bluuebunny/crossref_metadata_2025 huggingface dataset:
Angiotensin II induces apoptosis in human and rat alveolar epithelial cells
Authors: Wang Rongqi , Zagariya Alex , Ibarra-Sunga Olivia , Gidea Claudia , Ang Edmund , Deshmukh Sonali , Chaudhary Gaurav , Baraboutis John , Filippatos Gerasimos , Uhal Bruce D.
Published: December 2017

Recent work from this laboratory demonstrated potent inhibition of apoptosis in human alveolar epithelial cells (AECs) by the angiotensin-converting enzyme inhibitor captopril [B. D. Uhal, C. Gidea, R. Bargout, A. Bifero, O. Ibarra-Sunga, M. Papp, K. Flynn, and G. Filippatos. Am. J. Physiol. 275 ( Lung Cell. Mol. Physiol. 19): L1013–L1017, 1998]. On this basis, we hypothesized that apoptosis in this cell type might be induced by angiotensin II (ANG II) through its interaction with the ANG II receptor. Purified ANG II induced dose-dependent apoptosis in both the human AEC-derived A549 cell line and in primary type II pneumocytes isolated from adult Wistar rats as detected by nuclear and chromatin morphology, caspase-3 activity, and increased binding of annexin V. Apoptosis also was induced in primary rat AECs by purified angiotensinogen. The nonselective ANG II-receptor antagonist saralasin completely abrogated both ANG II- and angiotensinogen-induced apoptosis at a concentration of 50 μg/ml. With RT-PCR, both cell types expressed the ANG II-receptor subtypes 1 and 2 and angiotensin-converting enzyme (ACE). The nonthiol ACE inhibitor lisinopril blocked apoptosis induced by angiotensinogen, but not apoptosis induced by purified ANG II. These data demonstrate the presence of a functional ANG II-dependent pathway for apoptosis in human and rat AECs and suggest a role for the ANG II receptor and ACE in the induction of AEC apoptosis in vivo.
Acute hypoxia increases alveolar macrophage tumor necrosis factor activity and alters NF-κB expression
Authors: Leeper-Woodford Sandra K. , Detmer Kristina
Published: December 2017

Alterations in alveolar macrophage (AM) function during sepsis-induced hypoxia may influence tumor necrosis factor (TNF) secretion and the progression of acute lung injury. Nuclear factor (NF)-κB is thought to regulate the expression of endotoxin [lipopolysaccharide (LPS)]-induced inflammatory cytokines such as TNF, and NF-κB may also be influenced by changes in O2tension. It is thus proposed that acute changes in O2tension surrounding AMs alter NF-κB activation and TNF secretion in these lung cells. AM-derived TNF secretion and NF-κB expression were determined after acute hypoxic exposure of isolated Sprague-Dawley rat AMs. Adhered AMs (106/ml) were incubated (37°C at 5% CO2) for 2 h with LPS ( Pseudomonas aeruginosa, 1 μg/ml) in normoxia (21% O2-5% CO2) or hypoxia (1.8% O2-5% CO2). AM-derived TNF activity was measured with a TNF-specific cytotoxicity assay. Electrophoretic mobility shift and supershift assays were used to determine NF-κB activation and to identify NF-κB isoforms in AM extracts. In addition, mRNAs for selected AM proteins were determined with RNase protection assays. LPS-exposed AMs in hypoxia had higher levels of TNF ( P < 0.05) and enhanced expression of NF-κB ( P < 0.05); the predominant isoforms were p65 and c-Rel. Increased mRNA bands for TNF-α, interleukin-1α, and interleukin-1β were also observed in the hypoxic AMs. These results suggest that acute hypoxia in the lung may induce enhanced NF-κB activation in AMs, which may result in increased production and release of inflammatory cytokines such as TNF.
Cholinomimetic action of macrolide antibiotics on airway gland electrolyte secretion
Authors: Irokawa Toshiya , Sasaki Tsukasa , Shimura Sanae , Sasamori Kan , Oshiro Takako , Nara Masayuki , Tamada Tsutomu , Shirato Kunio
Published: December 2017

We investigated the acute effects of erythromycin (EM) and its derivatives on ionic currents in airway glands from feline tracheae. Therapeutic concentrations of EM or clarithromycin (CAM) attenuated the whole cell currents evoked by ACh in a competitive manner. The maximally stimulated inward Cl−currents were reduced to 54 and 83% and the outward K+currents to 55 and 84% of control values by EM and CAM, respectively, whereas the responses induced by phenylephrine, norepinephrine, caffeine, or ionomycin were unaffected by EM, CAM, or EM523, a synthetic derivative of EM. K+channels in excised outside-out patches were not influenced by macrolides. Although therapeutic concentrations of macrolides showed no effect on the baseline currents, high concentrations of macrolides alone evoked currents mimicking the ACh response, which were abolished completely by atropine. We concluded that macrolides act as a partial agonist on cholinergic receptors, resulting in a reduction of Cl−secretion at pharmacological doses of the agents, which may exhibit a pronounced effectiveness on hypertrophied and/or cholinergically sensitized submucosal glands in pathological airways.
Role of alveolar epithelial cell intercellular adhesion molecule-1 in host defense againstKlebsiella pneumoniae
Authors: O’Brien Aidan D. , Standiford Theodore J. , Bucknell Kathy A. , Wilcoxen Steven E. , Paine Robert
Published: December 2017

Intercellular adhesion molecule-1 (ICAM-1) is expressed at high levels on type I alveolar epithelial cells (AEC) in the normal alveolar space. We postulate that AEC ICAM-1 enhances the antimicrobial activity of macrophages and neutrophils in the alveolar space. Wild-type and mutant mice deficient in ICAM-1 were inoculated intratracheally with Klebsiella pneumoniae. After 10 days, 43% of the ICAM-1 mutant mice had died compared with 14% of the wild-type controls ( P = 0.003). Significantly more bacteria were isolated from lungs of ICAM-1 mutant mice than controls 24 h after inoculation (log colony-forming units 5.14 ± 0.21 vs. 3.46 ± 0.16, P = 0.001). However, neutrophil recruitment to the lung was not different. In similar experiments in the rat, inhibition of alveolar ICAM-1 by intratracheal administration of antibody resulted in significantly impaired clearance of K. pneumoniae. The role of phagocyte interactions with AEC ICAM-1 for antimicrobial activity was investigated in vitro using primary cultures of rat AEC that express abundant ICAM-1. Alveolar macrophage phagocytosis and killing of K. pneumoniae were increased significantly in the presence of AEC; these effects were inhibited significantly (47.5 and 52%, respectively) when AEC ICAM-1 was blocked. Similarly, neutrophil phagocytic activity for K. pneumoniae in the presence of AEC in vitro was decreased when ICAM-1 on the AEC surface was blocked. Thus in the absence of ICAM-1, there is impaired ability to clear K. pneumoniae from the lungs, resulting in increased mortality. These studies indicate that AEC ICAM-1 plays an important role in host defense against K. pneumoniae by determining the antimicrobial activity of phagocytes within the lung.
Expression of the Na+/H+and Cl−/ HCO 3 − exchanger isoforms in proximal and distal human airways
Authors: Dudeja P. K. , Hafez N. , Tyagi S. , Gailey C. A. , Toofanfard M. , Alrefai W. A. , Nazir T. M. , Ramaswamy K. , Al-Bazzaz F. J.
Published: December 2017

Recent studies have indicated the presence of Na+/H+and Cl−/[Formula: see text]exchange activities in lung alveolar and tracheal tissues of various species. To date, the identity of the Na+/H+(NHE) and Cl−/[Formula: see text](AE) exchanger isoforms and their regional distribution in human airways are not known. Molecular species of the NHE and AE gene families and their relative abundance in the human airway regions were assessed utilizing RT-PCR and the RNase protection assay, respectively. Organ donor lung epithelia from various bronchial regions (small, medium, and large bronchi and trachea) were harvested for RNA extraction. Gene-specific primers for the human NHE and AE isoforms were utilized for RT-PCR. Our results demonstrated that NHE1, AE2, and brain AE3 isoforms were expressed in all regions of the human airways, whereas NHE2, NHE3, AE1, and cardiac AE3 were not detected. RNase protection studies for NHE1 and AE2, utilizing glyceraldehyde-3-phosphate dehydrogenase as an internal standard, demonstrated that there were regional differences in the NHE1 mRNA levels in human airways. In contrast, the levels of AE2 mRNA remained unchanged. Differential expression of these isoforms in the human airways may have functional significance related to the airway absorption and secretion of electrolytes.
Establishment of an immortalized fetal intrapulmonary artery endothelial cell line
Authors: Pace Margaret C. , Chambliss Ken L. , German Zohre , Yuhanna Ivan S. , Mendelsohn Michael E. , Shaul Philip W.
Published: December 2017

The investigation of fetal pulmonary endothelial cell gene expression and function has been limited by the requirement for primary cells. In an effort to establish an immortalized cell line, ovine fetal pulmonary artery endothelial cells (PAECs; passage 5) were permanently transfected with the E6 and E7 open reading frames of human papillomavirus type 16, and phenotypes related to nitric oxide (NO) production were evaluated up to passage 28. Acetylated low-density lipoprotein uptake, endothelial NO synthase (eNOS) expression, and proliferation rates were unaltered by immortalization. Acetylcholine-stimulated eNOS activity was 218–255% above basal levels in immortalized cells, and this was comparable to the 250% increase seen in primary PAECs ( passage 6). eNOS was also acutely activated by estradiol to levels 197–309% above basal, paralleling the stimulation obtained in primary cells. In addition, the expression of estrogen receptor-α, which has recently been shown to mediate the acute response in primary PAECs, was conserved. Thus fetal PAECs transfected with E6 and E7 show no signs of senescence with passage, and mechanisms of NO production, including those mediated by estradiol, are conserved. Immortalized PAECs will provide an excellent model for further studies of eNOS gene expression and function in fetal pulmonary endothelium.
Retrovirus-mediated HO gene transfer into endothelial cells protects against oxidant-induced injury
Authors: Yang Liming , Quan Shuo , Abraham Nader G.
Published: December 2017

Heme oxygenase (HO)-1 is a stress protein that has been implicated in defense mechanisms against agents that may induce oxidative injury, such as endotoxins, heme, and cytokines. Overexpression of HO-1 in cells might, therefore, protect against oxidative stress produced by certain agents, specifically heme, by catalyzing its degradation to bilirubin, which by itself has antioxidant properties. We report for the first time the successful transduction of human HO-1 gene into rat lung microvessel endothelium using replication-defective retroviral vector. Cells transduced with human HO-1 gene exhibited a 2.1-fold increase in HO-1 protein level, which was associated with a 2.3-fold elevation in enzyme activity compared with that in nontransduced cells. The cGMP content in transduced endothelial cells was increased by 2.9-fold relative to that in nontransduced cells. Moreover, human HO-1 gene-transduced endothelial cells acquired substantial resistance to toxicity produced by exposure to heme and H2O2compared with that in nontransduced cells. The protective effect of enhancement of HO-1 activity against heme and H2O2was reversed by pretreatment with stannic mesoporphyrin, a competitive inhibitor of HO. These data demonstrate that the induction of HO-1 in response to injurious stimuli represents an important mechanism for moderating the severity of cell damage. Regulation of HO activity in this manner may have clinical applications.
Fatty acid translocase/CD36 mediates the uptake of palmitate by type II pneumocytes
Authors: Guthmann Florian , Haupt Renate , Looman A. Cornelis , Spener Friedrich , Rüstow Bernd
Published: December 2017

Type II pneumocytes, which synthesize, store, and secrete pulmonary surfactant, require exogenous fatty acids, in particular palmitic acid, for maximum surfactant synthesis. The uptake of palmitate by type II pneumocytes is thought to be protein mediated, but the protein involved has not been characterized. Here we show by RT-PCR and Northern blot analysis that rat type II pneumocytes express the mRNA for fatty acid translocase (FAT/CD36), a membrane-associated protein that is known to facilitate the uptake of fatty acids into adipocytes. The deduced amino acid sequence from rat type II pneumocytes reveals 98% identity to the FAT/CD36 sequence obtained from rat adipocytes. The uptake of palmitate by type II pneumocytes follows Michaelis-Menten kinetics (Michaelis-Menten constant = 11.9 ± 1.8 nM; maximum velocity = 62.7 ± 5.8 pmol ⋅ min−1⋅ 5 × 105pneumocytes−1) and decreases reversibly under conditions of ATP depletion to 35% of control uptake. Incubation of cells at 0°C inhibited the uptake of palmitate almost completely, whereas depletion of potassium was without effect. Preincubation of the cells with bromobimane or phloretin decreases the uptake of palmitate significantly as does preincubation with sulfo- N-succinimidyl oleate, the specific inhibitor of FAT/CD36 (C. M. Harmon, P. Luce, A. H. Beth, and N. A. Abumrad. J. Membr. Biol. 121: 261–268, 1991). From these data, we conclude that FAT/CD36 is expressed in type II pneumocytes and mediates the uptake of palmitate in a saturable and energy-dependent manner. The data suggest that the uptake process is independent of the formation of coated pits and endocytotic vesicles.
Intracellular calcium oscillations induced by ATP in airway epithelial cells
Authors: Evans John H. , Sanderson Michael J.
Published: December 2017

In airway epithelial cells, extracellular ATP (ATPo) stimulates an initial transient increase in intracellular Ca2+concentration that is followed by periodic increases in intracellular Ca2+concentration (Ca2+oscillations). The characteristics and mechanism of these ATP-induced Ca2+responses were studied in primary cultures of rabbit tracheal cells with digital video fluorescence microscopy and the Ca2+-indicator dye fura 2. The continual presence of ATPoat concentrations of 0.1–100 μM stimulated Ca2+oscillations that persisted for 20 min. The frequency of the Ca2+oscillations was found to be dependent on both ATPoconcentration and intrinsic sensitivity of each cell to ATPo. Cells exhibited similar Ca2+oscillations to extracellular UTP (UTPo), but the oscillations typically occurred at lower UTPoconcentrations. The ATP-induced Ca2+oscillations were abolished by the phospholipase C inhibitor U-73122 and by the endoplasmic reticulum Ca2+-pump inhibitor thapsigargin but were maintained in Ca2+-free medium. These results are consistent with the hypothesis that in airway epithelial cells ATPoand UTPoact via P2U purinoceptors to stimulate Ca2+oscillations by the continuous production of inositol 1,4,5-trisphosphate and the oscillatory release of Ca2+from internal stores. ATP-induced Ca2+oscillations of adjacent individual cells occurred independently of each other. By contrast, a mechanically induced intercellular Ca2+wave propagated through a field of Ca2+-oscillating cells. Thus Ca2+oscillations and propagating Ca2+waves are two fundamental modes of Ca2+signaling that exist and operate simultaneously in airway epithelial cells.
Enhancement of tumoricidal activity of alveolar macrophages via CD40-CD40 ligand interaction
Authors: Imaizumi Kazuyoshi , Kawabe Tsutomu , Ichiyama Satoshi , Kikutani Hitoshi , Yagita Hideo , Shimokata Kaoru , Hasegawa Yoshinori
Published: December 2017

CD40-CD40 ligand (CD40L) interaction was originally defined as important molecules for the development of humoral immunity. Thereafter, some investigations have focused on its essential roles for the induction of cell-mediated immunity in host defenses. Here we investigated the antitumor activity of murine alveolar macrophages through CD40-CD40L interaction. The CD40L gene was transfected into murine lung cancer cells (3LLSA), and CD40L-expressing clones (3LLSA-CD40L) were established. Stimulation of CD40 molecules on the surface of alveolar macrophages with 3LLSA-CD40L cells induced the production of nitric oxide, tumor necrosis factor-α, and interleukin-12 and the tumoricidal activity of alveolar macrophages in the presence of interferon-γ, which increased the surface expression of CD40 molecules on alveolar macrophages. These findings were not observed when alveolar macrophages were obtained from CD40-deficient mice. On the other hand, interleukin-6 production by alveolar macrophages did not depend on CD40-CD40L interaction. We also established a murine melanoma cell line expressing CD40L (B16 4A5-CD40L) that could induce tumoricidal activity of alveolar macrophages. Furthermore, when spleen cells were cocultivated with 3LLSA-CD40L cells, specific cytotoxic T lymphocytes for wild-type 3LLSA cells could be induced. These results suggest that CD40L gene transfer into tumor cells may induce antitumor immunity in a tumor-bearing host and may offer a new strategy for cancer gene therapy.
Infection of human respiratory submucosal glands with rhinovirus: effects on cytokine and ICAM-1 production
Authors: Yamaya Mutsuo , Sekizawa Kiyohisa , Suzuki Tomoko , Yamada Norihiro , Furukawa Masayuki , Ishizuka Satoshi , Nakayama Katsutoshi , Terajima Masanori , Numazaki Yoshio , Sasaki Hidetada
Published: December 2017

To further understand the early biochemical events that occur in infected surface epithelium, we developed for the first time a model in which a respiratory submucosal gland cell population can be infected with rhinovirus (RV). Viral infection was confirmed by demonstrating with PCR that viral titers in supernatants and lysates from infected cells increased with time. Infection by RV14 upregulated the expression of intercellular adhesion molecule-1 (ICAM-1) mRNA, the major RV receptor, on submucosal gland cells, and it increased production of interleukin (IL)-1α, IL-1β, IL-6, IL-8, tumor necrosis factor-α, and granulocyte-macrophage colony-stimulating factor in supernatants. Antibodies to ICAM-1 inhibited RV infection of submucosal gland cells and decreased the production of cytokines after RV infection. Both IL-1α and IL-1β upregulated ICAM-1 mRNA expression and increased susceptibility to RV infection, whereas other cytokines failed to alter ICAM-1 mRNA expression. Furthermore, neutralizing antibodies to IL-1α and IL-1β significantly decreased the viral titers in supernatants and ICAM-1 mRNA expression after RV infection, but a neutralizing antibody to tumor necrosis factor-α was without effect. These findings suggest that respiratory submucosal gland cells play an important role in the initial stages of inflammation and provide useful insights into the pathogenesis of RV infection.
Ozone-induced acute lung injury: genetic analysis of F2mice generated from A/J and C57BL/6J strains
Authors: Prows Daniel R. , Daly Mark J. , Shertzer Howard G. , Leikauf George D.
Published: December 2017

Acute lung injury (or acute respiratory distress syndrome) is a devastating and often lethal condition. This complex disease (trait) may be associated with numerous candidate genes. To discern the major gene(s) controlling mortality from acute lung injury, two inbred mouse strains displaying contrasting survival times to 10 parts/million ozone were identified. A/J (A) mice were sensitive [6.6 ± 1 (SE) h] and C57BL/6J (B) were resistant (20.6 ± 1 h). The designation for these phenotypes was 13 h, a point that clearly separated their survival time distributions. Our prior segregation studies suggested that survival time to ozone-induced acute lung injury was a quantitative trait, and genetic analysis identified three linked loci [acute lung injury-1, -2, and -3 ( Ali1–3, respectively)]. In this report, acute lung injury in A or B mice was characterized histologically and by measuring lung wet-to-dry weight ratios at death. Ozone produced comparable effects in both strains. To further delineate genetic loci associated with reduced survival, a genomewide scan was performed with F2mice generated from the A and B strains. The results strengthen and extend our initial findings and firmly establish that Ali1 on mouse chromosome 11 has significant linkage to this phenotype. Ali3 was suggestive of linkage, supporting previous recombinant inbred analysis, whereas Ali2 showed no linkage. Together, our findings support the fact that several genes, including Ali1 and Ali3, control susceptibility to death after acute lung injury. Identification of these loci should allow a more focused effort to determine the key events leading to mortality after oxidant-induced acute lung injury.
Adenovirus-mediated decorin gene transfer prevents TGF-β-induced inhibition of lung morphogenesis
Authors: Zhao Jingsong , Sime Patricia J. , Bringas Pablo , Gauldie Jack , Warburton David
Published: December 2017

Excessive transforming growth factor (TGF)-β signaling has been implicated in pulmonary hypoplasia associated with bronchopulmonary dysplasia, a chronic lung disease of human prematurity featuring pulmonary fibrosis. This implies that inhibitors of TGF-β could be useful therapeutic agents. Because exogenous TGF-β ligands are known to inhibit lung branching morphogenesis and cytodifferentiation in mouse embryonic lungs in ex vivo culture, we examined the capacity of a naturally occurring inhibitor of TGF-β activity, the proteoglycan decorin, to overcome the inhibitory effects of exogenous TGF-β. Intratracheal microinjection of a recombinant adenovirus containing decorin cDNA resulted in overexpression of the exogenous decorin gene in airway epithelium. Although exogenous TGF-β efficiently decreased epithelial lung branching morphogenesis in control cultures, TGF-β-induced inhibition of lung growth was abolished after epithelial transfer of the decorin gene. Additionally, exogenous TGF-β-induced antiproliferative effects as well as the downregulation of surfactant protein C were abrogated by decorin in cultured embryonic lungs. Moreover, lung branching inhibition by TGF-β could be restored by the addition of decorin antisense oligodeoxynucleotides in culture, indicating that decorin is both specifically and directly involved in suppressing TGF-β-mediated negative regulation of lung morphogenesis. Our findings suggest that decorin can antagonize bioactive TGF-β during lung growth and differentiation, establishing the rationale for decorin as a candidate therapeutic approach to ameliorate excessive levels of TGF-β signaling in the developing lung.
Vitamin A decreases the incidence and severity of nitrofen-induced congenital diaphragmatic hernia in rats
Authors: Thébaud Bernard , Tibboel Dick , Rambaud Caroline , Mercier Jean-Christophe , Bourbon Jacques R. , Dinh-Xuan A. Tuan , Archer Stephen L.
Published: December 2017

Congenital diaphragmatic hernia (CDH) is a major cause of refractory respiratory failure in the newborn. Pulmonary hypoplasia often limits survival. Vitamin A (Vit A) is an important signal for lung growth. We hypothesized that antenatal treatment with Vit A would stimulate lung growth and decrease mortality in experimental CDH induced in rats by ingestion of the herbicide nitrofen (2,4-dichlorophenyl- p-nitrophenyl ether). Nitrofen was administered to pregnant rats on day 12 of gestation (term 22 days). Rats were assigned to five groups: three groups received one dose of oral antenatal Vit A (15,000 IU) before ( day 10), concomitant with ( day 12), or after ( day 14) nitrofen administration; one group received only nitrofen; and a control group received vehicle (olive oil). The incidence of CDH was markedly lower in all groups receiving Vit A ( day 10, 44%; day 12, 20%; and day 14, 40%) compared with the nitrofen-treated group (84%; P < 0.05). The 72-h survival was higher in all 3 Vit A-treated groups ( day 10, 40%; day 12, 58%; and day 14, 70%) compared with the nitrofen-treated group (16%; P< 0.05). Lung-to-body weight ratio and radial saccular count were significantly increased by Vit A. Antenatal treatment with Vit A lowers the incidence and severity of experimental CDH and increases lung growth and maturation.
Chronic hypoxia alters effects of endothelin and angiotensin on K+ currents in pulmonary arterial myocytes
Authors: Shimoda Larissa A. , Sylvester J. T. , Sham James S. K.
Published: December 2017

We tested the hypothesis that chronic hypoxia alters the regulation of K+ channels in intrapulmonary arterial smooth muscle cells (PASMCs). Charybdotoxin-insensitive, 4-aminopyridine-sensitive voltage-gated K+(KV,CI) and Ca2+-activated K+(KCa) currents were measured in freshly isolated PASMCs from rats exposed to 21 or 10% O2 for 17–21 days. In chronically hypoxic PASMCs, KV,CIcurrent was reduced and KCacurrent was enhanced. 4-Aminopyridine (10 mM) depolarized both normoxic and chronically hypoxic PASMCs, whereas charybdotoxin (100 nM) had no effect in either group. The inhibitory effect of endothelin (ET)-1 (10−7 M) on KV,CI current was significantly reduced in PASMCs from chronically hypoxic rats, whereas inhibition by angiotensin (ANG) II (10−7M) was enhanced. Neither ET-1 nor ANG II altered KCa current in normoxic PASMCs; however, both stimulated KCacurrent at positive potentials in chronically hypoxic PASMCs. These results suggest that although modulation of KV,CI and KCa channels by ET-1 and ANG II is altered by chronic hypoxia, the role of these channels in the regulation of resting membrane potential was not changed.
Angiotensin II activates MAPK and stimulates growth of human pulmonary artery smooth muscle via AT1receptors
Authors: Morrell Nicholas W. , Upton Paul D. , Kotecha Sailesh , Huntley Alyson , Yacoub Magdi H. , Polak Julia M. , Wharton John
Published: December 2017

To determine a potential role for the renin-angiotensin system in the growth of human pulmonary artery (PA) smooth muscle, we studied the localization of angiotensin (ANG) II-receptor subtypes by autoradiography in sections of human PA and in cultured PA smooth muscle cells (PASMCs) and examined the growth responses to ANG II in vitro. Specific125I-labeled [Sar1,Ile8]ANG II binding was demonstrated within the pulmonary arterial media, but binding to cultured cells varied between isolates. Binding in tissues and cells was inhibited by the ANG II type 1 (AT1) receptor antagonist losartan but not by the type 2 (AT2) receptor antagonist PD-123319. Microautoradiographic studies indicated that cultured PASMCs exhibit heterogeneity with regard to ANG II binding sites. Addition of ANG II to serum-deprived PASMCs, exhibiting a relatively high level of125I-[Sar1,Ile8]ANG II binding, led to a dose-dependent stimulation of DNA synthesis at 24 h and protein synthesis at 48 h. ANG II led to an increase in cell size without an increase in cell number. These effects were inhibited by losartan but not by PD-123319. In addition, ANG II led to rapid activation of mitogen-activated protein kinase (MAPK), and ANG II-stimulated DNA synthesis was inhibited by the specific inhibitor of MAPK PD-98059. We conclude that the AT1receptor is expressed by human PASMCs in vivo and in vitro and is coupled to activation of MAPK and increased DNA and protein synthesis in vitro. These results are consistent with the hypothesis that ANG II may be involved in human pulmonary vascular remodeling.
Role of soluble metals in oil fly ash-induced airway epithelial injury and cytokine gene expression
Authors: Dye Janice A. , Adler Kenneth B. , Richards Judy H. , Dreher Kevin L.
Published: December 2017

Particulate matter (PM) metal content and bioavailability have been hypothesized to play a role in the health effects epidemiologically associated with PM exposure, in particular that associated with emission source PM. Using rat tracheal epithelial cells in primary culture, the present study compared and contrasted the acute airway epithelial effects of an emission source particle, residual oil fly ash (ROFA), with that of its principal constitutive transition metals, namely iron, nickel, and vanadium. Over a 24-h period, exposure to ROFA, vanadium, or nickel plus vanadium, but not to iron or nickel, resulted in increased epithelial permeability, decreased cellular glutathione, cell detachment, and lytic cell injury. Treatment of vanadium-exposed cells with buthionine sulfoximine further increased cytotoxicity. Conversely, treatment with the radical scavenger dimethylthiourea inhibited the effects in a dose-dependent manner. RT-PCR analysis of RNA isolated from ROFA-exposed rat tracheal epithelial cells demonstrated significant macrophage inflammatory protein-2 and interleukin-6 gene expression as early as 6 h after exposure, whereas gene expression of inducible nitric oxide synthase was maximally increased 24 h postexposure. Again, vanadium (not nickel) appeared to be mediating the effects of ROFA on gene expression. Treatment with dimethylthiourea inhibited both ROFA- and vanadium-induced gene expression in a dose-dependent manner. Corresponding effects were observed in interleukin-6 and macrophage inflammatory protein-2 synthesis. In summary, generation of an oxidative stress was critical to induction of the ROFA- or vanadium-induced effects on airway epithelial gene expression, cytokine production, and cytotoxicity.
Effect of acrolein on human alveolar macrophage NF-κB activity
Authors: Li Li , Hamilton Raymond F. , Holian Andrij
Published: December 2017

Acrolein is an environmental pollutant that is known to suppress respiratory host defense against infections; however, the mechanism of the decrease in host defense is not yet clear. We have previously reported that acrolein inhibited endotoxin-induced cytokine release and induced apoptosis in human alveolar macrophages, suggesting that the inhibition of cytokine release and/or cytotoxicity to alveolar macrophages may, in part, be responsible for acrolein-induced immunosuppression in the lung. Because nuclear factor-κB (NF-κB) is an important transcription factor for a number of cytokine genes and is also an important regulator of apoptosis, the effect of acrolein on NF-κB activity was examined by electrophoresis mobility shift assay. Acrolein caused a dose-dependent inhibition of endotoxin-induced NF-κB activation as well as an inhibition of basal level NF-κB activity. Because IκB is a principal regulator of NF-κB activity in the nucleus, changes in IκB were determined by Western blotting. Acrolein-inhibited IκB phosphorylation leads to an increase in cellular IκB levels preventing NF-κB nuclear translocation and is likely the mechanism of acrolein-induced inhibition of NF-κB activity. The role of basal level NF-κB in acrolein-induced apoptosis was also examined. An NF-κB inhibitor (MG-132) also induced apoptosis in human alveolar macrophages, suggesting that a certain basal level NF-κB activity may be required for macrophage cell survival. Taken together, our results suggest that the acrolein-inhibited endotoxin-induced NF-κB activation decreased the basal level NF-κB activity, which may be responsible for the inhibition of cytokine release and the induction of apoptosis in human alveolar macrophages.
Potassium channels modulate canine pulmonary vasoreactivity to protein kinase C activation
Authors: Barman Scott A.
Published: December 2017

The role of Ca2+-activated K+-channel, ATP-sensitive K+-channel, and delayed rectifier K+-channel modulation in the canine pulmonary vascular response to protein kinase C (PKC) activation was determined in the isolated blood-perfused dog lung. Pulmonary vascular resistances and compliances were measured with vascular occlusion techniques. The PKC activators phorbol 12-myristate 13-acetate (PMA; 10−7M) and thymeleatoxin (THX; 10−7M) significantly increased pulmonary arterial and pulmonary venous resistances and pulmonary capillary pressure and decreased total vascular compliance by decreasing both microvascular and large-vessel compliances. The Ca2+-activated K+-channel blocker tetraethylammonium ions (1 mM), the ATP-sensitive K+-channel inhibitor glibenclamide (10−5M), and the delayed rectifier K+-channel blocker 4-aminopyridine (10−4M) potentiated the pressor response to both PMA and THX on the arterial and venous segments and also further decreased pulmonary vascular compliance. In contrast, the ATP-sensitive K+-channel opener cromakalim (10−5M) attenuated the vasoconstrictor effect of PMA and THX on both the arterial and venous vessels. In addition, membrane depolarization by 30 mM KCl elicited an increase in the pressor response to PMA. These results indicate that pharmacological activation of PKC elicits pulmonary vasoconstriction. Closure of the Ca2+-activated K+channels, ATP-sensitive K+channels, and delayed rectifier K+channels as well as direct membrane depolarization by KCl potentiated the response to PMA and THX, indicating that K+channels modulate the canine pulmonary vasoconstrictor response to PKC activation.
LPS-induced depolymerization of cytoskeleton and its role in TNF-α production by rat pneumocytes
Authors: Isowa Noritaka , Xavier Alexandre M. , Dziak Ewa , Opas Michal , McRitchie Donna I. , Slutsky Arthur S. , Keshavjee Shaf H. , Liu Mingyao
Published: December 2017

Lipopolysaccharide (LPS) polymerizes microfilaments and microtubules in macrophages and monocytes. Disrupting microfilaments or microtubules with cytochalasin D (CytoD) or colchicine can suppress LPS-induced tumor necrosis factor-α (TNF-α) gene expression and protein production from these cells. We have recently demonstrated that primary cultured rat alveolar epithelial cells can produce TNF-α on LPS stimulation. In the present study, we found that the LPS-induced increase in TNF-α mRNA level and protein production in alveolar epithelial cells was not inhibited by CytoD or colchicine (1 nM to 10 μM). In fact, LPS-induced TNF-α production was further enhanced by CytoD (1–10 μM) and inhibited by jasplakinolide, a polymerizing agent for microfilaments. Immunofluorescent staining and confocal microscopy showed that LPS (10 μg/ml) depolymerized microfilaments and microtubules within 15 min, which was prolonged until 24 h for microfilaments. These results suggest that the effects of LPS on the cytoskeleton and the role of the cytoskeleton in mediating TNF-α production in alveolar epithelial cells are opposite to those in immune cells. This disparity may reflect the different roles between nonimmune and immune cells in host defense.
Enhanced expression of inducible nitric oxide synthase without vasodilator effect in chronically infected lungs
Authors: Cadogan Elaine , Hopkins Natalie , Giles Shay , Bannigan John G. , Moynihan John , McLoughlin Paul
Published: December 2017

We hypothesized that abnormal ventilation-perfusion matching in chronically infected lungs was in part due to excess nitric oxide (NO) production after upregulation of inducible NO synthase (iNOS) expression. Rats were anesthetized and inoculated intratracheally with Pseudomonas aeruginosa incorporated into agar beads (chronically infected) or with sterile agar beads (placebo inoculated) and killed 10–15 days later. Immunohistochemistry demonstrated increased expression of iNOS and reduced expression of endothelial NOS (eNOS) in chronically infected compared with placebo-inoculated or noninoculated lungs. In isolated lungs from chronically infected rats, NOS inhibition with Nω-nitro-l-arginine methyl ester increased the mean perfusion pressure (14.4 ± 2.7 mmHg) significantly more than in the placebo-inoculated (4.8 ± 1.0 mmHg) or noninoculated (5.3 ± 0.8 mmHg) lungs ( P < 0.01). Although the chronically infected lungs were more sensitive to NOS inhibition, further evidence suggested that the increased iNOS expression was not associated with enhanced iNOS activity. Selective inhibitors of iNOS did not produce an increase in vascular resistance similar to that produced by nonselective inhibitors. Accumulation of nitrate/nitrite in the perfusate of isolated lungs was unchanged by chronic infection. Thus although iNOS expression was increased in chronic pulmonary infection, iNOS activity in the intact lung was not. Nonetheless, endogenous NO production was essential to maintain normal vascular resistance in these lungs.
Collagen accumulation is decreased in SPARC-null mice with bleomycin-induced pulmonary fibrosis
Authors: Strandjord Thomas P. , Madtes David K. , Weiss Daniel J. , Sage E. Helene
Published: December 2017

Secreted protein acidic and rich in cysteine (SPARC) has been shown to be coexpressed with type I collagen in tissues undergoing remodeling and wound repair. We speculated that SPARC is required for the accumulation of collagen in lung injury and that its absence would attenuate collagen accumulation. Accordingly, we have assessed levels of collagen in SPARC-null mice in an intratracheal bleomycin-injury model of pulmonary fibrosis. Eight- to ten-week-old SPARC-null and wild-type (WT) mice received bleomycin (0.0035 U/g) or saline intratracheally and were subsequently killed after 14 days. Relative levels of SPARC mRNA were increased 2.7-fold ( P < 0.001) in bleomycin-treated WT lungs in comparison with saline-treated lungs. Protein from bleomycin-treated WT lung contained significantly more hydroxyproline (191.9 μg/lung) than protein from either bleomycin-treated SPARC-null lungs or saline-treated WT and SPARC-null lungs (147.4 μg/lung, 125.4 μg/lung, and 113.0 μg/lung, respectively; P < 0.03). These results indicate that SPARC is increased in response to lung injury and that accumulation of collagen, as indicated by hydroxyproline content, is attenuated in the absence of SPARC. The properties of SPARC as a matricellular protein associated with cell proliferation and matrix turnover are consistent with its participation in the development of pulmonary fibrosis.
Dexfenfluramine increases pulmonary artery smooth muscle intracellular Ca2+, independent of membrane potential
Authors: Reeve Helen L. , Archer Stephen L. , Soper Marjorie , Weir E. Kenneth
Published: December 2017

The anorexic agent dexfenfluramine causes the development of primary pulmonary hypertension in susceptible patients by an unknown mechanism that may include changes in K+-channel activity and intracellular Ca2+concentration ([Ca2+]i). We investigated the dose-dependent effects of dexfenfluramine on [Ca2+]i, K+current, and membrane potential in freshly dispersed rat pulmonary artery smooth muscle cells. Dexfenfluramine caused a dose-dependent (1–1,000 μM) increase in [Ca2+]i, even at concentrations lower than those necessary to inhibit K+currents (10 μM) and cause membrane depolarization (100 μM). The [Ca2+]iresponse to 1 and 10 μM dexfenfluramine was completely abolished by pretreatment of the cells with 0.1 μM thapsigargin, whereas the response to 100 μM dexfenfluramine was reduced. CoCl2(1 mM), removal of extracellular Ca2+, and pretreatment with caffeine (1 mM) reduced but did not abolish the response to 100 μM dexfenfluramine. We conclude that dexfenfluramine increases [Ca2+]iin rat pulmonary artery smooth muscle cells by both release of Ca2+from the sarcoplasmic reticulum and influx of extracellular Ca2+.
Abnormal lung growth and the development of pulmonary hypertension in the Fawn-Hooded rat
Authors: le Cras Timothy D. , Kim Dug-Ha , Gebb Sarah , Markham Neil E. , Shannon John M. , Tuder Rubin M. , Abman Steven H.
Published: December 2017

The Fawn-Hooded rat (FHR) strain develops accelerated and severe pulmonary hypertension when exposed to slight decreases in alveolar[Formula: see text]. 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.
Modeling the interactions of particulates with epithelial lining fluid antioxidants
Authors: Zielinski Henryk , Mudway Ian S. , Bérubé Kelly A. , Murphy Samantha , Richards Roy , Kelly Frank J.
Published: December 2017

Oxidative stress may be a fundamental mode of injury associated with inspired particles. To examine this, we determined the ability of three carbon black particles (CBPs; M120, M880, and R250) and two forms of silicon dioxide, amorphous (Cabosil) and crystalline (DQ12) quartz, to deplete epithelium lining fluid antioxidant defenses. Single and composite antioxidant solutions of uric acid, ascorbic acid (AA), and reduced glutathione (GSH) were examined in the presence of particle concentrations of 150 μg/ml. Uric acid was not depleted by any particle considered. AA was depleted in a near-linear fashion with time by the three different CBPs; however, AA depletion rates varied markedly with CBP type and decreased in the presence of metal chelators. An initially high GSH depletion rate was noted with all CBPs, and this was always accompanied by the appearance of oxidized glutathione. Exposure to Cabosil or DQ12 did not result in the loss of GSH. Together, these data demonstrate that particle type, size, and surface area are all important factors when considering particle-antioxidant interactions in the airways.
Keratinocyte growth factor stimulates bronchial epithelial cell proliferation in vitro and in vivo
Authors: Michelson Peter H. , Tigue Margaret , Panos Ralph J. , Sporn Peter H. S.
Published: December 2017

Airway epithelial cell (AEC) proliferation is crucial to the maintenance of an intact airway surface and the preservation of host defenses. The factors that regulate AEC proliferation are not known. Keratinocyte growth factor (KGF), also known as FGF-7, is a member of the fibroblast growth factor family and a known epithelial cell mitogen. We studied the influence of KGF on the growth of cultured human bronchial epithelial cells and on bronchial cells of rats treated with KGF in vivo. First, we demonstrated the mRNA for the KGF receptor (KGFR) in both normal human bronchial epithelial (NHBE) cells and BEAS-2B cells (a human bronchial epithelial cell line). KGF caused a dose-dependent increase in DNA synthesis, as assessed by thymidine incorporation, in both cell types, with a maximal twofold increase in NHBE cells after 50 ng/ml KGF ( P < 0.001). KGF also induced a doubling in NHBE cell number at 10 ng/ml ( P < 0.001). Finally, we determined the effect of intratracheal administration of KGF to rats on proliferation of AEC in vivo. Measuring bromodeoxyuridine (BrdU) incorporation in AEC nuclei, KGF increased BrdU labeling of rat AEC in both large and small airways by approximately threefold compared with PBS-treated controls ( P < 0.001). Thus KGF induces proliferation of bronchial epithelial cells both in vitro and in vivo.
Critical role of GSH in silica-induced oxidative stress, cytotoxicity, and genotoxicity in alveolar macrophages
Authors: Zhang Zhuo , Shen Han-Ming , Zhang Qi-Feng , Ong Choon-Nam
Published: December 2017

The main objective of this study was to evaluate the critical role of glutathione (GSH) in silica-induced oxidative stress, cytotoxicity, and genotoxicity in rat alveolar macrophages (AMs). Silica-induced superoxide radical and hydrogen peroxide formation were determined with lucigenin-dependent chemiluminescence and 2′,7′-dichlorofluorescin diacetate fluorescence test, respectively. The cytotoxicity of silica was estimated by lactate dehydrogenase leakage, and a comet assay was used for examining silica-induced DNA damage in AMs. The intracellular GSH content was modulated by N-acetylcysteine, a GSH precursor, and buthionine sulfoximine, a specific GSH synthesis inhibitor. It was found that silica led to a dose- and time-dependent decrease in GSH content in AMs. N-acetylcysteine increased intracellular GSH level and protected against silica-induced reactive oxygen species formation, lactate dehydrogenase leakage, and DNA strand breaks in AMs. In contrast, buthionine sulfoximine pretreatment depleted cellular GSH and enhanced the susceptibility of AMs to the cytotoxic and genotoxic effects of silica. It thus appears that GSH plays a critical role in protecting against silica-induced cell injury, most probably through its antioxidant activity.
Surfactant protein A enhances alveolar macrophage phagocytosis of a live, mucoid strain ofP. aeruginosa
Authors: Mariencheck William I. , Savov Jordan , Dong Qun , Tino Michael James , Wright Jo Rae
Published: December 2017

In this study, we investigate the interaction between surfactant protein A (SP-A) and a live, mucoid strain of Pseudomonas aeruginosa and identify a mechanism of clearance of this organism by alveolar macrophages.125I-labeled SP-A bound live, but not heat-killed, P. aeruginosaorganisms in a concentration-dependent manner. Unlabeled SP-A bound live bacteria, protein isolated from whole organisms, and specific proteins of the P. aeruginosa outer membrane. The binding of SP-A to P. aeruginosa and outer membrane components was inhibited by either EDTA or mannose. Phagocytosis assays with fluorescent microscopy demonstrated that the percentage of macrophages with internalized FITC-labeled P. aeruginosa was increased 1.8-fold (19 vs. 35%) by pretreating the live bacteria with SP-A. This finding was confirmed by direct visualization of ingested bacteria by electron microscopy. Adhering macrophages to SP-A-coated surfaces attenuated the increased uptake of P. aeruginosa pretreated with SP-A, suggesting that SP-A acts as an opsonin to stimulate macrophage phagocytosis of this strain of P. aeruginosa.
Increased expression of calreticulin is linked to ANG IV-mediated activation of lung endothelial NOS
Authors: Patel Jawaharlal M. , Li Yong D. , Zhang Jianliang , Gelband Craig H. , Raizada Mohan K. , Block Edward R.
Published: December 2017

This study demonstrates that ANG IV-induced activation of lung endothelial cell nitric oxide synthase (ecNOS) is mediated through mobilization of Ca2+concentration and by increased expression and release of the Ca2+binding protein calreticulin in pulmonary artery endothelial cells (PAEC). In Ca2+-free medium and in the presence of the ANG II AT1and AT2receptor antagonists losartan and PD-123319 (1 μM each), respectively, ANG IV (5, 50, and 500 nM) significantly increased intracellular Ca2+release in PAEC ( P < 0.05 for all concentrations). In contrast, ANG IV-mediated activation of ecNOS was abolished by the intracellular Ca2+chelator 1,2-bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid-AM. ANG IV stimulation resulted in significantly increased expression of calreticulin in cells as well as release of calreticulin into the medium of cells as early as 2 h after ANG IV stimulation ( P < 0.05). Catalytic activity of purified ecNOS in the absence of calmodulin was increased in a concentration-dependent fashion by calreticulin. Immunocoprecipitation studies revealed that ecNOS and calreticulin were coprecipitated in ANG IV-stimulated PAEC. These results demonstrate that ANG IV-mediated activation of ecNOS is regulated by intracellular Ca2+mobilization and by increased expression of calreticulin, which appears to involve interaction of ecNOS and calreticulin proteins in PAEC.