birth and increases the risk of adverse outcomes such as bronchopulmonary dysplasia (BPD). Transforming growth factor (TGF)-beta1 is a key regulator of lung development, airway remodeling, lung fibrosis, and regulation of inflammation, and all these processes contribute to the development of BPD. Connective tissue growth factor (CTGF) is a downstream mediator of some of the profibrotic effects of TGF-beta1, vascular remodeling, and angiogenesis. TGF-beta1-induced CTGF expression can be blocked by TNF-alpha. We asked whether chorioamnionitis-associated antenatal inflammation would regulate TGF-beta1, the TGF-beta1 signaling pathway, and CTGF in preterm lamb lungs. Fetal sheep were exposed to 4 mg of intra-amniotic endotoxin or saline for 5 h, 24 h, 72 h, or 7 days before preterm delivery at 125 days gestation (full term = 150 days). Intra-amniotic endotoxin increased lung TGF-beta1 mRNA and protein expression. Elevated TGF-beta1 levels were associated with TGF-beta1-induced phosphorylation of Smad2. CTGF was selectively expressed in lung endothelial cells in control lungs, and intra-amniotic endotoxin caused CTGF expression to decrease to 30% of control values and TNF-alpha protein to increase. The antenatal inflammation-induced TGF-beta1 expression and Smad signaling in the fetal lamb lung may contribute to impaired lung alveolarization and reduced lung inflammation. Decreased CTGF expression may inhibit vascular development or remodeling and limit lung fibrosis during remodeling. These effects may contribute to the impaired alveolar and pulmonary vascular development that is the hallmark of the new form of BPD.     

Dynamics of metalloproteinase-2 and -9, TGF-beta,and uPA activities during normoxic vs. hyperoxic alveolarization

The final stage of lung development, alveolarization, continues after birth in humans and rodents. Clinical interventions, such as oxygen therapy, in the first week of life can adversely impact alveolar formation. We compared alveolarization in the rat neonate under normal vs. hyperoxic conditions, examining gelatinase, transforming growth factor (TGF)-beta, and the protease urokinase-type plasminogen activator (uPA) activities in whole lung and cultured type II alveolar epithelial cells (AEC2). The dynamic induction of gelatinase, TGF-beta, and uPA activities seen in neonatal lungs during the first days of life was significantly impacted by hyperoxia. In whole lung, gelatinase and TGF-beta activities were increased, while uPA activity was decreased. At the level of the epithelium, AEC2 isolated from hyperoxic rat pups early in life secreted less active TGF-beta, less active gelatinases, and less active uPA than control neonatal AEC2. AEC2 from hyperoxic pups also expressed increased levels of proliferating cell nuclear antigen early in life compared with control neonatal AEC2, suggesting that oxygen-induced proliferation and/or repair were occurring. The developmental profile of neonatal lung was perturbed within a day of initiating oxygen treatment, suggesting that putative palliative treatments should be coadministered with oxygen therapy.     

Curcumin augments lung maturation, preventing neonatal lung injury by inhibiting TGF-β signaling

There is no effective intervention to prevent or treat bronchopulmonary dysplasia (BPD). Curcumin has potent antioxidant and anti-inflammatory properties, and it modulates signaling of peroxisome proliferator-activated receptor-γ (PPARγ), an important molecule in the pathobiology of BPD. However, its role in the prevention of BPD is not known. We determined 1) if curcumin enhances neonatal lung maturation, 2) if curcumin protects against hyperoxia-induced neonatal lung injury, and 3) if this protection is mediated by blocking TGF-β. Embryonic day 19 fetal rat lung fibroblasts were exposed to 21% or 95% O(2) for 24 h following 1 h of treatment with curcumin. Curcumin dose dependently accelerated e19 fibroblast differentiation [increased parathyroid hormone-related protein (PTHrP) receptor, PPARγ, and adipocyte differentiation-related protein (ADRP) levels and triolein uptake] and proliferation (increased thymidine incorporation). Pretreatment with curcumin blocked the hyperoxia-induced decrease (PPARγ and ADRP) and increase (α-smooth muscle actin and fibronectin) in markers of lung injury/repair, as well as the activation of TGF-β signaling. In a separate set of experiments, neonatal Sprague-Dawley rat pups were exposed to 21% or 95% O(2) for 7 days with or without intraperitoneal administration of curcumin. Analysis for markers of lung injury/repair [PTHrP receptor, PPARγ, ADRP, fibronectin, TGF-β receptor (activin receptor-like kinase 5), and Smad3] and lung morphology (radial alveolar count) demonstrated that curcumin effectively blocks TGF-β activation and hyperoxia-induced lung injury. Therefore, curcumin accelerates lung maturation by stimulating key alveolar epithelial-mesenchymal interactions and prevents hyperoxia-induced neonatal lung injury, possibly by blocking TGF-β activation, suggesting that it is a potential intervention against BPD.     

Recombinant human VEGF treatment enhances alveolarization after hyperoxic lung injury in neonatal rats

VEGF signaling inhibition decreases alveolar and vessel growth in the developing lung, suggesting that impaired VEGF signaling may contribute to decreased lung growth in bronchopulmonary dysplasia (BPD). Whether VEGF treatment improves lung structure in experimental models of BPD is unknown. The objective was to determine whether VEGF treatment enhances alveolarization in infant rats after hyperoxia. Two-day-old Sprague-Dawley rats were placed into hyperoxia or room air (RA) for 12 days. At 14 days, rats received daily treatment with rhVEGF-165 or saline. On day 22, rats were killed. Tissue was collected. Morphometrics was assessed by radial alveolar counts (RAC), mean linear intercepts (MLI), and skeletonization. Compared with RA controls, hyperoxia decreased RAC (6.1 +/- 0.4 vs. 11.3 +/- 0.4, P < 0.0001), increased MLI (59.2 +/- 1.8 vs. 44.0 +/- 0.8, P < 0.0001), decreased nodal point density (447 +/- 14 vs. 503 +/- 12, P < 0.0004), and decreased vessel density (11.7 +/- 0.3 vs. 18.9 +/- 0.3, P < 0.001), which persisted despite RA recovery. Compared with hyperoxic controls, rhVEGF treatment after hyperoxia increased RAC (11.8 +/- 0.5, P < 0.0001), decreased MLI (42.2 +/- 1.2, P < 0.0001), increased nodal point density (502 +/- 7, P < 0.0005), and increased vessel density (23.2 +/- 0.4, P < 0.001). Exposure of neonatal rats to hyperoxia impairs alveolarization and vessel density, which persists despite RA recovery. rhVEGF treatment during recovery enhanced vessel growth and alveolarization. We speculate that lung structure abnormalities after hyperoxia may be partly due to impaired VEGF signaling.     

Retinoic acid attenuates O2-induced inhibition of lung septation

Exposure of the newborn lung to hyperoxia is associated with impaired alveolar development. In newborn rats exposed to hyperoxia and studied at day 14 of life, retinoic acid (RA) treatment improved survival and increased lung collagen but did not improve alveolar development. To determine whether RA treatment during exposure to hyperoxia results in late improvement in alveolarization, we treated newborn rats with RA and hyperoxia from day 3 to day 14 and then weaned O2 to room air by day 20, and studied the animals on day 42. O2-exposed animals had larger mean lung volumes, larger alveoli, and decreased gas-exchange tissue relative to air-exposed animals, whereas RA-treated O2-exposed animals were not statistically different from air-exposed controls. Relative to control animals, elastin staining at day 14 was decreased in hyperoxia-exposed lung independent of RA treatment, and, at day 42, elastin staining was similar in all treatment groups. At day 14, elastin gene expression was similar in all treatment groups, whereas at day 42 lung previously exposed to hyperoxia showed increased elastin signal independent of RA treatment. These results indicate that RA treatment during hyperoxia exposure promotes septal formation without evidence of effects on elastin gene expression after 4 wk of recovery.     

Hyperoxia reduces bone marrow, circulating, and lung endothelial progenitor cells in the developing lung: implications for the pathogenesis of bronchopulmonary dysplasia

Hyperoxia disrupts vascular and alveolar growth of the developing lung and contributes to the development of bronchopulmonary dysplasia (BPD). Endothelial progenitor cells (EPC) have been implicated in repair of the vasculature, but their role in lung vascular development is unknown. Since disruption of vascular growth impairs lung structure, we hypothesized that neonatal hyperoxia impairs EPC mobilization and homing to the lung, contributing to abnormalities in lung structure. Neonatal mice (1-day-old) were exposed to 80% O(2) at Denver's altitude (= 65% at sea level) or room air for 10 days. Adult mice were also exposed for comparison. Blood, lung, and bone marrow were harvested after hyperoxia. Hyperoxia decreased pulmonary vascular density by 72% in neonatal but not adult mice. In contrast to the adult, hyperoxia simplified distal lung structure neonatal mice. Moderate hyperoxia reduced EPCs (CD45-/Sca-1+/CD133+/VEGFR-2+) in the blood (55%; P < 0.03), bone marrow (48%; P < 0.01), and lungs (66%; P < 0.01) of neonatal mice. EPCs increased in bone marrow (2.5-fold; P < 0.01) and lungs (2-fold; P < 0.03) of hyperoxia-exposed adult mice. VEGF, nitric oxide (NO), and erythropoietin (Epo) contribute to mobilization and homing of EPCs. Lung VEGF, VEGF receptor-2, endothelial NO synthase, and Epo receptor expression were reduced by hyperoxia in neonatal but not adult mice. We conclude that moderate hyperoxia decreases vessel density, impairs lung structure, and reduces EPCs in the circulation, bone marrow, and lung of neonatal mice but increases EPCs in adults. This developmental difference may contribute to the increased susceptibility of the developing lung to hyperoxia and may contribute to impaired lung vascular and alveolar growth in BPD.     

LRP5 Regulates Development of Lung Microvessels and Alveoli through the Angiopoietin-Tie2 Pathway

Angiogenesis is crucial for lung development. Although there has been considerable exploration, the mechanism by which lung vascular and alveolar formation is controlled is still not completely understood. Here we show that low-density lipoprotein receptor-related protein 5 (LRP5), a component of the Wnt ligand-receptor complex, regulates angiogenesis and alveolar formation in the lung by modulating expression of the angiopoietin (Ang) receptor, Tie2, in vascular endothelial cells (ECs). Vascular development in whole mouse lungs and in cultured ECs is controlled by LRP5 signaling, which is, in turn, governed by a balance between the activities of the antagonistic Tie2 ligands, Ang1 and Ang2. Under physiological conditions when Ang1 is dominant, LRP5 knockdown decreases Tie2 expression and thereby, inhibits vascular and alveolar development in the lung. Conversely, when Ang2 dominates under hyperoxia treatment in neonatal mice, high LRP5 and Tie2 expression suppress angiogenesis and lung development. These findings suggest that the LRP5-Tie2-Ang signaling axis plays a central role in control of both angiogenesis and alveolarization during postnatal lung development, and that deregulation of this signaling mechanism might lead to developmental abnormalities of the lung, such as are observed in bronchopulmonary dysplasia (BPD).     

Effects of hyperoxia on VEGF,its receptors,and HIF-2alpha in the newborn rat lung

Signaling through the hypoxia inducible factor (HIF)-VEGF-VEGF receptor system (VEGF signaling system) leads to angiogenesis and epithelial cell proliferation and is a key mechanism regulating alveolarization in lungs of newborn rats. Hyperoxia exposure (>95% O2 days 4-14) arrests lung alveolarization and may do so through suppression of the VEGF signaling system. Lung tissue mRNA levels of HIF-2alpha and VEGF increased from days 4-14 in normoxic animals, but hyperoxia suppressed these increases. Levels of HIF-2alpha and VEGF mRNA were correlated in the air but not the O2-treated group, suggesting that the low levels of HIF-2alpha observed at high O2 concentrations are not stimulating VEGF expression. VEGF164 protein levels increased with developmental age, and with hyperoxia to day 9, but continuing hyperoxia decreased levels by day 12. VEGFR1 and VEGFR2 mRNA expression also increased in air-exposed animals, and these, too, were significantly decreased by hyperoxia by day 9 and day 12, respectively. Receptor protein levels did not increase with development; however, O2 did decrease protein to less than air values. Hyperoxic suppression of VEGF signaling from days 9-14 may be one mechanism by which alveolarization is arrested.     

Inhaled nitric oxide attenuates pulmonary inflammation and fibrin deposition and prolongs survival in neonatal hyperoxic lung injury

Administration of inhaled nitric oxide (iNO) is a potential therapeutic strategy to prevent bronchopulmonary dysplasia (BPD) in premature newborns with respiratory distress syndrome. We evaluated this approach in a rat model, in which premature pups were exposed to room air, hyperoxia, or a combination of hyperoxia and NO (8.5 and 17 ppm). We investigated the anti-inflammatory effects of prolonged iNO therapy by studying survival, histopathology, fibrin deposition, and differential mRNA expression (real-time RT-PCR) of key genes involved in the development of BPD. iNO therapy prolonged median survival 1.5 days (P = 0.0003), reduced fibrin deposition in a dosage-dependent way up to 4.3-fold (P < 0.001), improved alveolar development by reducing septal thickness, and reduced the influx of leukocytes. Analysis of mRNA expression revealed an iNO-induced downregulation of genes involved in inflammation (IL-6, cytokine-induced neutrophilic chemoattractant-1, and amphiregulin), coagulation, fibrinolysis (plasminogen activator inhibitor 1 and urokinase-type plasminogen activator receptor), cell cycle regulation (p21), and an upregulation of fibroblast growth factor receptor-4 (alveolar formation). We conclude that iNO therapy improves lung pathology and prolongs survival by reducing septum thickness, inhibiting inflammation, and reducing alveolar fibrin deposition in premature rat pups with neonatal hyperoxic lung injury.     

TGF-beta-neutralizing antibodies improve pulmonary alveologenesis and vasculogenesis in the injured newborn lung

Pulmonary injury is associated with the disruption of alveologenesis in the developing lung and causes bronchopulmonary dysplasia (BPD) in prematurely born infants. Transforming growth factor (TGF)-beta is an important regulator of cellular differentiation and early lung development, and its levels are increased in newborn lung injury. Although overexpression of TGF-beta in the lungs of newborn animals causes pathological features that are consistent with BPD, the role of endogenous TGF-beta in the inhibition of the terminal stage of lung development is incompletely understood. In this investigation, the hypothesis that O(2)-induced injury of the maturing lung is associated with TGF-beta-mediated disruption of alveologenesis and microvascular development was tested using a murine model of BPD. Here we report that treatment of developing mouse lungs with TGF-beta-neutralizing antibodies attenuates the increase in pulmonary cell phospho-Smad2 nuclear localization, which is indicative of augmented TGF-beta signaling, associated with pulmonary injury induced by chronic inhalation of 85% oxygen. Importantly, the neutralization of the abnormal TGF-beta activity improves quantitative morphometric indicators of alveologenesis, extracellular matrix assembly, and microvascular development in the injured developing lung. Furthermore, exposure to anti-TGF-beta antibodies is associated with improved somatic growth in hyperoxic mouse pups and not with an increase in pulmonary inflammation. These studies indicate that excessive pulmonary TGF-beta signaling in the injured newborn lung has an important role in the disruption of the terminal stage of lung development. In addition, they suggest that anti-TGF-beta antibodies may be an effective therapy for preventing some important developmental diseases of the newborn lung.     

Hyperoxic inhibition of newborn rat lung development: protection by deferoxamine.

Prolonged exposure to hyperoxia markedly inhibits normal lung development (alveolarization and respiratory surface area expansion) in immature animals. Since (a) hyperoxia results in excess hydroxyl radical (OH.) formation, (b) (OH.) is implicated in O2-induced lipid peroxidation and DNA alterations, and (c) both OH. formation and its interaction with DNA are Fe++ dependent; chelation of Fe++ should act to protect against pulmonary O2 toxicity and hyperoxic inhibition of lung development. We therefore treated litters of newborn rats with the iron chelator Deferoxamine mesylate (DES) (150 mg/kg/day) during a 10-day exposure to greater than 95% O2. Morphometric analysis demonstrated that compared to the mean airspace size in air control rat pups (Lm = 44.5 microns), hyperoxic exposure resulted in a 34% larger mean air space diameter in O2-saline rat lungs (59.5 microns) versus only an 11% enlargement in O2-DES lungs (51.1 microns*). Lung internal surface area (cm2) per 100-g body weight were air control = 4480, O2-saline = 3570 (decreases 20.3%), and O2-DES = 4125* (decreases 7.9%) (*p less than 0.05 versus O2-saline group). DES-treated animals also had significantly decreased lung conjugated diene levels during hyperoxic exposure and increased lung elastin content (reflective of preserved lung alveolar formation) compared to O2-saline rats. These results indicate that DES treatment substantially ameliorated the inhibitory effects of neonatal hyperoxic exposure on normal lung development.     

Functional roles of the bone morphogenetic protein system in thyrotropin signaling in porcine thyroid cells

We uncovered a new regulation of thyrocyte function by bone morphogenetic protein (BMP) under the influence of thyrotropin (TSH) using primary culture of porcine thyrocytes. The BMP type I receptors, ALK-2 (ActRIA), -3 (BMPRIA), and -6 (BMPRIB), were expressed in porcine thyrocytes, while ALK-6 was not detected in human thyroid. Treatment with BMP-2, -4, -6, -7, and TGF-beta1 exhibited a dose-dependent suppression of DNA synthesis by porcine thyrocytes. BMP-2, -4, -6, -7, and TGF-beta1 suppressed TSH receptor mRNA expression on thyrocytes, which was consistent with their suppressive effect on TSH-induced cAMP synthesis and TSH-induced insulin-like growth factor-1 expression. Activin exhibited minimal suppression of thyrocyte DNA synthesis and did not exhibit suppressive effects on TSH receptor mRNA expression. Phosphorylated Smad1/5/8 was detected in the lysates of porcine thyrocytes treated with BMP-2, -4, -6, and -7. However, in the presence of TSH, BMP-6 and -7 failed to activate Smad1/5/8 phosphorylation and 3TP-reporter activity, whereas BMP-2 and -4 maintained clear activation of the BMP signaling regardless of the presence of TSH. This diverged regulation of thyroid BMP system by TSH is most likely due to the reduction of ALK-6 expression caused by TSH. Thus, the thyroid BMP system is functionally linked to TSH actions through modulating TSH receptor expression and TSH, in turn, selectively inhibits BMP signaling. Given that BMP system is present in human thyroid and the expression pattern of ALK-2 and BMPRII is different between follicular adenomas and normal thyroid tissues, the endogenous BMP system may be involved in regulating thyrocyte growth and TSH sensitivity of human thyroid adenomas.     

Selective inhibitory effects of Smad6 on bone morphogenetic protein type I receptors

The inhibitory Smads, Smad6 and Smad7, play pivotal roles in negative regulation of transforming growth factor-beta (TGF-beta) family signaling as feedback molecules as well as mediators of cross-talk with other signaling pathways. Whereas Smad7 acts as a ubiquitous inhibitor of Smad signaling, Smad6 has been shown to effectively inhibit bone morphogenetic protein (BMP) signaling but only weakly TGF-beta/activin signaling. In the present study, we have found that Smad6 inhibits signaling from the activin receptor-like kinase (ALK)-3/6 subgroup in preference to that from the ALK-1/2 subgroup of BMP type I receptors. The difference is attributable to the interaction of Smad6 with these BMP type I receptors. The amino acid residues responsible for Smad6 sensitivity of ALK-3 were identified as Arg-238, Phe-264, Thr-265, and Ala-269, which map to the N-terminal lobe of the ALK-3 kinase domain. Although Smad6 regulates BMP signaling through multiple mechanisms, our findings suggest that interaction with type I receptors is a critical step in the function of Smad6.     

Disruption of NO-cGMP signaling by neonatal hyperoxia impairs relaxation of lung parenchyma

Exposure of immature lungs to hyperoxia for prolonged periods contributes to neonatal lung injury and airway hyperreactivity. We studied the role of disrupted nitric oxide-guanosine 3',5'-cyclic monophosphate (NO-cGMP) signaling in impairing the relaxant responses of lung tissue from hyperoxia-exposed rat pups. Pups were exposed to >/=95% O(2) or room air for 7 days starting from days 1, 5, or 14. The animals were killed, lungs were removed, and 1-mm-thick lung parenchymal strips were prepared. Lung parenchymal strips of room air or hyperoxic pups were preconstricted using bethanechol and then graded electrical field stimulation (EFS) was applied to induce relaxation. EFS-induced relaxation of lung parenchymal strips was greater at 7 and 12 days than at 21 days in room air-exposed rat pups. Hyperoxic exposure significantly reduced relaxation at 7 and 12 days but not 21 days compared with room air exposure. NO synthase blockade with N(omega)-nitro-l-arginine methyl ester diminished relaxant responses in room air but not in hyperoxic pups at 12 days. After incubation with supplemental l-arginine, the relaxation response of hyperoxic strips was restored. cGMP, a key mediator of the NO signaling pathway, also decreased in strips from hyperoxic vs. room air pups and cGMP levels were restored after incubation with supplemental l-arginine. In addition, arginase activity was significantly increased in hyperoxic lung parenchymal strips compared with room air lung parenchymal strips. These data demonstrate disruption of NO-cGMP signaling in neonatal rat pups exposed to hyperoxia and show that bioavailability of the substrate l-arginine is implicated in the predisposition of this model to airway hyperreactivity.     

Transforming growth factor-beta signaling mediates hypoxia-induced pulmonary arterial remodeling and inhibition of alveolar development in newborn mouse lung

Hypoxia causes abnormal neonatal pulmonary artery remodeling (PAR) and inhibition of alveolar development (IAD). Transforming growth factor (TGF)-beta is an important regulator of lung development and repair from injury. We tested the hypothesis that inhibition of TGF-beta signaling attenuates hypoxia-induced PAR and IAD. Mice with an inducible dominant-negative mutation of the TGF-beta type II receptor (DNTGFbetaRII) and nontransgenic wild-type (WT) mice were exposed to hypoxia (12% O(2)) or air from birth to 14 days of age. Expression of DNTGFbetaRII was induced by 20 microg/g ZnSO(4) given intraperitoneally daily from birth. PAR, IAD, cell proliferation, and expression of extracellular matrix (ECM) proteins were assessed. In WT mice, hypoxia led to thicker, more muscularized resistance pulmonary arteries and impaired alveolarization, accompanied by increases in active TGF-beta and phosphorylated Smad2. Hypoxia-induced PAR and IAD were greatly attenuated in DNTGFbetaRII mice given ZnSO(4) compared with WT control mice and DNTGFbetaRII mice not given ZnSO(4). The stimulatory effects of hypoxic exposure on pulmonary arterial cell proliferation and lung ECM proteins were abrogated in DNTGFbetaRII mice given ZnSO(4). These data support the conclusion that TGF-beta plays an important role in hypoxia-induced pulmonary vascular adaptation and IAD in the newborn animal model.