Neonatal lung side population cells demonstrate endothelial potential and are altered in response to hyperoxia-induced lung simplification.

Lung side population (SP) cells are resident lung precursor cells with both epithelial and mesenchymal potential that are believed to play a role in normal lung development and repair. Neonatal hyperoxic exposure impairs lung development leading to a long-term decrease in gas exchange surfaces. The hypothesis that lung SP cells are altered during impaired lung development has not been studied. To address this issue, we characterized the endothelial potential of neonatal lung SP and subsets of lung SP from neonatal mice following hyperoxic exposure during room air recovery. Lung SP cells were isolated and sorted on the basis of their capacity to efflux Hoechst 33342. The lung SP was further sorted based on expression of Flk-1 and CD45. In vitro, both CD45(pos)/Flk-1(pos) and CD45(neg)/Flk-1(pos) bind isolectin B4 and incorporate LDL and form networks in matrigel, indicating that these populations have endothelial cell characteristics. Hyperoxic exposure of neonatal mice resulted in subtle changes in vascular and alveolar density on P13, which persisted with room air recovery to P41. During room air recovery, a decrease in lung SP cells was detected in the hyperoxic-exposed group on postnatal day 13 followed by an increase on day 41. Within this group, the lung SP subpopulation of cells expressing CD45 increased on day 21, 41, and 55. Here, we show that lung SP cells demonstrate endothelial potential and that the population distribution changes in number as well as composition following hyperoxic exposure. The hyperoxia-induced changes in lung SP cells may limit their ability to effectively contribute to tissue morphogenesis during room air recovery.

Effects of chronic estrogen-receptor blockade on ovine perinatal pulmonary circulation.

Prolonged infusions of 17beta-estradiol reduce fetal pulmonary vascular resistance (PVR), but the effects of endogenous estrogens in the fetal pulmonary circulation are unknown. To test the hypothesis that endogenous estrogen promotes pulmonary vasodilation at birth, we studied the hemodynamic effects of prolonged estrogen-receptor blockade during late gestation and at birth in fetal lambs. We treated chronically prepared fetal lambs with ICI-182,780 (ICI, a specific estrogen-receptor blocker, n = 5) or 1% DMSO (CTRL, n = 5) for 7 days and then measured pulmonary hemodynamic responses to ventilation with low- and high-fraction inspired oxygen (FI(O(2))). Treatment with ICI did not change basal fetal PVR or arterial blood gas tensions. However, treatment with ICI abolished the vasodilator response to ventilation with low FI(O(2)) [change in PVR -30 +/- 6% (CTRL) vs. +10 +/- 13%, (ICI), P < 0.05] without reducing the vasodilator response to ventilation with high FI(O(2)) [change in PVR, -73 +/- 3% (CTRL) vs. -77 +/- 4%, (ICI); P = not significant]. ICI treatment reduced prostacyclin synthase (PGIS) expression by 33% (P < 0.05) without altering expression of endothelial nitric oxide synthase or cyclooxygenase-1 and -2. In situ hybridization and immunohistochemistry revealed that PGIS is predominantly expressed in the airway epithelium of late gestation fetal lambs. We conclude that prolonged estrogen-receptor blockade inhibits the pulmonary vasodilator response at birth and that this effect may be mediated by downregulation of PGIS. We speculate that estrogen exposure during late gestation prepares the pulmonary circulation for postnatal adaptation.

Endothelin B receptor deficiency potentiates ET-1 and hypoxic pulmonary vasoconstriction.

Endothelin (ET)-1 contributes to the regulation of pulmonary vascular tone by stimulation of the ET(A) and ET(B) receptors. Although activation of the ET(A) receptor causes vasoconstriction, stimulation of the ET(B) receptors can elicit either vasodilation or vasoconstriction. To examine the physiological role of the ET(B) receptor in the pulmonary circulation, we studied a genetic rat model of ET(B) receptor deficiency [transgenic(sl/sl)]. We hypothesized that deficiency of the ET(B) receptor would predispose the transgenic(sl/sl) rat lung circulation to enhanced pulmonary vasoconstriction. We found that the lungs of transgenic(sl/sl) rats are ET(B) deficient because they lack ET(B) mRNA in the pulmonary vasculature, have minimal ET(B) receptors as determined with an ET-1 radioligand binding assay, and lack ET-1-mediated pulmonary vasodilation. The transgenic(sl/sl) rats have higher basal pulmonary arterial pressure and vasopressor responses to brief hypoxia or ET-1 infusion. Plasma ET-1 levels are elevated and endothelial nitric oxide synthase protein content and nitric oxide production are diminished in the transgenic(sl/sl) rat lung. These findings suggest that the ET(B) receptor plays a major physiological role in modulating resting pulmonary vascular tone and reactivity to acute hypoxia. We speculate that impaired ET(B) receptor activity can contribute to the pathogenesis of pulmonary hypertension.

Lung hypoplasia in the nitrofen model of congenital diaphragmatic hernia occurs early in development.

The teratogen nitrofen produces a congenital diaphragmatic hernia (CDH) and pulmonary hypoplasia in rodent fetuses that closely parallel observations made in humans. We hypothesized that these changes may be due to primary pulmonary hypoplasia and not herniation of the abdominal contents. Timed-pregnant rats were given nitrofen on day 9, and fetuses were harvested on days 13 through 21. Initial evagination of lung buds on gestational day 11 was not delayed in nitrofen-treated fetuses. On gestational day 13, however, there was a significant decrease in the number of terminal end buds in the lungs of nitrofen-exposed fetuses vs. controls. Thymidine-labeled lung epithelial and mesenchymal cells were significantly decreased in nitrofen-treated lungs. Lungs from nitrofen-treated fetuses exhibited wide septae with disorganized, compacted tissue, particularly around the air spaces. Expression of surfactant protein B and C mRNAs was significantly decreased in the nitrofen litters. In situ hybridization of fetal lung tissue at all gestational ages showed no difference in the expression of vascular endothelial growth factor, Flk-1, or Flt-1 mRNAs. Because closure of the diaphragm is completed on gestational day 16 in the rat, our results suggest that lung hypoplasia in this model of CDH is due at least in part to a primary effect of nitrofen on the developing lung.

Inhibition of angiogenesis decreases alveolarization in the developing rat lung.

To determine whether angiogenesis is necessary for normal alveolarization, we studied the effects of two antiangiogenic agents, thalidomide and fumagillin, on alveolarization during a critical period of lung growth in infant rats. Newborn rats were treated with daily injections of fumagillin, thalidomide, or vehicle during the first 2 wk of life. Compared with control treatment, fumagillin and thalidomide treatment reduced lung weight-to-body weight ratio and pulmonary arterial density by 20 and 36%, respectively, and reduced alveolarization by 22%. Because these drugs potentially have nonspecific effects on lung growth, we also studied the effects of Su-5416, an inhibitor of the vascular endothelial growth factor receptor known as kinase insert domain-containing receptor/fetal liver kinase (KDR/flk)-1. As observed with the other antiangiogenic agents, Su-5416 treatment decreased alveolarization and arterial density. We conclude that treatment with three different antiangiogenic agents attenuated lung vascular growth and reduced alveolarization in the infant rat. We speculate that angiogenesis is necessary for alveolarization during normal lung development and that injury to the developing pulmonary circulation during a critical period of lung growth can contribute to lung hypoplasia.

Endothelial monocyte activating polypeptide II inhibits lung neovascularization and airway epithelial morphogenesis.

Neovascularization is crucial to lung development and is mediated through a variety of angiogenic and anti-angiogenic factors. Herein, we show that excess Endothelial Monocyte Activating Polypeptide (EMAP) II, an anti-angiogenic protein, not only inhibits fetal lung neovascularization, but also significantly alters airway epithelial morphogenesis. In a murine allograft model of lung neovascularization and morphogenesis, embryonic lungs transplanted under the skin of immunocompromised mice receiving intraperitoneal EMAP II, had a 56% reduction in vessel density (P<0.0001) compared to control. EMAP II treated lung transplants also exhibited a marked alteration in lung morphogenesis, including lack of type II alveolar cell formation, determined by markedly decreased expression of surfactant protein C, and increased apoptosis. In contrast, lung implants in animals receiving an EMAP II blocking antibody had an increase in vessel density of 50% (P<0.0001) and increased expression of surfactant protein C mRNA in distal epithelium. These studies demonstrate that EMAP II negatively modulates lung neovascularization as well as leading to the arrest of lung airway epithelial morphogenesis and apoptosis.

Tissue interactions mediate early events in pulmonary vasculogenesis.

Extensive study has provided considerable insight into the mechanisms governing branching morphogenesis and developmental maturation of the pulmonary epithelium. The process by which the vascular tree arises in the mesodermal mesenchyme of the developing lung, however, is not known. Because normal epithelial branching and differentiation have been shown to be dependent on interactions with the lung mesenchyme, we hypothesized that the developing pulmonary vasculature is dependent on a reciprocal interaction with pulmonary epithelium. In this study we have defined the temporal and spatial expression of flk-1 mRNA, which encodes an endothelial cell-specific vascular endothelial growth factor (VEGF) receptor, in fetal and neonatal rat lung. Flk-1-positive cells were observed in the lung at every prenatal stage from fetal day 11 through birth, demonstrating that vascularization has been initiated as soon as the lung evaginates from the foregut epithelium. The spatial distribution of vascular precursors was distinct and consistent in early lung (fetal days 11-16): clusters of flk-1-positive cells were localized in the mesenchyme closely apposed to the developing epithelium. This spatial relationship between vascular precursors and the developing epithelium suggested that vascular development in the lung may be dependent on interactions between the two tissue types. To investigate this possibility, day-13 distal lung mesenchyme was cultured in the presence and absence of lung epithelium. Lung mesenchyme cultured in the absence of epithelium degenerated significantly, and few flk-1-positive cells were maintained. In contrast, lung mesenchyme recombined with lung epithelium contained abundant flk-1-positive cells, and their spatial distribution mimicked that observed in vivo. These studies provide the first detailed information regarding the temporal and spatial pattern of pulmonary vascularization in early development and suggest that tissue interactions play an important role in growth and maintenance of the developing lung vasculature.

Abnormal lung growth and the development of pulmonary hypertension in the Fawn-Hooded rat.

The Fawn-Hooded rat (FHR) strain develops accelerated and severe pulmonary hypertension when exposed to slight decreases in alveolar PO(2). We recently observed that adult FHR lungs showed a striking pattern of disrupted alveolarization and hypothesized that abnormalities in lung growth in the perinatal period predisposes the FHR to the subsequent development of pulmonary hypertension. We found a reduction in lung weight in the fetus and 1-day- and 1-wk-old FHR compared with a normal rat strain (Sprague-Dawley). Alveolarization was reduced in infant and adult FHR lungs. In situ hybridization showed similar patterns of expression of two epithelial markers, surfactant protein C and 10-kDa Clara cell secretory protein, suggesting that the FHR lung is not characterized by global delays in epithelial maturation. Barium-gelatin angiograms demonstrated reduced background arterial filling and density in adult FHR lungs. Perinatal treatment of FHR with supplemental oxygen increased alveolarization and reduced the subsequent development of right ventricular hypertrophy in adult FHR. We conclude that the FHR strain is characterized by lung hypoplasia with reduced alveolarization and increased risk for developing pulmonary hypertension. We speculate that altered oxygen sensing may cause impaired lung alveolar and vascular growth in the FHR.

Induction of alveolar type II cell differentiation in embryonic tracheal epithelium in mesenchyme-free culture.

We have previously shown that fetal lung mesenchyme can reprogram embryonic rat tracheal epithelium to express a distal lung phenotype. We have also demonstrated that embryonic rat lung epithelium can be induced to proliferate and differentiate in the absence of lung mesenchyme. In the present study we used a complex growth medium to induce proliferation and distal lung epithelial differentiation in embryonic tracheal epithelium. Day-13 embryonic rat tracheal epithelium was separated from its mesenchyme, enrobed in growth factor-reduced Matrigel, and cultured for up to 7 days in medium containing charcoal-stripped serum, insulin, epidermal growth factor, hepatocyte growth factor, cholera toxin, fibroblast growth factor 1 (FGF1), and keratinocyte growth factor (FGF7). The tracheal epithelial cells proliferated extensively in this medium, forming lobulated structures within the extracellular matrix. Many of the cells differentiated to express a type II epithelial cell phenotype, as evidenced by expression of SP-C and osmiophilic lamellar bodies. Deletion studies showed that serum, insulin, cholera toxin, and FGF7 were necessary for maximum growth. While no single deletion abrogated expression of SP-C, deleting both FGF7 and FGF1 inhibited growth and prevented SP-C expression. FGF7 or FGF1 as single additions to the medium, however, were unable to induce SP-C expression, which required the additional presence of serum or cholera toxin. FGF10, which binds the same receptor as FGF7, did not support transdifferentiation when used in place of FGF7. These data indicate that FGF7 is necessary, but not sufficient by itself, to induce the distal rat lung epithelial phenotype, and that FGF7 and FGF10 play distinct roles in lung development.

Mesenchyme specifies epithelial differentiation in reciprocal recombinants of embryonic lung and trachea.

Normal lung morphogenesis and cytodifferentiation require interactions between epithelium and mesenchyme. We have previously shown that distal lung mesenchyme (LgM) is capable of reprogramming tracheal epithelium (TrE) from day 13-14 rat fetuses to branch in a lung-like pattern and express a distal lung epithelial phenotype. In the present study, we have assessed the effects of tracheal mesenchyme (TrM) on branching and cytodifferentiation of distal lung epithelium (LgE). Tracheae and distal lung tips from day 13 rat fetuses were separated into purified epithelial and mesenchymal components, then recombined as homotypic (LgM + LgE or TrM + TrE) or heterotypic (LgM + TrE or TrM + LgE) recombinants and cultured for 5 days; unseparated lung tips and tracheae served as controls. Control lung tips, LgM + LgE, and LgM + TrE recombinants all branched in an identical pattern. Epithelial cells, including those from the induced TrE, contained abundant glycogen deposits and lamellar bodies, and expressed surfactant protein C (SP-C) mRNA. Trachea controls, and both TrM + TrE, and TrM + LgE recombinants did not branch, but instead formed cysts. The epithelium contained ciliated and mucous secretory cells; importantly, no cells containing lamellar bodies were observed, nor was SP-C mRNA detected. Mucin immunostaining showed copious production of mucous in both LgE and TrE when recombined with TrM. These results demonstrate that epithelial differentiation in the recombinants appears to be wholly dependent on the type of mesenchyme used, and that the entire respiratory epithelium has significant plasticity in eventual phenotype at this stage in development.

Selective and transient in vitro effects of heat shock on alveolar type II cell gene expression.

The heat shock response is a highly conserved stress response that can transiently inhibit non-heat shock protein gene expression. Although heat shock protects against acute lung injury, its effects on lung cell gene expression are not known. We studied the in vitro effects of heat shock on the expression of several genes important to alveolar type II cells. Prior induction of heat shock transiently inhibited cytokine-mediated inducible nitric oxide synthase gene expression and cytokine-mediated manganese-superoxide dismutase mRNA expression in murine lung epithelium. In contrast, heat shock had no effect on expression of surfactant protein (SP) A or B mRNA, or SP-B peptide synthesis. Cell survival studies indicated that the inhibitory effects were not secondary to cytotoxicity. Previous heat shock also modestly enhanced the ability of cells to withstand oxidant stress. We conclude that in vitro heat shock has selective and transient inhibitory effects on alveolar type II cell gene expression. Transient inhibition of cytokine-inducible genes, with concomitant conservation of genes required for normal respiratory function (SP) may explain, in part, the mechanism by which heat shock protects during acute lung injury.

Redox regulation of manganese superoxide dismutase.

The importance of reactive oxygen species (ROS) or changes in cellular redox state in signal transduction and gene regulation is becoming increasingly evident. In this study, we tested the hypothesis that ROS are directly involved in the induction of the mitochondrial antioxidant manganese superoxide dismutase (MnSOD) and mediate the induction of MnSOD by tumor necrosis factor-alpha (TNF-alpha). Pretreatment of human pulmonary adenocarcinoma cells H441 with the antioxidants N-acetyl-L-cysteine (NAC) and nordihydroguaiaretic acid (NDGA) blocked MnSOD induction by TNF-alpha, implicating ROS as a signaling agent in this pathway. Treatment of H441 cells with the exogenous oxidants hydrogen peroxide (H2O2) and diamide increased MnSOD mRNA, supporting the hypothesis that ROS directly affect expression of MnSOD. The temporal pattern of MnSOD induction differed for TNF-alpha and H2O2, suggesting distinct signaling pathways. DNA binding of two redox-sensitive transcription factors, NF-kappa B and activator protein (AP)-1, was evaluated. TNF-alpha increased nuclear factor (NF)-kappa B-DNA binding, an effect blocked by pretreatment with NAC. H2O2 did not alter NF-kappa B-DNA binding. There was no evidence of AP-1 binding in cells treated with either TNF-alpha or H2O2. We conclude that ROS directly alter MnSOD expression and are involved in the induction of MnSOD by TNF-alpha.

Pulmonary capillary diameters and recruitment characteristics in subpleural and interior networks.

In vivo microscopic observations of pulmonary capillaries are limited to subpleural networks that are less dense than interior networks. In addition to the density difference, subpleural and interior capillary diameters may differ, although there are conflicting data on this point. We measured the diameters of subpleural and interior capillaries in rats and dogs. Subpleural diameters were 30% larger in rats and 20% larger in dogs. Because diameter and density differences might cause differences in recruitment between subpleural and interior networks, we measured subpleural and interior recruitment by counting the number of red blood cells per 10 microns of alveolar wall in histological cross sections of rapidly frozen rat lungs. Lung inflation pressures of 4, 12, and 25 cmH2O created a wide range of capillary recruitment in different groups of animals. Red blood cell counts for interior and subpleural capillaries moved in parallel and progressively increased as inflation pressures were reduced. These data demonstrate that recruitment in subpleural capillaries accurately reflect recruitment in interior capillaries and validate the use of in vivo microscopic observations of subpleural capillaries to investigate pulmonary capillary recruitment in general.

Distribution of pulmonary capillary red blood cell transit times.

In theory, red blood cells can pass through the pulmonary capillaries too rapidly to be completely saturated with oxygen during exercise. This idea has not been directly tested because the transit times of the fastest red blood cells are unknown. We report the first measurements of the entire transit time distribution for red blood cells crossing single subpleural capillary networks of canine lung using in vivo fluorescence videomicroscopy and compare those times with the distribution of plasma transit times in the same capillary networks. On average, plasma took 1.4 times longer than red blood cells to pass through the capillary bed. Decreased transit times with increased cardiac output were mitigated by both capillary recruitment and a narrowing of the transit time distribution. This design feature of the pulmonary capillary bed kept the shortest times from falling below the theoretical minimum time for complete oxygenation.

Sites of leukocyte sequestration in the pulmonary microcirculation.

The location and mechanisms of leukocyte sequestration in the pulmonary circulation have been investigated by using high-magnification in vivo videomicroscopy to record the passage of unlabeled native leukocytes through canine pulmonary capillaries. Of 650 leukocytes traversing capillary networks, 46 +/- 6% (SE) of the leukocytes passed through without stopping, 42 +/- 9% stopped in segments between junctions, and 12 +/- 4% stopped in junctions. Leukocytes rolling along arteriolar walls were nearly spherical, as 94% had aspect ratios (major axis divided by minor axis) < or = 1.25. To pass through the capillary bed, the leukocytes deformed into elongated shapes. Many leukocytes remained elongated after entering the venules (53% had aspect ratios > or = 1.25). Venular rolling was blocked by fucoidin (blocking both L- and P-selectin) but not by anti-P-selectin antibodies alone, indicating that rolling leukocytes adhered to the venular endothelium by L-selectin. These observations demonstrate that leukocytes deform to transit the capillary bed, that they stop more frequently in segments than in junctions, and that rolling leukocytes in the venular marginated pool adhere via L-selectin.

Acetylated polyamines in lungs from rats with monocrotaline-induced pneumotoxicity.

Multiple lines of evidence implicate the polyamines, putrescine, spermidine, and spermine in the lung injury and hypertensive pulmonary vascular disease produced in rats by the pyrrolizidine alkaloid monocrotaline. While increases in lung polyamine content evoked by monocrotaline can be attributed in part to induction of the two rate-limiting enzymes in de novo polyamine synthesis, ornithine decarboxylase and S-adenosylmethionine decarboxylase, little attention has been paid to the role that catabolic interconversion processes might play in lung polyamine accumulation. Accordingly, the present study evaluated dose (10-60 mg/kg)- and time (0-21 days)-dependent effects of monocrotaline on lung contents of acetylated polyamines and on the activity of spermidine/spermine acetyltransferase (SAT), the enzyme affecting spermidine acetylation. A single subcutaneous injection of monocrotaline produced dose- and time-dependent increases in the lung content of N1-acetylspermidine. Neither N1-acetylspermine nor N1-acetylputrescine could be detected in lungs from control rats or from rats treated with monocrotaline. SAT activity also was increased in monocrotaline-treated rat lungs in a dose- and time-dependent manner that was closely related to increases in the lung burden of N1-acetylspermidine. As expected, monocrotaline also caused dose- and time-dependent elevations in the lung contents of the primary polyamines, putrescine, spermidine, and spermine. Right ventricular hypertrophy, an index of sustained pulmonary hypertension, did not develop in animals treated with 10 or 20 mg/kg monocrotaline despite elevations in the lung contents of putrescine and N1-acetylspermidine and increases in the activity of SAT. In contrast, 30 and 60 mg/kg monocrotaline provoked right ventricular hypertrophy accompanied by elevations in the primary polyamines, N1-acetyl spermidine and SAT activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Polyamine synthesis in rat lungs injured with alpha-naphthylthiourea.

The diamine, putrescine, and polyamines, spermidine and spermine, are low molecular weight organic cations with documented regulatory roles in cell growth and differentiation. Multiple lines of direct and indirect evidence suggest that these organic cations also may function in stimulus-response coupling processes regulating cellular injury and repair. For example, recent studies in monocrotaline-treated rats, hyperoxic rats, and in cultured pulmonary endothelial cells suggest that polyamines regulate pulmonary endothelial integrity and may thus participate in development and/or regression of acute edematous lung injury. To determine if the polyamines are involved in a well-characterized animal model of acute lung injury, the present experiments assessed the relation between changes in polyamine synthesis and development of edema in lungs from rats treated with alpha-naphthylthiourea (ANTU). ANTU caused dose- and time-dependent increases in the lung activity of the initial and rate-limiting enzyme in polyamine biosynthesis, ornithine decarboxylase (ODC) and in the lung contents of the polyamines putrescine, spermidine, and spermine. ANTU also caused dose- and time-dependent increases in the lung wet-to-dry weight ratio indicative of pulmonary edema formation. Changes in lung polyamine biosyntheic activity after ANTU did not relate temporally to changes in the lung wet-to-dry weight ratio: ODC activity was depressed during the 3-h period immediately following ANTU administration, a period when the wet-to-dry weight ratio was increasing, and markedly elevated at 18 h after ANTU administration when the wet-to-dry weight ratio had returned to control levels. Pretreatment of the animals with alpha-difluoromethylornithine, a highly specific inhibitor of ODC, failed to attenuate ANTU-induced increases in lung wet-to-dry weight ratio. These observations indicate polyamine synthesis is enhanced in rat lungs with ANTU-induced pulmonary edema but, unlike certain other models of lung injury and pulmonary edema, accumulation of polyamines probably is not essential for development of edematous lung injury. It is conceivable that in this animal model polyamines play a role in lung repair processes or some longer-term consequence of lung injury.