bluuebunny/crossref_metadata_2025 huggingface dataset:
Binding and uptake of surfactant protein D by freshly isolated rat alveolar type II cells
Authors: Herbein Joel F. , Savov Jordan , Wright Jo Rae
Published: December 2017

Alveolar type II cells secrete, internalize, and recycle pulmonary surfactant, a lipid and protein complex that increases alveolar compliance and participates in pulmonary host defense. Surfactant protein (SP) D, a collagenous C-type lectin, has recently been described as a modulator of surfactant homeostasis. Mice lacking SP-D accumulate surfactant in their alveoli and type II cell lamellar bodies, organelles adapted for recycling and secretion of surfactant. The goal of current study was to characterize the interaction of SP-D with rat type II cells. Type II cells bound SP-D in a concentration-, time-, temperature-, and calcium-dependent manner. However, SP-D binding did not alter type II cell surfactant lipid uptake. Type II cells internalized SP-D into lamellar bodies and degraded a fraction of the SP-D pool. Our results also indicated that SP-D binding sites on type II cells may differ from those on alveolar macrophages. We conclude that, in vitro, type II cells bind and recycle SP-D to lamellar bodies, but SP-D may not directly modulate surfactant uptake by type II cells.
Tyrosine kinase-dependent calcium signaling in airway smooth muscle cells
Authors: Tolloczko Barbara , Tao Florence C. , Zacour Mary E. , Martin James G.
Published: December 2017

Contractile agonists may stimulate mitogenic responses in airway smooth muscle by mechanisms that involve tyrosine kinases. The role of contractile agonist-evoked activation of tyrosine kinases in contractile signaling is not clear. We addressed this issue using cultured rat airway smooth muscle cells. In these cells, serotonin (5-HT, 1 μM) caused contraction (quantitated by a decrease in cell area), which was blocked by the tyrosine kinase inhibitor genistein (40 μM). Genistein and tyrphostin 23 (40 and 10 μM, respectively) significantly decreased 5-HT-evoked peak Ca2+responses, and the effect of genistein could be observed in the absence of extracellular Ca2+. The specific inhibitor of mitogen-activated protein kinase kinase PD-98059 (30 μM) had no significant effect on peak Ca2+levels. Western analysis of cell extracts revealed that 5-HT caused a significant increase in tyrosine phosphorylation of proteins with molecular masses of ∼70 kDa within 10 s of stimulation but no measurable tyrosine phosphorylation of the γ isoform of phospholipase C (PLC-γ). Tyrosine phosphorylation was inhibited by genistein. Furthermore, genistein (40 μM) significantly attenuated 5-HT-induced inositol phosphate production. We conclude that in airway smooth muscle contractile agonists acting on G protein-coupled receptors may activate tyrosine kinase(s), which in turn modulate calcium signaling by affecting, directly or indirectly, PLC-β activity. It is unlikely that PLC-γ or the mitogen-activated protein kinase pathway is involved in Ca2+signaling to 5-HT.
Increased lung expansion alters the proportions of type I and type II alveolar epithelial cells in fetal sheep
Authors: Flecknoe S. , Harding R. , Maritz G. , Hooper S. B.
Published: December 2017

Type I and type II alveolar epithelial cells (AECs) are derived from the same progenitor cell, but little is known about the factors that regulate their differentiation into separate phenotypes. An alteration in lung expansion alters the proportion type II AECs in the fetal lung, indicating that this may be a regulatory factor. Our aim was to quantify the changes in the proportion of type I and type II AECs caused by increased fetal lung expansion and to provide evidence for transdifferentiation of type II into type I cells. Lung tissue samples were collected from ovine fetuses exposed to increased lung expansion induced by 2, 4, or 10 days of tracheal obstruction (TO). The identities and proportions of AEC types were determined with electron microscopy. The proportion of type II cells was reduced from 28.5 ± 2.2% in control fetuses to 9.4 ± 2.3% at 2 days of TO and then to 1.9 ± 0.8% at 10 days. The proportion of type I AECs was not altered at 2 days of TO (63.1 ± 2.3%) compared with that of control cells (64.8 ± 0.5%) but was markedly elevated (to 89.4 ± 0.9%) at 10 days of TO. The proportion of an intermediate AEC type, which displayed characteristics of both type I and type II cells, increased from 5.7 ± 1.3% in control fetuses to 23.8 ± 5.1% by 2 days of TO and was similar to control values at 10 days of TO (7.7 ± 0.9%). Our data show that increases in fetal lung expansion cause time-dependent changes in the proportion of AEC types, including a transient increase in an intermediate cell type. These data provide the first evidence to support the hypothesis that increases in fetal lung expansion induce differentiation of type II into type I AECs via an intermediate cell type.
Brief hypoxia differentially regulates LPS-induced IL-1β and TNF-α gene transcription in RAW 264.7 cells
Authors: Ndengele Michael M. , Bellone Clifford J. , Lechner Andrew J. , Matuschak George M.
Published: December 2017

Episodes of tissue hypoxia and reoxygenation frequently occur during gram-negative bacteremia that progresses to septic shock. However, few studies have evaluated modulation by hypoxia and reoxygenation of the proinflammatory cytokine gene expression that is normally induced by gram-negative bacteremia or endotoxemia. In buffer-perfused organs, hypoxia downregulates Escherichia coli-induced expression of tumor necrosis factor (TNF)-α and interleukin (IL)-1β in the liver but upregulates these cytokines in the lungs. To identify molecular mechanisms underlying these events, we investigated the effects of brief (1.5-h) hypoxia on TNF-α and IL-1β expression in cultured RAW 264.7 cells during their continuous exposure to lipopolysaccharide (LPS) endotoxin derived from E. coli (serotype 055:B5) for up to 24 h. IL-1β and TNF-α concentrations in cell lysates and culture supernatants were measured by ELISA, and steady-state mRNA was measured by Northern analysis. LPS-induced IL-1β synthesis was downregulated by hypoxia at both the protein and mRNA levels despite no change in cellular redox status as measured by levels of GSH. In contrast, LPS-induced TNF-α production was unaffected by hypoxia as assessed by cell lysate mRNA and lysate and supernatant protein levels. Nuclear runoff analysis showed that downregulation of IL-1β gene expression by hypoxia occurred transcriptionally. Allopurinol or catalase treatment did not alter modulation of LPS-induced IL-1β expression by hypoxia, suggesting that this suppression was not caused by reactive oxygen species. Cycloheximide pretreatment suggested that hypoxia-induced downregulation of IL-1β expression did not require de novo protein synthesis.
Parathyroid hormone-related protein reduces alveolar epithelial cell proliferation during lung injury in rats
Authors: Hastings Randolph H. , Berg John T. , Summers-Torres Daphne , Burton Douglas W. , Deftos Leonard J.
Published: December 2017

Parathyroid hormone-related protein (PTHrP) is a growth inhibitor for alveolar type II cells and could be a regulatory factor for alveolar epithelial cell proliferation after lung injury. We investigated lung PTHrP expression in rats exposed to 85% oxygen. Lung levels of PTHrP were significantly decreased between 4 and 8 days of hyperoxia, concurrent with increased expression of proliferating cell nuclear antigen and increased incorporation of 5-bromo-2′-deoxyuridine (BrdU) into DNA in lung corner cells. PTHrP receptor was present in both normal and hyperoxic lung. To test whether the fall in PTHrP was related to cell proliferation, we instilled PTHrP into lungs on the fourth day of hyperoxia. Eight hours later, BrdU labeling in alveolar corner cells was 3.2 ± 0.4 cells/high-power field in hyperoxic PBS-instilled rats compared with 0.5 ± 0.3 cells/high-power field in PTHrP-instilled rats ( P < 0.01). Thus PTHrP expression changes in response to lung injury due to 85% oxygen and may regulate cell proliferation.
Phenotypic characterization of alveolar monocyte recruitment in acute respiratory distress syndrome
Authors: Rosseau Simone , Hammerl Peter , Maus Ulrich , Walmrath Hans-Dieter , Schütte Hartwig , Grimminger Friedrich , Seeger Werner , Lohmeyer Jürgen
Published: December 2017

In 49 acute respiratory distress syndrome (ARDS) patients, the phenotype of alveolar macrophages (AMs) was analyzed by flow cytometry. Bronchoalveolar lavage (BAL) was performed within 24 h after intubation and on days 3– 5, 9– 12, and 18– 21 of mechanical ventilation. The 27E10high/CD11bhigh/CD71low/ 25F9low/HLA DRlow/RM3/1lowAM population in the first BAL indicated extensive monocyte influx into the alveolar compartment. There was no evidence of increased local AM proliferation as assessed by nuclear Ki67 staining. Sequential BAL revealed two distinct patient groups. In one, a decrease in 27E10 and CD11b and an increase in CD71, 25F9, HLA DR, and RM3/1 suggested a reduction in monocyte influx and maturation of recruited cells into AMs, whereas the second group displayed sustained monocyte recruitment. In the first BAL from all patients, monocyte chemoattractant protein (MCP)-1 was increased, and AMs displayed elevated MCP-1 gene expression. In sequential BALs, a decrease in MCP-1 coincided with the disappearance of monocyte-like AMs, whereas persistent upregulation of MCP-1 paralleled ongoing monocyte influx. A highly significant correlation between BAL fluid MCP-1 concentration, the predominance of monocyte-like AMs, and the severity of respiratory failure was noted.
Interactions between hypoxia and hypercapnic acidosis on calcium signaling in carotid body type I cells
Authors: Dasso Leonardo L. T. , Buckler Keith J. , Vaughan-Jones Richard D.
Published: December 2017

The effects of hypercapnic acidosis and hypoxia on intracellular Ca2+concentration ([Ca2+]i) were determined with Indo 1 in enzymatically isolated single type I cells from neonatal rat carotid bodies. Type I cells responded to graded hypoxic stimuli with graded [Ca2+]i rises. The percentage of cells responding was also dependent on the severity of the hypoxic stimulus. Raising CO2 from 5 to 10 or 20% elicited a significant increase in [Ca2+]i in the same cells as those that responded to hypoxia. Thus both stimuli can be sensed by each individual cell. When combinations of hypoxic and acidic stimuli were given simultaneously, the responses were invariably greater than the response to either stimulus given alone. Indeed, in most cases, the response to hypercapnia was slightly potentiated by hypoxia. These data provide the first evidence that the classic synergy between hypoxic and hypercapnic stimuli observed in the intact carotid body may, in part, be an inherent property of the type I cell.
Surfactant protein A enhances mycobacterial killing by rat macrophages through a nitric oxide-dependent pathway
Authors: Weikert Laura F. , Lopez Joseph P. , Abdolrasulnia Rasul , Chroneos Zissis C. , Shepherd Virginia L.
Published: December 2017

Surfactant-associated protein A (SP-A) is involved in surfactant homeostasis and host defense in the lung. We have previously demonstrated that SP-A specifically binds to and enhances the ingestion of bacillus Calmette-Guerin (BCG) organisms by macrophages. In the current study, we investigated the effect of SP-A on the generation of inflammatory mediators induced by BCG and the subsequent fate of ingested BCG organisms. Rat macrophages were incubated with BCG in the presence and absence of SP-A. Noningested BCG organisms were removed, and the release of tumor necrosis factor-α (TNF-α) and nitric oxide were measured at varying times. TNF-α and nitric oxide production induced by BCG were enhanced by SP-A. In addition, SP-A enhanced the BCG-induced increase in the level of inducible nitric oxide synthase protein. Addition of antibodies directed against SPR210, a specific macrophage SP-A receptor, inhibited the SP-A-enhanced mediator production. BCG in the absence of SP-A showed increased growth over a 5-day period, whereas inclusion of SP-A dramatically inhibited BCG growth. Inhibition of nitric oxide production blocked BCG killing in the presence and absence of SP-A. These results demonstrate that ingestion of SP-A-BCG complexes by rat macrophages leads to production of inflammatory mediators and increased mycobacterial killing.
β1-Integrins are involved in migration of human fetal tracheal epithelial cells and tubular morphogenesis
Authors: Coraux Christelle , Zahm Jean-Marie , Puchelle Edith , Gaillard Dominique
Published: December 2017

Development of human fetal airways requires interaction of the respiratory epithelium and the extracellular matrix through integrins. Nevertheless, the specific roles of β1-integrins during development and tubular morphogenesis are still unknown. To analyze β1-integrin localization and influence during migration, we developed a model of human fetal tracheal explants growing on collagen and overlaid with a second layer of collagen to form a sandwich. In this configuration, cord and tubule formation proceeded normally but were inhibited by incubation with anti-β1-integrin subunit antibodies. On a collagen matrix, β1-integrins were immunolocalized on the entire plasma membrane of migrating epithelial cells and almost exclusively on the basal plasma membrane of nonmigratory epithelial cells. In a sandwich configuration, β1-integrins became detectable in the cytoplasm of epithelial cells. Coating cultures with collagen transiently altered the morphology of migrating cells and their speed and direction of migration, whereas incubation with anti-β1-integrin subunit antibodies irreversibly altered these parameters. These observations suggest that the matrix environment, by modulating β1-integrin expression patterns, plays a key role during tubular morphogenesis of human fetal tracheal epithelium, principally by modulating epithelial cell migration.
K+channel inhibition, calcium signaling, and vasomotor tone in canine pulmonary artery smooth muscle
Authors: Doi Shouzaburoh , Damron Derek S. , Ogawa Koji , Tanaka Satoru , Horibe Mayumi , Murray Paul A.
Published: December 2017

We investigated the role of K+channels in the regulation of baseline intracellular free Ca2+concentration ([Ca2+]i), α-adrenoreceptor-mediated Ca2+signaling, and capacitative Ca2+entry in pulmonary artery smooth muscle cells (PASMCs). Inhibition of voltage-gated K+channels with 4-aminopyridine (4-AP) increased the membrane potential and the resting [Ca2+]ibut attenuated the amplitude and frequency of the [Ca2+]ioscillations induced by the α-agonist phenylephrine (PE). Inhibition of Ca2+-activated K+channels (with charybdotoxin) and inhibition (with glibenclamide) or activation of ATP-sensitive K+channels (with lemakalim) had no effect on resting [Ca2+]ior PE-induced [Ca2+]ioscillations. Thapsigargin was used to deplete sarcoplasmic reticulum Ca2+stores in the absence of extracellular Ca2+. Under these conditions, 4-AP attenuated the peak and sustained components of capacitative Ca2+entry, which was observed when extracellular Ca2+was restored. Capacitative Ca2+entry was unaffected by charybdotoxin, glibenclamide, or lemakalim. In isolated pulmonary arterial rings, 4-AP increased resting tension and caused a leftward shift in the KCl dose-response curve. In contrast, 4-AP decreased PE-induced contraction, causing a rightward shift in the PE dose-response curve. These results indicate that voltage-gated K+channel inhibition increases resting [Ca2+]iand tone in PASMCs but attenuates the response to PE, likely via inhibition of capacitative Ca2+entry.
Heparin-like molecules inhibit pulmonary vascular pericyte proliferation in vitro
Authors: Khoury Joseph , Langleben David
Published: December 2017

Proliferation of vascular pericytes (PCs), smooth muscle-like cells found in the distal microvasculature, contributes to vascular remodeling in pulmonary hypertension. The factors controlling lung PC quiescence in normal states are poorly understood. We demonstrate that exogenous heparin and heparan sulfate proteoglycans inhibit rat lung PC proliferation in vitro as does pulmonary vascular subendothelial matrix, particularly its heparan sulfate component. Heparin inhibits the intracellular alkalinization essential to proliferation, and we show that inhibition of alkalinization by 5-( N,N-dimethyl)amiloride also reduces PC proliferation. As shown by DNA staining and fluorescence-activated cell sorting analysis, heparin does not induce apoptosis in PCs. However, heparin maintains lung PCs in the G0/G1growth phase. Heparin induces production of p21, a potent inhibitor of cyclin-dependent kinases, thereby potentially identifying a fundamental mechanism by which heparin inhibits proliferation in smooth muscle-like cells. These studies establish additional similarities between lung PCs and smooth muscle cells and provide further understanding of growth control in the lung microvasculature. They also further support the rationale that heparin-like molecules might be therapeutically beneficial in pulmonary hypertension.
Expression ofN-deacetylase/sulfotransferase and 3-O-sulfotransferase in rat alveolar type II cells
Authors: Li Zhong-Yuan , Hirayoshi Kazunori , Suzuki Yasuhiro
Published: December 2017

Basal laminae beneath alveolar type I cells are suggested to contain highly sulfated heparan sulfate-containing proteoglycans (PGs), and cultured type II cells accumulate highly sulfated matrices. To characterize the regulation of PG synthesis during the transition from type II cells to type I cells, we examined mRNA expression of N-deacetylase/sulfotransferase (NST) and 3- O-sulfotransferase (3-OST), two enzymes specific for heparan sulfate synthesis. We found that both freshly isolated and cultured type II cells expressed NST and 3-OST as shown by in situ hybridization. Expression of surfactant-associated protein A, B, and C mRNAs, determined by semiquantitative PCR, decreased during culture. Expression of type I cell marker T1α mRNA increased except in cells cultured on an Engelbrecht-Holm-Swarm gel. Expression of NST was dependent on cell density and matrix and was intense in conditions where cells spread fully, whereas 3-OST expression was unchanged in the conditions examined. The PG sulfation inhibitor sodium chlorate significantly inhibited cultured type II cell spreading, and this inhibition was reversed by sodium sulfate. These results suggest that highly sulfated PGs modified by NST are necessary for the spreading of cells during transdifferentiation of type II cells to mature type I cells.
TNF-α increases transcription of Gαi-2in human airway smooth muscle cells
Authors: Hotta Kunihisa , Hirshman Carol A. , Emala Charles W.
Published: December 2017

Tumor necrosis factor-α (TNF-α) is a proinflammatory cytokine that has an important role in the regulation of airway smooth muscle tone and reactivity. We have shown previously that TNF-α upregulates the expression of Gαi-2protein without significantly increasing Gsα protein and enhances adenylyl cyclase inhibition by carbachol in cultured human airway smooth muscle cells (Hotta K, Emala CW, and Hirshman CA. Am J Physiol Lung Cell Mol Physiol 276: L405–L411, 1999). The present study was designed to investigate the molecular mechanisms by which TNF-α upregulates Gαi-2protein in these cells. TNF-α pretreatment for 48 h increased the expression of Gαi-2protein without significantly altering the Gαi-2protein half-life (41.0 ± 8.2 h for control and 46.8 ± 5.2 h for TNF-α-treated cells). Inhibition of new protein synthesis by cycloheximide blocked the increase in Gαi-2protein induced by TNF-α. Furthermore, TNF-α treatment for 12–24 h increased the steady-state level of Gαi-2mRNA without significantly altering Gαi-2mRNA half-life (9.0 ± 0.75 h for control and 8.9 ± 1.1 h for TNF-α-treated cells). The transcription inhibitor actinomycin D blocked the increase in Gαi-2mRNA induced by TNF-α. These observations indicate that the increase in Gαi-2protein induced by TNF-α is due to an increased rate of Gαi-2protein synthesis, most likely as a consequence of the transcriptional increase in the steady-state levels of its mRNA.
Quantitative trait locus mapping of genes regulating pulmonary PKC activity and PKC-α content
Authors: Dwyer-Nield Lori D. , Paigen Beverly , Porter Stephanie E. , Malkinson Alvin M.
Published: December 2017

Strain A/J mice, which are predisposed to experimentally induced asthma and adenocarcinoma, have the lowest pulmonary protein kinase (PK) C activity and content among 22 inbred mouse strains. PKC in neonatal A/J mice is similar to that in other strains, so this difference reflects strain-dependent postnatal regulation. PKC activity is 60% higher in C57BL/6J (B6) than in A/J lungs, and the protein and mRNA concentrations of PKC-α, the major pulmonary PKC isozyme, are two- to threefold higher in B6 mice. These differences result from more than a single gene as assessed in F1, F2, and backcross progeny of B6 and A/J parents. Quantitative trait locus (QTL) analysis of 23 A×B and B×A recombinant inbred strains derived from B6 and A/J progenitors indicates a major locus regulating lung PKC-α content that maps near the Pkcα structural gene on chromosome 11 ( D11MIT333; likelihood ratio statistic = 12.5) and a major locus controlling PKC activity that maps on chromosome 3 ( D3MIT19; likelihood ratio statistic = 15.4). The chromosome 11 QTL responsible for low PKC-α content falls within QTLs for susceptibilities to lung tumorigenesis and ozone-induced toxicity.
Lung redox homeostasis: emerging concepts
Authors: Merker Marilyn P. , Pitt Bruce R. , Choi Augustine M. , Hassoun Paul M. , Dawson Christopher A. , Fisher Aron B.
Published: December 2017

This symposium was organized to present some aspects of current research pertaining to lung redox function. Focuses of the symposium were on roles of pulmonary endothelial NADPH oxidase, xanthine oxidase (XO)/xanthine dehydrogenase (XDH), heme oxygenase (HO), transplasma membrane electron transport (TPMET), and the zinc binding protein metallothionein (MT) in the propagation and/or protection of the lung or other organs from oxidative injury. The presentations were chosen to reflect the roles of both intracellular (metallothionein, XO/XDH, and HO) and plasma membrane (NADPH oxidase, XO/XDH, and unidentified TPMET) redox proteins in these processes. Although the lung endothelium was the predominant cell type under consideration, at least some of the proposed mechanisms operate in or affect other cell types and organs as well.
Thrombin stimulates the expression of PDGF in lung epithelial cells
Authors: Shimizu Shino , Gabazza Esteban C. , Hayashi Tatsuya , Ido Masaru , Adachi Yukihiko , Suzuki Koji
Published: December 2017

Several growth factors, including platelet-derived growth factor (PDGF), have been implicated in the mechanism of lung and airway remodeling. In the present study, we evaluated whether thrombin may promote lung and airway remodeling by increasing PDGF production from lung and airway epithelial cells. Conditioned medium (CM) was prepared by treating epithelial cells with increasing concentrations of thrombin; before use in the assays, CM was treated with hirudin until complete inhibition of thrombin activity. CM from epithelial cells stimulated the proliferation of lung fibroblasts and bronchial smooth muscle cells. Anti-PDGF antibody significantly inhibited this CM proliferative activity, implicating PDGF in this effect. Enzyme immunoassay and RT-PCR demonstrated that thrombin induced the secretion and expression of PDGF from bronchial and alveolar epithelial cells. RT-PCR showed that epithelial cells express the thrombin receptors protease-activated receptor (PAR)-1, PAR-3, and PAR-4. The PAR-1 agonist peptide was also found to induce PDGF secretion from epithelial cells, suggesting that the cellular effect of thrombin occurs via a PAR-1-mediated mechanism. Overall, this study showed for the first time that thrombin may play an important role in the process of lung and airway remodeling by stimulating the expression of PDGF via its cellular receptor, PAR-1.
Regulation of diaphragmatic nitric oxide synthase expression during hypobaric hypoxia
Authors: Javeshghani Danesh , Sakkal Dalia , Mori Mastaka , Hussain Sabah N. A.
Published: December 2017

Nitric oxide (NO) is normally synthesized inside skeletal muscle fibers by both endothelial (eNOS) and neuronal (nNOS) nitric oxide synthases. In this study, we evaluated the influence of hypobaric hypoxia on the expression of NOS isoforms, argininosuccinate synthetase (AS), argininosuccinate lyase (AL), and manganese superoxide dismutase (Mn SOD) in the ventilatory muscles. Rats were exposed to hypobaric hypoxia (∼95 mmHg) from birth for 60 days or 9–11 mo. Age-matched control groups of rats also were examined. Sixty days of hypoxia elicited approximately two- and ninefold increases in diaphragmatic eNOS and nNOS protein expression (evaluated by immunoblotting), respectively, and about a 50% rise in diaphragmatic NOS activity. In contrast, NOS activity and the expression of these proteins declined significantly in response to 9 mo of hypoxia. Hypoxia elicited no significant alterations in AS, AL and Mn SOD protein expression. Moreover, the inducible NOS (iNOS) was not detected in normoxic and hypoxic diaphragmatic samples. We conclude that diaphragmatic NOS expression and activity undergo significant adaptations to hypobaric hypoxia and that iNOS does not participate in this response.
TIMP-1, -2, -3, and -4 in idiopathic pulmonary fibrosis. A prevailing nondegradative lung microenvironment?
Authors: Selman Moises , Ruiz Victor , Cabrera Sandra , Segura Lourdes , Ramírez Remedios , Barrios Roberto , Pardo Annie
Published: December 2017

Fibroblast proliferation and extracellular matrix accumulation characterize idiopathic pulmonary fibrosis (IPF). We evaluated the presence of tissue inhibitor of metalloproteinase (TIMP)-1, -2, -3, and -4; collagenase-1, -2, and -3; gelatinases A and B; and membrane type 1 matrix metalloproteinase (MMP) in 12 IPF and 6 control lungs. TIMP-1 was found in interstitial macrophages and TIMP-2 in fibroblast foci. TIMP-3 revealed an intense staining mainly decorating the elastic lamina in vessels. TIMP-4 was expressed in IPF lungs by epithelial and plasma cells. TIMP-2 colocalized with Ki67 in fibroblasts, whereas TIMP-3 colocalized with p27 in inflammatory and epithelial cells. Collagenase-1 was localized in macrophages and alveolar epithelial cells, collagenase-2 was localized in a few neutrophils, and collagenase-3 was not detected. MMP-9 was found in neutrophils and subepithelial myofibroblasts. Myofibroblast expression of MMP-9 was corroborated in vitro by RT-PCR. MMP-2 was noticed in myofibroblasts, some of them close to areas of basement membrane disruption, and membrane type 1 MMP was noticed in interstitial macrophages. These findings suggest that in IPF there is higher expression of TIMPs compared with collagenases, supporting the hypothesis that a nondegrading fibrillar collagen microenvironment is prevailing.
Genetic susceptibility to irritant-induced acute lung injury in mice
Authors: Wesselkamper Scott C. , Prows Daniel R. , Biswas Pratim , Willeke Klaus , Bingham Eula , Leikauf George D.
Published: December 2017

Recent studies suggest that genetic variability can influence irritant-induced lung injury and inflammation. To begin identifying genes controlling susceptibility to inhaled irritants, seven inbred mouse strains were continuously exposed to nickel sulfate (NiSO4), polytetrafluoroethylene, or ozone (O3), and survival time was recorded. The A/J (A) mouse strain was sensitive, the C3H/He (C3) strain was intermediate, and the C57BL/6 (B6) strain was resistant to NiSO4-induced acute lung injury. The B6AF1 offspring were also resistant. The strain sensitivity pattern for NiSO4 exposure was similar to that of polytetrafluoroethylene or ozone (O3). Pulmonary pathology was comparable for A and B6 mice. In the A strain, 15 μg/m3 of NiSO4 produced 20% mortality. The strain sensitivity patterns for lavage fluid proteins (B6 > C3 > A) and neutrophils (A ≥ B6 > C3) differed from those for acute lung injury. This phenotype discordance suggests that these traits are not causally linked (i.e., controlled by independent arrays of genes). As in acute lung injury, B6C3F1 offspring exhibited phenotypes (lavage fluid proteins and neutrophils) resembling those of the resistant parental strain. Agreement of acute lung injury strain sensitivity patterns among irritants suggested a common mechanism, possibly oxidative stress, and offspring resistance suggested that sensitivity is inherited as a recessive trait.
eNOS-deficient mice show reduced pulmonary vascular proliferation and remodeling to chronic hypoxia
Authors: Quinlan Timothy R. , Li Dechun , Laubach Victor E. , Shesely Edward G. , Zhou Nan , Johns Roger A.
Published: December 2017

Pulmonary hypertension is characterized by structural and morphological changes to the lung vasculature. To determine the potential role of nitric oxide in the vascular remodeling induced by hypoxia, we exposed wild-type [WT(+/+)] and endothelial nitric oxide synthase (eNOS)-deficient [(−/−)] mice to normoxia or hypoxia (10% O2) for 2, 4, and 6 days or for 3 wk. Smooth muscle α-actin and von Willebrand factor immunohistochemistry revealed significantly less muscularization of small vessels in hypoxic eNOS(−/−) mouse lungs than in WT(+/+) mouse lungs at early time points, a finding that correlated with decreases in proliferating vascular cells (5-bromo-2′-deoxyuridine positive) at 4 and 6 days of hypoxia in the eNOS(−/−) mice. After 3 wk of hypoxia, both mouse types exhibited similar percentages of muscularized small vessels; however, only the WT(+/+) mice exhibited an increase in the percentage of fully muscularized vessels and increased vessel wall thickness. eNOS protein expression was increased in hypoxic WT(+/+) mouse lung homogenates at all time points examined, with significantly increased percentages of small vessels expressing eNOS protein after 3 wk. These results indicate that eNOS deficiency causes decreased muscularization of small pulmonary vessels in hypoxia, likely attributable to the decrease in vascular cell proliferation observed in these mice.
Percutaneous in vivo gene transfer to the peripheral lungs using plasmid-liposome complexes
Authors: Saito Hiroshi , Nakamura Hidenori , Kato Suichi , Inoue Sumito , Inage Minoru , Ito Minoru , Tomoike Hitonobu
Published: December 2017

The purpose of this study was to investigate a new method of in vivo gene transfer to the lung parenchyma by the percutaneous approach. The plasmid that contains the gene for firefly luciferase driven by a cytomegalovirus (CMV) promoter (pCMVL) in combination with cationic lipids was percutaneously injected into the lung parenchyma. Luciferase activities were localized to the lobes of the lung where the plasmids with cationic lipids were injected. Percutaneous injection of the plasmid containing the human endothelin-1 (hET-1) gene driven by a CMV promoter (pRc/CMVhET-1) in combination with cationic lipids into the lungs caused pulmonary fibrosis localized to the injection site in the peripheral lungs. We concluded that percutaneous in vivo gene transfer to the lungs is a unique and important approach to introduce exogenous gene expression in the limited area of the lung parenchyma. This method of gene transfer will be applicable for human gene therapy for targeted areas of peripheral lung and will also be useful to assess the function of the proteins expressed by a gene in the local area of the lungs.
Adenosine induces endothelial apoptosis by activating protein tyrosine phosphatase: a possible role of p38α
Authors: Harrington Elizabeth O. , Smeglin Anthony , Parks Nancy , Newton Julie , Rounds Sharon
Published: December 2017

Endothelial cell (EC) apoptosis is important in vascular injury, repair, and angiogenesis. Homocysteine and/or adenosine exposure of ECs causes apoptosis. Elevated homocysteine or adenosine occurs in disease states such as homocysteinuria and tissue necrosis, respectively. We examined the intracellular signaling mechanisms involved in this pathway of EC apoptosis. Inhibition of protein tyrosine phosphatase (PTPase) attenuated homocysteine- and/or adenosine-induced apoptosis and completely blocked apoptosis induced by the inhibition of S-adenosylhomocysteine hydrolase with MDL-28842. Consistent with this finding, the tyrosine kinase inhibitor genistein enhanced apoptosis in adenosine-treated ECs. Adenosine significantly elevated the PTPase activity in the ECs. Mitogen-activated protein kinase activities were examined to identify possible downstream targets for the upregulated PTPase(s). Extracellular signal-regulated kinase (ERK) 1 activity was slightly elevated in adenosine-treated ECs, whereas ERK2, c-Jun NH2-terminal kinase-1, or p38β activities differed little. The mitogen-activated protein kinase-1 inhibitor PD-98059 enhanced DNA fragmentation, suggesting that increased ERK1 activity is a result but not a cause of apoptosis in adenosine-treated ECs. Adenosine-treated ECs had diminished p38α activity compared with control cells; this effect was blunted on PTPase inhibition. These results indicate that PTPase(s) plays an integral role in the induction of EC apoptosis upon exposure to homocysteine and/or adenosine, possibly by the attenuation of p38α activity.
Structural determinants of antiproliferative activity of heparin on pulmonary artery smooth muscle cells
Authors: Garg Hari G. , Thompson B. Taylor , Hales Charles A.
Published: December 2017

In addition to its anticoagulant properties, heparin (HP), a complex polysaccharide covalently linked to a protein core, inhibits proliferation of several cell types including pulmonary artery smooth muscle cells (PASMCs). Commercial lots of HP exhibit varying degrees of antiproliferative activity on PASMCs that may due to structural differences in the lots. Fractionation of a potent antiproliferative HP preparation into high and low molecular weight components does not alter the antiproliferative effect on PASMCs, suggesting that the size of HP is not the major determinant of this biological activity. The protein core of HP obtained by cleaving the carbohydrate-protein linkage has no growth inhibition on PASMCs, demonstrating that the antiproliferative activity resides in the glycosaminoglycan component. Basic sugar residues of glucosamine can be replaced with another basic sugar, i.e., galactosamine, without affecting growth inhibition of PASMCs. N-sulfonate groups on these sugar residues of HP are not essential for growth inhibition. However, O-sulfonate groups on both sugar residues are essential for the antiproliferative activity on PASMCs. In whole HP, in contrast to an earlier finding based on a synthetic pentasaccharide of HP, 3-O-sulfonation is not critical for the antiproliferative activity against PASMCs. The amounts and distribution of sulfonate groups on both sugar residues of the glycosaminoglycan chain are the major determinant of antiproliferative activity.
Characterization of rabbit SP-B promoter region responsive to downregulation by tumor necrosis factor-α
Authors: Berhane Kiflu , Margana Ramgopal K. , Boggaram Vijayakumar
Published: December 2017

Surfactant protein B (SP-B) is essential for the maintenance of biophysical properties and physiological function of pulmonary surfactant. Tumor necrosis factor-α (TNF-α), an important mediator of lung inflammation, inhibits surfactant phospholipid and surfactant protein synthesis in the lung. In the present study, we investigated the TNF-α inhibition of rabbit SP-B promoter activity in a human lung adenocarcinoma cell line (NCI-H441). Deletion experiments indicated that the TNF-α response elements are located within −236 bp of SP-B 5′-flanking DNA. The TNF-α response region contained binding sites for nuclear factor-κB (NF-κB), Sp1/Sp3, thyroid transcription factor (TTF)-1, and hepatocyte nuclear factor (HNF)-3 transcription factors. Inhibitors of NF-κB activation such as dexamethasone and N-tosyl-l-phenylalanine chloromethyl ketone and mutation of the NF-κB element did not reverse TNF-α inhibition of SP-B promoter, indicating that TNF-α inhibition of SP-B promoter activity occurs independently of NF-κB activation. TNF-α treatment decreased the binding activities of TTF-1 and HNF-3 elements without altering the nuclear levels of TTF-1 and HNF-3α proteins. Pretreatment of cells with okadaic acid reversed TNF-α inhibition of SP-B promoter activity. Taken together these data indicated that in NCI-H441 cells 1) TNF-α inhibition of SP-B promoter activity may be caused by decreased binding activities of TTF-1 and HNF-3 elements, 2) the decreased binding activities of TTF-1 and HNF-3α are not due to decreased nuclear levels of the proteins, and 3) okadaic acid-sensitive phosphatases may be involved in mediating TNF-α inhibition of SP-B promoter activity.
Store-operated calcium entry and increased endothelial cell permeability
Authors: Norwood Natalie , Moore Timothy M. , Dean David A. , Bhattacharjee Rakesh , Li Ming , Stevens Troy
Published: December 2017

We hypothesized that myosin light chain kinase (MLCK) links calcium release to activation of store-operated calcium entry, which is important for control of the endothelial cell barrier. Acute inhibition of MLCK caused calcium release from inositol trisphosphate-sensitive calcium stores and prevented subsequent activation of store-operated calcium entry by thapsigargin, suggesting that MLCK serves as an important mechanism linking store depletion to activation of membrane calcium channels. Moreover, in voltage-clamped single rat pulmonary artery endothelial cells, thapsigargin activated an inward calcium current that was abolished by MLCK inhibition. F-actin disruption activated a calcium current, and F-actin stabilization eliminated the thapsigargin-induced current. Thapsigargin increased endothelial cell permeability in the presence, but not in the absence, of extracellular calcium, indicating the importance of calcium entry in decreasing barrier function. Although MLCK inhibition prevented thapsigargin from stimulating calcium entry, it did not prevent thapsigargin from increasing permeability. Rather, inhibition of MLCK activity increased permeability that was especially prominent in low extracellular calcium. In conclusion, MLCK links store depletion to activation of a store-operated calcium entry channel. However, inhibition of calcium entry by MLCK is not sufficient to prevent thapsigargin from increasing endothelial cell permeability.
Hypoxia modifies the effect of PDGF on glycosaminoglycan synthesis by primary human lung cells
Authors: Papakonstantinou Eleni , Karakiulakis George , Tamm Michael , Perruchoud André P. , Roth Michael
Published: December 2017

Hypoxia, a consequence of interstitial lung diseases, may lead to secondary pulmonary hypertension and pulmonary vascular remodeling. Hypoxia induces activation and proliferation of lung cells and enhances the deposition of extracellular matrix including glycosaminoglycans (GAGs). To elucidate the cell biological mechanisms underlying the development of secondary pulmonary hypertension, we studied the effect of hypoxia on GAG synthesis by human lung cells. GAG synthesis was measured by incorporation of [3H]glucosamine; GAGs were isolated, purified, and characterized with GAG-degrading enzymes. Fibroblasts and vascular smooth muscle cells (VSMCs) synthesized hyaluronic acid, heparan sulfate, and chondroitin sulfates, whereas dermatan sulfate was found only in fibroblasts. Hypoxia did not influence the size or charge of the individual GAGs. However, hypoxia inhibited platelet-derived growth factor-induced [3H]glucosamine incorporation in secreted GAGs, especially hyaluronic acid, in VSMCs. In contrast, it stimulated GAG secretion, specifically heparan sulfate, by fibroblasts. Our results indicate that hypoxia induces modifications in GAG synthesis by human lung VSMCs and fibroblasts that may be correlated to pathophysiological manifestations in lung diseases causing hypoxia.
Chronic intrauterine pulmonary hypertension decreases calcium-sensitive potassium channel mRNA expression
Authors: Cornfield David N. , Resnik Ernesto R. , Herron Jean M. , Abman Steven H.
Published: December 2017

Calcium-sensitive potassium (KCa) channels play a critical role in mediating perinatal pulmonary vasodilation. Because infants with persistent pulmonary hypertension of the newborn (PPHN) have blunted vasodilator responses to birth-related stimuli, we hypothesized that lung KCachannel gene expression is decreased in PPHN. To test this hypothesis, we measured KCa channel gene expression in distal lung homogenates from both fetal lambs with severe pulmonary hypertension caused by prolonged compression of the ductus arteriosus and age-matched, sham-operated animals (controls). After at least 9 days of compression of the ductus arteriosus, fetal lambs were killed. To determine lung KCa channel mRNA levels, primers were designed against the known sequence of the KCa channel and used in semiquantitative RT-PCR, with lung 18S rRNA content as an internal control. Compared to that in control lambs, lung KCa channel mRNA content in the PPHN group was reduced by 26 ± 6% ( P < 0.02), whereas lung voltage-gated K+ 2.1 mRNA content was unchanged. We conclude that lung KCa channel mRNA expression is decreased in an ovine model of PPHN. Decreased KCa channel gene expression may contribute to the abnormal pulmonary vascular reactivity associated with PPHN.
Hoxa-5 in mouse developing lung: cell-specific expression and retinoic acid regulation
Authors: Kim Chiwan , Nielsen Heber C.
Published: December 2017

Hoxa-5 is a homeobox gene that is highly expressed in the developing mouse lung. However, little is known about the molecular mechanisms controlling expression. We characterized the ontogeny of Hoxa-5 gene and protein expressions during lung development and then studied the cell-specific effects of retinoic acid (RA) on Hoxa-5 mRNA in fetal lung fibroblasts and MLE-12 mouse lung epithelial cells. Strong but constant Hoxa-5 gene and protein expressions were detected from mouse lung on embryonic day 13.5 to postnatal day 2. At baseline, the gene was strongly expressed in the fibroblasts of day 17.5 fetal mouse lungs. A very weak but reproducible expression was present in the MLE-12 cells. RA stimulated gene expression in both cell types in a time- and dose-dependent manner. Peak expression occurred much later in the MLE-12 cells compared with that in fibroblasts. Cycloheximide and actinomycin D treatment studies suggested that the differences in RA effect on each cell type may involve the presence of a repressor that can be overcome by RA.
Inhibition of KCa channels restores blunted hypoxic pulmonary vasoconstriction in rats with cirrhosis
Authors: Carter Ethan P. , Sato Koichi , Morio Yoshiteru , McMurtry Ivan F.
Published: December 2017

Rats with liver cirrhosis exhibit the hepatopulmonary syndrome composed of blunted hypoxic pulmonary vasoconstriction and arterial hypoxemia. The purpose of this study was to investigate the roles of nitric oxide (NO) and endothelin-1 (ET-1) in the blunted hypoxic pressor response (HPR) in rats with common bile duct ligation (CBDL). Lungs from CBDL rats exhibited markedly blunted HPR, increased endothelial NO synthase (NOS) protein expression, and decreased ET-1 mRNA and peptide expression. The blunted HPR was not reversed by sequential NOS and soluble guanylyl cyclase inhibition by nitro-l-arginine and 1 H-[1,2,4]oxadiazolo[4,3- a]quinoxaline-1-one (ODQ), respectively, or by NOS inhibition combined with ET-1 addition. The blunted HPR was not due to a generalized inability to vasoconstrict because perfusion pressure was equally elevated by increased perfusate KCl in CBDL and sham lungs. After KCl vasoconstriction, HPR was potentiated and did not differ between CBDL and sham lungs. Blunted HPR was also completely restored in CBDL lungs treated with nitro-l-arginine, ODQ, and the Ca2+-activated K+ channel blockers apamin and charybdotoxin. These results indicate that although CBDL-induced liver cirrhosis is associated with increased NO and decreased ET-1 in the lung, the blunted HPR is a result of additional factors and appears to involve Ca2+-activated K+ channel activation.
Selectivity properties of a Na-dependent amino acid cotransport system in adult alveolar epithelial cells
Authors: Jiang Xinpo , Ingbar David H. , O'Grady Scott M.
Published: December 2017

We investigated the amino acid specificity of a Na-dependent amino acid cotransport system that contributes to transepithelial Na absorption in the apical membrane of cultured adult rat alveolar epithelial cell monolayers. Short-circuit current was increased by basic, uncharged polar, and nonpolar amino acids but not by l-aspartic acid or l-proline. EC50 values for l-lysine andl-histidine were 0.16 and 0.058 mM, respectively. Thel-lysine-stimulated short-circuit current was Na dependent, with a concentration causing a half-maximal stimulation by Na of 44.24 mM. l-Serine, l-glutamine, andl-cysteine had EC50 values of 0.095, 0.25, and 0.12 mM, respectively. l-Alanine had the highest affinity, with an EC50 of 0.027 mM. We conclude that monolayer cultures of adult rat alveolar epithelial cells possess a broad-specificity Na-dependent amino acid cotransport system with properties consistent with system B0,+. We suggest that this cotransport system plays a critical role in recycling of constituent amino acids that make up glutathione, thus ensuring efficient replenishment of this important antioxidant within the alveolar fluid.
Vitronectin adsorption to chrysotile asbestos increases fiber phagocytosis and toxicity for mesothelial cells
Authors: Wu J. , Liu W. , Koenig K. , Idell S. , Broaddus V. C.
Published: December 2017

Biological modification of asbestos fibers can alter their interaction with target cells. We have shown that vitronectin (VN), a major adhesive protein in serum, adsorbs to crocidolite asbestos and increases fiber phagocytosis by mesothelial cells via integrins. Because chrysotile asbestos differs significantly from crocidolite in charge and shape, we asked whether VN would also adsorb to chrysotile asbestos and increase its toxicity for mesothelial cells. We found that VN, either from purified solutions or from serum, adsorbed to chrysotile but at a lower amount per surface area than to crocidolite. Nevertheless, VN coating increased the phagocytosis of chrysotile as well as of crocidolite asbestos. VN coating of both chrysotile and crocidolite, but not of glass beads, increased intracellular oxidation and apoptosis of mesothelial cells. The additional apoptosis could be blocked by integrin-ligand blockade with arginine-glycine-aspartic acid peptides, confirming a role for integrins in the fiber-induced toxicity. We conclude that VN increases the phagocytosis of chrysotile as well as of crocidolite asbestos and that phagocytosis is important in fiber-induced toxicity for mesothelial cells.