bluuebunny/crossref_metadata_2025 huggingface dataset:
Endotoxin responsiveness and subchronic grain dust-induced airway disease
Authors: George Caroline L. S. , Jin Hong , Wohlford-Lenane Christine L. , O'Neill Marsha E. , Phipps John C. , O'Shaughnessy Patrick , Kline Joel N. , Thorne Peter S. , Schwartz David A.
Published: December 2017

Endotoxin is one of the principal components of grain dust that causes acute reversible airflow obstruction and airway inflammation. To determine whether endotoxin responsiveness influences the development of chronic grain dust-induced airway disease, physiological and airway inflammation remodeling parameters were evaluated after an 8-wk exposure to corn dust extract (CDE) and again after a 4-wk recovery period in a strain of mice sensitive to (C3H/HeBFeJ) and one resistant to (C3H/HeJ) endotoxin. After the CDE exposure, both strains of mice had equal airway hyperreactivity to a methacholine challenge; however, airway hyperreactivity persisted only in the C3H/HeBFeJ mice after the recovery period. Only the C3H/HeBFeJ mice showed significant inflammation of the lower airway after the 8-wk exposure to CDE. After the recovery period, this inflammatory response completely resolved. Lung stereological measurements indicate that an 8-wk exposure to CDE resulted in persistent expansion of the airway submucosal cross-sectional area only in the C3H/HeBFeJ mice. Collagen type III and an influx of cells into the subepithelial area participated in the expansion of the submucosa. Our findings demonstrate that subchronic inhalation of grain dust extract results in the development of chronic airway disease only in mice sensitive to endotoxin but not in mice that are genetically hyporesponsive to endotoxin, suggesting that endotoxin is important in the development of chronic airway disease.
Expression and localization of lung surfactant protein B in Eustachian tube epithelium
Authors: Paananen Reija , Glumoff Virpi , Sormunen Raija , Voorhout Wim , Hallman Mikko
Published: December 2017

Surfactant protein (SP) B is an essential component of the pulmonary surfactant complex, which participates in reducing the surface tension across the alveolar air-liquid interface. The Eustachian tube (ET) connects the upper respiratory tract to the middle ear, serving as an intermittent airway between the pharynx and the middle ear. Recently, we described the expression of SP-A and SP-D in the ET, suggesting their role in middle ear host defense. Our present aim was to detect whether the expression of SP-B is evident in the porcine ET. With Northern blot analysis, RT-PCR, and in situ hybridizations, SP-B mRNA was identified and localized in the ET epithelium. The cellular localization of SP-B was revealed with immunohistochemistry, electron microscopy, and immunoelectron microscopy. The protein was found in the secretory granules of epithelial cells and also attached to the microvilli at the luminal side of these cells. The SP-B immunoreactivity of aggregates isolated from ET lavage fluid was similar to that isolated from bronchoalveolar lavage fluid. We conclude that there are specialized cells in the ET epithelium expressing and secreting SP-B and propose that SP-B may facilitate normal opening of the tube and mucociliary transport.
Requirement for Ca2+signaling in the mechanism of thrombin-induced increase in endothelial permeability
Authors: Sandoval Raudel , Malik Asrar B. , Naqvi Tabassum , Mehta Dolly , Tiruppathi Chinnaswamy
Published: December 2017

We compared the thrombin-activated responses in human umbilical vein endothelial cells (HUVECs) and a HUVEC-derived cell line, ECV304. Thrombin induced a 40–50% decrease in transendothelial monolayer electrical resistance and a twofold increase in125I-albumin permeability in HUVECs, whereas it failed to alter the endothelial barrier function in ECV304 cells. Thrombin produced a brisk intracellular Ca2+concentration transient and phosphorylation of 20-kDa myosin light chain in HUVECs but not in ECV304 cells. Thrombin-induced phosphoinositide hydrolysis was comparable in ECV304 cells and HUVECs, indicating the activation of thrombin receptors in both cell types. La3+reduced both the thrombin-induced decrease in endothelial monolayer electrical resistance and the increase in125I-albumin permeability in HUVECs. Because the absence of Ca2+signaling could explain the impairment in the permeability response in ECV304 cells, we studied the effect of increasing intracellular Ca2+concentration in ECV304 cells with thapsigargin. Exposure of ECV304 cells to thapsigargin caused decreased endothelial monolayer electrical resistance and increased125I-albumin permeability. These results indicate that Ca2+influx and activation of Ca2+-dependent signaling pathways are important determinants of the thrombin-induced increase in endothelial permeability.
The NO donor molsidomine reduces endothelin-1 gene expression in chronic hypoxic rat lungs
Authors: Blumberg Friedrich C. , Wolf Konrad , Sandner Peter , Lorenz Cornelia , Riegger Günter A. J. , Pfeifer Michael
Published: December 2017

We investigated the effects of the nitric oxide (NO) donor molsidomine and the nitric oxide synthase inhibitor N-nitro-l-arginine methyl ester (l-NAME) on pulmonary endothelin (ET)-1 gene expression and ET-1 plasma levels in chronic hypoxic rats. Two and four weeks of hypoxia (10% O2) significantly increased right ventricular systolic pressure, the medial cross-sectional vascular wall area of the pulmonary arteries, and pulmonary ET-1 mRNA expression (2-fold and 3.2-fold, respectively). ET-1 plasma levels were elevated after 4 wk of hypoxia. In rats exposed to 4 wk of hypoxia, molsidomine (15 mg · kg−1· day−1) given either from the beginning or after 2 wk of hypoxia significantly reduced pulmonary hypertension, pulmonary vascular remodeling, pulmonary ET-1 gene expression, and ET-1 plasma levels.l-NAME administration (45 mg · kg−1· day−1) in rats subjected to 2 wk of hypoxia did not modify these parameters. Our findings suggest that in chronic hypoxic rats, exogenously administered NO acts in part by suppressing the formation of ET-1. In contrast, inhibition of endogenous NO production exerts only minor effects on the pulmonary circulation and pulmonary ET-1 synthesis in these animals.
Cytokines decrease sGC in pulmonary artery smooth muscle cells via NO-dependent and NO-independent mechanisms
Authors: Takata Masao , Filippov Galina , Liu Heling , Ichinose Fumito , Janssens Stefan , Bloch Donald B. , Bloch Kenneth D.
Published: December 2017

Exposure of rat pulmonary artery smooth muscle cells (rPASMC) to cytokines leads to nitric oxide (NO) production by NO synthase 2 (NOS2). NO stimulates cGMP synthesis by soluble guanylate cyclase (sGC), a heterodimer composed of α1- and β1-subunits. Prolonged exposure of rPASMC to NO decreases sGC subunit mRNA and protein levels. The objective of this study was to determine whether levels of NO produced endogenously by NOS2 are sufficient to decrease sGC expression in rPASMC. Interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) increased NOS2 mRNA levels and decreased sGC subunit mRNA levels. Exposure of rPASMC to IL-1β and TNF-α for 24 h decreased sGC subunit protein levels and NO-stimulated sGC enzyme activity.l- N6-(1-iminoethyl)lysine (NOS2 inhibitor) or 1 H-[1,2,4]oxadiazolo-[4,3- a]quinoxalin-1-one (sGC inhibitor) partially prevented the cytokine-mediated decrease in sGC subunit mRNA levels. However, cytokines also decreased sGC subunit mRNA levels in PASMC derived from NOS2-deficient mice. These results demonstrate that levels of NO and cGMP produced in cytokine-exposed PASMC are sufficient to decrease sGC subunit mRNA levels. In addition, cytokines can decrease sGC subunit mRNA levels via NO-independent mechanisms.
Neutrophils induce apoptosis of lung epithelial cells via release of soluble Fas ligand
Authors: Serrao Karl L. , Fortenberry James D. , Owens Marilyn L. , Harris Frank L. , Brown Lou Ann S.
Published: December 2017

Neutrophils release soluble Fas ligand (sFasL), which can induce apoptosis in certain Fas-bearing cell types (Liles WC, Kiener PA, Ledbetter JA, Aruffo A, and Klebanoff SJ. J Exp Med 184: 429–440, 1996). We hypothesized that neutrophils could induce alveolar epithelial apoptosis via release of sFasL. A549 pulmonary adenocarcinoma cells expressed surface Fas and underwent cell death (10 ± 7% viability) and DNA fragmentation (354 ± 98% of control cells) when incubated with agonistic CD95/Fas monoclonal antibody ( P < 0.05). Coincubation with human neutrophils induced significant A549 cell death at 48 (51 ± 9% viability; P < 0.05) and 72 h (25 ± 10%; P < 0.05) and increased DNA fragmentation (178 ± 42% of control cells; P < 0.05), with morphological characteristics of apoptosis. The addition of antioxidants did not inhibit apoptosis. sFasL concentrations were maximally increased in coculture medium at 24 h (4.9 ± 0.7 ng/ml; P < 0.05). Neutrophil-induced A549 cell apoptosis was blocked by inhibitory anti-Fas (42 ± 6% of control cells; P < 0.05) and anti-FasL monoclonal antibodies (29 ± 3%; P < 0.05). Human neutrophils and Fas similarly affected murine primary alveolar epithelial cell bilayers, and caspase activation occurred in response to Fas exposure. We conclude that neutrophils undergoing spontaneous apoptosis induce A549 cell death and DNA fragmentation, independent of the oxidative burst, that is mediated by sFasL.
Attenuation of bleomycin-induced pneumopathy in mice by a caspase inhibitor
Authors: Kuwano Kazuyoshi , Kunitake Ritsuko , Maeyama Takashige , Hagimoto Naoki , Kawasaki Masayuki , Matsuba Tokuji , Yoshimi Michihiro , Inoshima Ichiro , Yoshida Koichiro , Hara Nobuyuki
Published: December 2017

Caspases have been implicated in the effector process of apoptosis in several systems including the Fas-Fas ligand pathway. We previously demonstrated that excessive apoptosis of lung epithelial cells and the Fas-Fas ligand pathway were essential in the pathogenesis of bleomycin-induced pneumopathy in mice. Therefore, the purpose of this study was to investigate whether a caspase inhibitor could prevent the development of this model. The expression of caspase-1 and caspase-3 was upregulated on lung epithelial cells, alveolar macrophages, and infiltrating inflammatory cells in this model. We demonstrated that a broad-spectrum caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, decreased the caspase-1- and caspase-3-like activity, the number of apoptotic cells, the pathological grade of lung inflammation and fibrosis, and the hydroxyproline content in lung tissues in this model. We conclude that caspase inhibitors could be a new therapeutic approach against lung injury and pulmonary fibrosis.
Regulation of PDGFR-α in rat pulmonary myofibroblasts by staurosporine
Authors: Lindroos Pamela M. , Wang Yi-Zhe , Rice Annette B. , Bonner James C.
Published: December 2017

Upregulation of the platelet-derived growth factor (PDGF) receptor-α (PDGFR-α) is a mechanism of myofibroblast hyperplasia during pulmonary fibrosis. We previously identified interleukin (IL)-1β as a major inducer of the PDGFR-α in rat pulmonary myofibroblasts in vitro. In this study, we report that staurosporine, a broad-spectrum kinase inhibitor, upregulates PDGFR-α gene expression and protein. A variety of other kinase inhibitors did not induce PDGFR-α expression. Staurosporine did not act via an IL-1β autocrine loop because the IL-1 receptor antagonist protein did not block staurosporine-induced PDGFR-α expression. Furthermore, staurosporine did not activate a variety of signaling molecules that were activated by IL-1β, including nuclear factor-κB, extracellular signal-regulated kinase, and c-Jun NH2-terminal kinase. However, both staurosporine- and IL-1β-induced phosphorylation of p38 mitogen-activated protein kinase and upregulation of PDGFR-α by these two agents was inhibited by the p38 inhibitor SB-203580. Finally, staurosporine inhibited basal and PDGF-stimulated mitogenesis over the same concentration range that induced PDGFR-α expression. Collectively, these data demonstrate that staurosporine is a useful tool for elucidating the signaling mechanisms that regulate PDGFR expression in lung connective tissue cells and possibly for evaluating the role of the PDGFR-α as a growth arrest-specific gene.
Arsenite stimulates plasma membrane NADPH oxidase in vascular endothelial cells
Authors: Smith Karol R. , Klei Linda R. , Barchowsky Aaron
Published: December 2017

Low-level arsenite treatment of porcine aortic endothelial cells (PAEC) stimulated superoxide accumulation that was attenuated by inhibitors of NAD(P)H oxidase. To demonstrate whether arsenite stimulated NADPH oxidase, intact PAEC were treated with arsenite for up to 2 h and membrane fractions were prepared to measure NADPH oxidase activity. Arsenite (5 μM) stimulated a twofold increase in activity by 1 h, which was inhibited by the oxidase inhibitor diphenyleneiodonium chloride. Direct treatment of isolated membranes with arsenite had no effect. Analysis of NADPH oxidase components revealed that p67phoxlocalized exclusively to membranes of both control and treated cells. In contrast, cytosolic Rac1 translocated to the membrane fractions of cells treated with arsenite or angiotensin II but not with tumor necrosis factor. Immunodepletion of p67phoxblocked oxidase activity stimulated by all three compounds. However, depleting Rac1 inhibited responses only to arsenite and angiotensin II. These data demonstrate that stimulus-specific activation of NADPH oxidase in endothelial cells was the source of reactive oxygen in endothelial cells after noncytotoxic arsenite exposure.
Cytoskeletal regulation of thel-arginine/NO pathway in pulmonary artery endothelial cells
Authors: Zharikov Sergey I. , Sigova Alla A. , Chen Sifeng , Bubb Michael R. , Block Edward R.
Published: December 2017

We investigated possible involvement of the actin cytoskeleton in the regulation of thel-arginine/nitric oxide (NO) pathway in pulmonary artery endothelial cells (PAEC). We exposed cultured PAEC to swinholide A (Swinh), which severs actin microfilaments, or jasplakinolide (Jasp), which stabilizes actin filaments and promotes actin polymerization, or both. After treatment, the state of the actin cytoskeleton,l-arginine uptake mediated by the cationic amino acid transporter-1 (CAT-1), Ca2+/calmodulin-dependent (endothelial) NO synthase (eNOS) activity and content, and NO production were examined. Jasp (50–100 nM, 2 h treatment) induced a reversible activation ofl-[3H]arginine uptake by PAEC, whereas Swinh (10–50 nM) decreased l-[3H]arginine uptake. The two drugs could abrogate the effect of each other onl-[3H]arginine uptake. The effects of both drugs on l-[3H]arginine transport were not related to changes in expression of CAT-1 transporters. Swinh (50 nM, 2 h) and Jasp (100 nM, 2 h) did not change eNOS activities and contents in PAEC. Detection of NO in PAEC by the fluorescent probe 4,5-diaminofluorescein diacetate showed that Swinh (50 nM) decreased and Jasp (100 nM) increased NO production by PAEC. The stimulatory effect of Jasp on NO production was dependent on the availability of extracellular l-arginine. Our results indicate that the state of actin microfilaments in PAEC regulates l-arginine transport and that this regulation can affect NO production by PAEC.
Proinflammatory cytokine responses toP. aeruginosainfection in human airway epithelial cell lines
Authors: Kube Dianne , Sontich Ute , Fletcher David , Davis Pamela B.
Published: December 2017

A tendency toward excessive inflammation in cystic fibrosis (CF) patients often accompanies lung infections with Pseudomonas aeruginosa. We tested the cytokine response to P. aeruginosa in two pairs of human airway epithelial cell lines matched except for CF transmembrane conductance regulator activity. The 9/HTEo−CF-phenotypic cell line produced significantly more interleukin (IL)-8, IL-6, and granulocyte-macrophage colony-stimulating factor but not regulated on activation normal T cell expressed and secreted (RANTES) in response to Pseudomonas than the 9/HTEo−control line, and the differences widened over time. Similarly, a 16HBE cell line lacking transmembrane conductance regulator activity showed enhanced IL-8 and IL-6 responses compared with the control cell line. The pharmacology of the cytokine response also differed because dexamethasone reduced cytokine production to similar levels in the matched cell lines. The protracted proinflammatory cytokine response of the CF-phenotypic cell lines suggests that the limiting mechanisms of normal cells are absent or attenuated. These results are consistent with in vivo observations in patients with CF and suggest that our novel cell lines may be useful for further investigation of the proinflammatory responses in CF airways.
Intra-amniotic endotoxin: chorioamnionitis precedes lung maturation in preterm lambs
Authors: Kallapur Suhas G. , Willet Karen E. , Jobe Alan H. , Ikegami Machiko , Bachurski Cindy J.
Published: December 2017

The inflammatory and lung maturational effects of intra-amniotic exposure to endotoxin were assessed in fetal lambs. Five hours to 25 days after intra-amniotic injection of endotoxin, preterm lambs were delivered at 119–125 days gestation. Intra-amniotic endotoxin caused an inflammatory cell infiltration in amnion/chorion at 5 h, which persisted for 25 days. At 5–15 h after endotoxin, amnion/chorion cytokine mRNAs increased [12- to 26-fold for interleukin (IL)-1β, IL-6, and IL-8 mRNA and 3-fold for tumor necrosis factor-α mRNA]. At 1–2 days after endotoxin, lung cytokine mRNAs increased 6- to 49-fold. Endotoxin caused modest changes in peripheral white blood cell counts and no significant cytokine mRNA responses in fetal liver, placenta, or jejunum. Lung maturation, as characterized by increased lung volumes and alveolar saturated phosphatidylcholine, occurred at 7 days and persisted for 25 days after endotoxin. We conclude that exposure to a single dose of intra-amniotic endotoxin causes inflammation and increases in cytokine mRNA in amnion/chorion and the fetal lung before lung maturation, consistent with the hypothesis that proinflammatory cytokines signal lung maturation.
B-cell isotype control in atopy and asthma assessed with cDNA array technology
Authors: Brutsche Martin H. , Brutsche Ingrid Carlen , Wood Peter , Mogulkoc Nesrin , Custovic Adnan , Egan Jim , Woodcock Ashley
Published: December 2017

B-cell isotype switching and the production of IgE is regulated by a variety of gene products through different mechanisms. A better understanding of these processes has the potential to identify markers of disease and new therapeutic targets. The aim of the study was to investigate human B-cell isotype control and IgE production in atopy and asthma with cDNA array technology. Eighteen atopic asthmatic, eight atopic nonasthmatic, and fourteen healthy control subjects were included. Peripheral blood mononuclear cells were separated by gradient centrifugation, mRNA was purified, and the reverse-transcribed probes were hybridized to cDNA membranes. Group differences were assessed with the Mann-Whitney U-test. Twenty-three of seventy-eight tested IgE-related genes had significantly altered expression in atopy and asthma compared with that in the healthy subjects. The differentially expressed genes include surface molecules involved in T- and B-cell interaction and activation, cytokines, intracellular signaling products, and transcription factors. In conclusion, both atopic nonasthmatic and atopic asthmatic individuals had activated proinflammatory pathways, a minimal requirement for B-cell isotype switching, and a clear net pro-IgE cytokine climate.
Differential expression of forkhead box transcription factors following butylated hydroxytoluene lung injury
Authors: Kalinichenko Vladimir V. , Lim Lorena , Shin Brian , Costa Robert H.
Published: December 2017

The forkhead box (Fox) proteins are a growing family of transcription factors that have important roles in cellular proliferation and differentiation and in organ morphogenesis. The Fox family members hepatocyte nuclear factor (HNF)-3β (Foxa2) and HNF-3/forkhead homolog (HFH)-8 (FREAC-1, Foxf1) are expressed in adult pulmonary epithelial and mesenchymal cells, respectively, but these cells display only low expression levels of the proliferation-specific HFH-11B gene (Trident, Foxm1b). The regulation of these Fox transcription factors in response to acute lung injury, however, has yet to be determined. We report here on the use of butylated hydroxytoluene (BHT)-mediated lung injury to demonstrate that HFH-11 protein and RNA levels were markedly increased throughout the period of lung repair. The maximum levels of HFH-11 were observed by day 2 following BHT injury when both bronchiolar and alveolar epithelial cells were undergoing extensive proliferation. Although BHT lung injury did not alter epithelial cell expression of HNF-3β, a 65% reduction in HFH-8 mRNA levels was observed during the period of mesenchymal cell proliferation. HFH-8-expressing cells were colocalized with platelet endothelial cell adhesion molecule-1-positive alveolar endothelial cells and with α-smooth muscle actin-positive peribronchiolar smooth muscle cells.
FGF-10 disrupts lung morphogenesis and causes pulmonary adenomas in vivo
Authors: Clark Jean C. , Tichelaar Jay W. , Wert Susan E. , Itoh Nobuyuki , Perl Anne-Karina T. , Stahlman Mildred T. , Whitsett Jeffrey A.
Published: December 2017

Transgenic mice in which fibroblast growth factor (FGF)-10 was expressed in the lungs of fetal and postnatal mice were generated with a doxycycline-inducible system controlled by surfactant protein (SP) C or Clara cell secretory protein (CCSP) promoter elements. Expression of FGF-10 mRNA in the fetal lung caused adenomatous malformations, perturbed branching morphogenesis, and caused respiratory failure at birth. When expressed after birth, FGF-10 caused multifocal pulmonary tumors. FGF-10-induced tumors were highly differentiated papillary and lepidic pulmonary adenomas. Epithelial cells lining the tumors stained intensely for thyroid transcription factor (TTF)-1 and SP-C but not CCSP, indicating that FGF-10 enhanced differentiation of cells to a peripheral alveolar type II cell phenotype. Withdrawal from doxycycline caused rapid regression of the tumors associated with rapid loss of the differentiation markers TTF-1, SP-B, and proSP-C. FGF-10 disrupted lung morphogenesis and induced multifocal pulmonary tumors in vivo and caused reversible type II cell differentiation of the respiratory epithelium.
Permeability of endothelial monolayers to albumin is increased by bradykinin and inhibited by prostaglandins
Authors: Farmer Pierre J. , Bernier Sylvie G. , Lepage Andrée , Guillemette Gaétan , Regoli Domenico , Sirois Pierre
Published: December 2017

Using monolayers of bovine aortic endothelial cells (BAEC) in modified Boyden chambers, we examined the role of prostaglandins (PGs) in the bradykinin (BK)-induced increase of albumin permeability. BK induced a concentration-dependent increase of the permeability of BAEC, which reached 49.9 ± 1% at the concentration of 10−8M. Two inhibitors of the prostaglandin G/H synthase, indomethacin (2.88 μM) and ibuprofen (10 μM), potentiated BK-induced permeability 1.8- and 3.9-fold, respectively. Exogenously administered PGE2and iloprost, a stable analog of prostacyclin, attenuated the effect of BK in a concentration-dependent manner. Butaprost equally reduced the effect of BK, suggesting the participation of the EP2receptor in this phenomenon. However, the EP4-selective antagonist AH-23848 did not significantly inhibit the protective effect of PGE2. The inhibitory effect of PGE2was reversed by the adenylate cyclase inhibitor MDL-12330A (10 μM). These results suggest that BK-induced increase of permeability of BAEC monolayer to125I-labeled albumin is negatively regulated by PGs. This postulated autocrine activity of PGs may involve an increase in the intracellular level of cAMP.
Role of LPS-induced microfilament depolymerization in MIP-2 production from rat pneumocytes
Authors: Isowa Noritaka , Liu Mingyao
Published: December 2017

We have previously demonstrated that lipopolysaccharide (LPS) induces production of macrophage inflammatory protein-2 (MIP-2), a C-X-C chemokine for neutrophil recruitment and activation, in primary cultured rat lung alveolar epithelial cells. We have also demonstrated that LPS depolymerizes microfilaments in rat alveolar epithelial cells. To determine whether the polymerization status of microfilaments affects LPS-induced MIP-2 production, we treated rat alveolar epithelial cells with cytochalasin D (CytoD), a microfilament-disrupting agent, before and during LPS stimulation. A lower concentration (0.1 μM) of CytoD inhibited LPS-induced MIP-2 production without affecting microfilament polymerization. In contrast, LPS-induced MIP-2 production was enhanced by a higher concentration (10 μM) of CytoD, which disrupted the filamentous structure of actin. Jasplakinolide (1 nM to 1 μM), a polymerizing agent for microfilaments, decreased LPS-induced MIP-2 secretion. Jasplakinolide (1 μM) also blocked LPS-induced depolymerization of microfilaments. These results suggest that, in alveolar epithelial cells, LPS-induced MIP-2 production is at least partially regulated by microfilament depolymerization.
Chronic hypoxia decreases KVchannel expression and function in pulmonary artery myocytes
Authors: Platoshyn Oleksandr , Yu Ying , Golovina Vera A. , McDaniel Sharon S. , Krick Stefanie , Li Li , Wang Jian-Ying , Rubin Lewis. J. , Yuan Jason X.-J.
Published: December 2017

Activity of voltage-gated K+(KV) channels regulates membrane potential ( Em) and cytosolic free Ca2+concentration ([Ca2+]cyt). A rise in [Ca2+]cytin pulmonary artery (PA) smooth muscle cells (SMCs) triggers pulmonary vasoconstriction and stimulates PASMC proliferation. Chronic hypoxia (Po230–35 mmHg for 60–72 h) decreased mRNA expression of KVchannel α-subunits (Kv1.1, Kv1.5, Kv2.1, Kv4.3, and Kv9.3) in PASMCs but not in mesenteric artery (MA) SMCs. Consistently, chronic hypoxia attenuated protein expression of Kv1.1, Kv1.5, and Kv2.1; reduced KVcurrent [ IK(V)]; caused Emdepolarization; and increased [Ca2+]cytin PASMCs but negligibly affected KVchannel expression, increased IK(V), and induced hyperpolarization in MASMCs. These results demonstrate that chronic hypoxia selectively downregulates KVchannel expression, reduces IK(V), and induces Emdepolarization in PASMCs. The subsequent rise in [Ca2+]cytplays a critical role in the development of pulmonary vasoconstriction and medial hypertrophy. The divergent effects of hypoxia on KVchannel α-subunit mRNA expression in PASMCs and MASMCs may result from different mechanisms involved in the regulation of KVchannel gene expression.
Blockade of CD49d inhibits allergic airway pathologies independent of effects on leukocyte recruitment
Authors: Borchers Michael T. , Crosby J. , Farmer S. , Sypek J. , Ansay T. , Lee N. A. , Lee J. J.
Published: December 2017

Lymphocyte and/or eosinophil recruitment is dependent on the sequential interactions between adhesion molecules expressed on activated endothelial cells and both leukocyte subtypes. Endothelial P- and E-selectins mediate tethering and rolling of leukocytes through interactions with P-selectin glycoprotein ligand-1 (PSGL-1), and diapedesis subsequently occurs by engagement of endothelial vascular cell adhesion molecule-1 and CD49d (α4-integrins). The anti-inflammatory potential of interfering with these adhesive interactions was assessed with an ovalbumin challenge mouse model of asthma. Administration of a soluble form of PSGL-1 reduced eosinophils (80%) and lymphocytes (50%) in bronchoalveolar lavage fluid without affecting epithelial changes or airway hyperreactivity (AHR). In contrast, although administration of anti-CD49d monoclonal antibodies (PS/2) resulted in similar reductions in eosinophils (75%) and lymphocytes (50%), PS/2 reduced and abolished mucous cell metaplasia and AHR, respectively. Administration of both PSGL-1 and PS/2 had the additive effect of eliminating eosinophils from the airways (96% decrease), with few or no additional reductions (relative to PS/2 administration alone) in lymphocyte recruitment, mucous cell metaplasia, or AHR. These data show that eosinophils and lymphocytes differentially utilize adhesive interactions during recruitment and that the inhibition of AHR is independent of this recruitment.
Endothelin B receptor deficiency potentiates ET-1 and hypoxic pulmonary vasoconstriction
Authors: Ivy D. Dunbar , McMurtry Ivan F. , Yanagisawa Masashi , Gariepy Cheryl E. , Le Cras Timothy D. , Gebb Sarah A. , Morris Kenneth G. , Wiseman Richard C. , Abman Steven H.
Published: December 2017

Endothelin (ET)-1 contributes to the regulation of pulmonary vascular tone by stimulation of the ETA and ETB receptors. Although activation of the ETA receptor causes vasoconstriction, stimulation of the ETB receptors can elicit either vasodilation or vasoconstriction. To examine the physiological role of the ETB receptor in the pulmonary circulation, we studied a genetic rat model of ETB receptor deficiency [transgenic( sl/ sl)]. We hypothesized that deficiency of the ETB receptor would predispose the transgenic( sl/ sl) rat lung circulation to enhanced pulmonary vasoconstriction. We found that the lungs of transgenic( sl/ sl) rats are ETBdeficient because they lack ETB mRNA in the pulmonary vasculature, have minimal ETB receptors as determined with an ET-1 radioligand binding assay, and lack ET-1-mediated pulmonary vasodilation. The transgenic( sl/ sl) rats have higher basal pulmonary arterial pressure and vasopressor responses to brief hypoxia or ET-1 infusion. Plasma ET-1 levels are elevated and endothelial nitric oxide synthase protein content and nitric oxide production are diminished in the transgenic( sl/ sl) rat lung. These findings suggest that the ETB receptor plays a major physiological role in modulating resting pulmonary vascular tone and reactivity to acute hypoxia. We speculate that impaired ETB receptor activity can contribute to the pathogenesis of pulmonary hypertension.
Isolation of pulmonary interstitial fluid in rabbits by a modified wick technique
Authors: Negrini Daniela , Passi Alberto , Bertin Katia , Bosi Federica , Wiig Helge
Published: December 2017

Interstitial fluid protein concentration (Cprotein) values in perivascular and peribronchial lung tissues were never simultaneously measured in mammals; in this study, perivascular and peribronchial interstitial fluids were collected from rabbits under control conditions and rabbits with hydraulic edema or lesional edema. Postmortem dry wicks were implanted in the perivascular and peribronchial tissues; after 20 min, the wicks were withdrawn and the interstitial fluid was collected to measure Cprotein and colloid osmotic pressure. Plasma, perivascular, and peribronchial Cproteinvalues averaged 6.4 ± 0.7 (SD), 3.7 ± 0.5, and 2.4 ± 0.7 g/dl, respectively, in control rabbits; 4.8 ± 0.7, 2.5 ± 0.6, and 2.4 ± 0.4 g/dl, respectively, in rabbits with hydraulic edema; and 5.1 ± 0.3, 4.3 ± 0.4 and 3.3 ± 0.6 g/dl, respectively, in rabbits with lesional edema. Contamination of plasma proteins from microvascular lesions during wick insertion was 14% of plasma Cprotein. In control animals, pulmonary interstitial Cprotein was lower than previous estimates from pre- and postnodal pulmonary lymph; furthermore, although the interstitium constitutes a continuum within the lung parenchyma, regional differences in tissue content seem to exist in the rabbit lung.
Hypoxic constriction of porcine distal pulmonary arteries: endothelium and endothelin dependence
Authors: Liu Q. , Sham J. S. K. , Shimoda L. A. , Sylvester J. T.
Published: December 2017

To determine the role of endothelium in hypoxic pulmonary vasoconstriction (HPV), we measured vasomotor responses to hypoxia in isolated seventh-generation porcine pulmonary arteries < 300 μm in diameter with (E+) and without endothelium. In E+ pulmonary arteries, hypoxia decreased the vascular intraluminal diameter measured at a constant transmural pressure. These constrictions were complete in 30–40 min; maximum at Po 2 of 2 mmHg; half-maximal at Po 2 of 40 mmHg; blocked by exposure to Ca2+-free conditions, nifedipine, or ryanodine; and absent in E+ bronchial arteries of similar size. Hypoxic constrictions were unaltered by indomethacin, enhanced by indomethacin plus N G-nitro-l-arginine methyl ester, abolished by BQ-123 or endothelial denudation, and restored in endothelium-denuded pulmonary arteries pretreated with 10−10 M endothelin-1 (ET-1). Given previous demonstrations that hypoxia caused contractions in isolated pulmonary arterial myocytes and that ET-1 receptor antagonists inhibited HPV in intact animals, our results suggest that full in vivo expression of HPV requires basal release of ET-1 from the endothelium to facilitate mechanisms of hypoxic reactivity in pulmonary arterial smooth muscle.
Synexin and GTP increase surfactant secretion in permeabilized alveolar type II cells
Authors: Chander Avinash , Sen Namita , Spitzer Alan R.
Published: December 2017

We have previously suggested that synexin (annexin VII), a Ca2+-dependent phospholipid binding protein, may have a role in surfactant secretion, since it promotes membrane fusion between isolated lamellar bodies (the surfactant-containing organelles) and plasma membranes. In this study, we investigated whether exogenous synexin can augment surfactant phosphatidylcholine (PC) secretion in synexin-deficient lung epithelial type II cells. Isolated rat type II cells were cultured for 20–22 h with [3H]choline to label cellular PC. The cells were then treated with β-escin, which forms pores in the cell membrane and releases cytoplasmic proteins including synexin. These cells, however, retained lamellar bodies. The permeabilized type II cells were evaluated for PC secretion during a 30-min incubation. Compared with PC secretion under basal conditions, the presence of Ca2+(up to 10 μM) did not increase PC secretion. In the presence of 1 μM Ca2+, synexin increased PC secretion in a concentration-dependent manner, which reached a maximum at ∼5 μg/ml synexin. The secretagogue effect of synexin was abolished when synexin was inactivated by heat treatment (30 min at 65°C) or by treatment with synexin antibodies. GTP or its nonhydrolyzable analog β:γ-imidoguanosine-5′-triphosphate also increased PC secretion in permeabilized type II cells. The PC secretion was further increased in an additive manner when a maximally effective concentration of synexin was added in the presence of 1 mM GTP, suggesting that GTP acts by a synexin-independent mechanism to increase membrane fusion. Thus our results support a direct role for synexin in surfactant secretion. Our study also suggests that membrane fusion during surfactant secretion may be mediated by two independent mechanisms.
Isoprostanes: an overview and putative roles in pulmonary pathophysiology
Authors: Janssen L. J.
Published: December 2017

Isoprostanes are produced during peroxidation of membrane lipids by free radicals and reactive oxygen species. Initially, they were recognized as being valuable markers of oxidative stress, and in the past 10 years, dozens of disease states and experimental conditions with diverse etiologies have been shown to be associated with marked increases in urinary, plasma, and tissue levels of isoprostanes. However, they are not just mere markers; they evoke important biological responses on virtually every cell type found within the lung, and these responses exhibit compound-, tissue-, and species-related variations. In fact, the isoprostanes may mediate many of the features of the disease states for which they are used as indicators. In this review, I describe the chemistry, metabolism, and pharmacology of isoprostanes, with a particular emphasis on pulmonary cell types, and the possible roles of isoprostanes in pulmonary pathophysiology.
Heterocellular gap junctional communication between alveolar epithelial cells
Authors: Abraham Valsamma , Chou Michael L. , George Philip , Pooler Patricia , Zaman Aisha , Savani Rashmin C. , Koval Michael
Published: December 2017

We analyzed the pattern of gap junction protein (connexin) expression in vivo by indirect immunofluorescence. In normal rat lung sections, connexin (Cx)32 was expressed by type II cells, whereas Cx43 was more ubiquitously expressed and Cx46 was expressed by occasional alveolar epithelial cells. In response to bleomycin-induced lung injury, Cx46 was upregulated by alveolar epithelial cells, whereas Cx32 and Cx43 expression were largely unchanged. Given that Cx46 may form gap junction channels with either Cx43 or Cx32, we examined the ability of primary alveolar epithelial cells cultured for 6 days, which express Cx43 and Cx46, to form heterocellular gap junctions with cells expressing other connexins. Day 6 alveolar epithelial cells formed functional gap junctions with other day 6 cells or with HeLa cells transfected with Cx43 (HeLa/Cx43), but they did not communicate with HeLa/Cx32 cells. Furthermore, day 6alveolar epithelial cells formed functional gap junction channels with freshly isolated type II cells. Taken together, these data are consistent with the notion that type I and type II alveolar epithelial cells communicate through gap junctions compatible with Cx43.
IL-8 activates endothelial cell CXCR1 and CXCR2 through Rho and Rac signaling pathways
Authors: Schraufstatter Ingrid U. , Chung Janice , Burger Meike
Published: December 2017

Stimulation of microvascular endothelial cells with interleukin (IL)-8 leads to cytoskeletal reorganization, which is mediated by combined activation of the CXCR1 and the CXCR2. In the early phase actin stress fibers appear, followed by cortical actin accumulation and cell retraction leading to gap formation between cells. The early response (between 1 and 5 min) is inhibited by an antibody that blocks the CXCR1. The later phase (from about 5 to 60 min), which is associated with cell retraction, is prevented by anti-CXCR2 antibody. Furthermore, anti-CXCR2, but not anti-CXCR1, antibody blocked IL-8-mediated haptotaxis of endothelial cells on collagen. The later phase of the IL-8-mediated actin response is inhibited by pertussis toxin, indicating that the CXCR2 couples to Gi. In contrast, the early phase is blocked by C3 botulinum toxin, which inactivates Rho, and by Y-27632, which inhibits Rho kinase, but not by pertussis toxin. Furthermore, the early CXCR1-mediated formation of stress fibers was prevented by dominant negative Rho. Dominant negative Rac on the other hand initially translocated to actin-rich filopodia after stimulation with IL-8 and later prevented cell retraction by blocking the CXCR2-mediated cytoskeletal response. These results indicate that IL-8 activates both the CXCR1 and the CXCR2 on microvascular endothelial cells, using different signal transduction cascades. The retraction of endothelial cells due to activation of the CXCR2 may contribute to the increased vascular permeability observed in acute inflammation and during the angiogenic response.
Upregulation of ET-1 and its receptors and remodeling in small pulmonary veins under hypoxic conditions
Authors: Takahashi Hideki , Soma Sanae , Muramatsu Masashi , Oka Masahiko , Fukuchi Yoshinosuke
Published: December 2017

Pulmonary veins show greater sensitivity to endothelin (ET)-1-induced vasoconstriction than pulmonary arteries, and remodeling was observed in pulmonary veins under hypoxic conditions. We examined, using an immunohistochemical method, the expression of Big ET-1, ET-converting enzyme (ECE), and ETAand ETBreceptors in rat pulmonary veins under normoxic and hypoxic conditions. In control rats, Big ET-1 and ECE were coexpressed in the intima and media of the pulmonary veins, with an even distribution along the axial pathway. ETAand ETBreceptors were expressed in the pulmonary veins, with a predominant distribution in the proximal segments. The expression of Big ET-1 was more abundant in the pulmonary veins than in the pulmonary arteries. After exposure to hypoxia for 7 or 14 days, the expression of Big ET-1, ECE, and ET receptors increased in small pulmonary veins. Increases in the medial thickness, wall thickness, and immunoreactivity for α-smooth muscle actin were also observed in the small pulmonary veins under hypoxic conditions. The upregulation of ET-1 and ET receptors in the small pulmonary veins is associated with vascular remodeling, which may lead to the development of hypoxic pulmonary hypertension.
Attenuation of bleomycin-induced pneumopathy in mice by monoclonal antibody to interleukin-12
Authors: Maeyama Takashige , Kuwano Kazuyoshi , Kawasaki Masayuki , Kunitake Ritsuko , Hagimoto Naoki , Hara Nobuyuki
Published: December 2017

We previously demonstrated essential roles of the Fas-Fas ligand (FasL) pathway in bleomycin-induced pneumopathy in mice. T lymphocytes and natural killer cells express FasL on activation and use it as a cytotoxic effector molecule. Because interleukin (IL)-12 is known to play a critical role in cell-mediated immunity, we investigated whether anti-IL-12 antibody treatment suppresses the development of this model. The anti-IL-12 antibody treatment decreased the number of apoptotic cells and the degree of inflammation and fibrosis in lung tissue. The results of RT-PCR showed that IL-12p40, IL-12 receptor (R) β2, interferon-γ, tumor necrosis factor-α and FasL mRNAs were upregulated after bleomycin instillation. The upregulation of FasL, IL-12Rβ2, and tumor necrosis factor-α mRNA expression in lung tissue was suppressed by anti-IL-12 antibody treatment. The results of enzyme-linked immunosorbent assay showed that the levels of IL-12p40, but not of IL-12p70, were increased in lung tissue after bleomycin instillation. Although the increase in IL-12Rβ2 mRNA levels suggests that the T helper type 1 cell response may participate in lung injury, the increase in IL-12p40 supports T helper type 2 cell predominance in the fibrotic process of this model. The administration of anti-IL-12 antibody could be a novel therapy against lung injury and pulmonary fibrosis.
GM-CSF mediates alveolar macrophage proliferation and type II cell hypertrophy in SP-D gene-targeted mice
Authors: Hawgood Samuel , Akiyama Jennifer , Brown Cynthia , Allen Lennell , Li Gordon , Poulain Francis R.
Published: December 2017

Mice deficient in surfactant protein (SP) D develop increased surfactant pool sizes and dramatic changes in alveolar macrophages and type II cells. To test the hypothesis that granulocyte-macrophage colony-stimulating factor (GM-CSF) mediates alveolar macrophage proliferation and activation and the type II cell hypertrophy seen in SP-D null mice, we bred SP-D and GM-CSF gene-targeted mice to obtain littermate double null, single null, and wild-type mice. Bronchoalveolar lavage levels of phospholipid, protein, SP-D, SP-A, and GM-CSF were measured from 1 to 4 mo. There was an approximately additive accumulation of phospholipid, total protein, and SP-A at each time point. Microscopy showed normal macrophage number and morphology in GM-CSF null mice, numerous giant foamy macrophages and hypertrophic type II cells in SP-D null mice, and large but not foamy macrophages and mostly normal type II cells in double null mice. These results suggest that the mechanisms underlying the alveolar surfactant accumulation in the SP-D-deficient and GM-CSF-deficient mice are different and that GM-CSF mediates some of the macrophage and type II cell changes seen in SP-D null mice.
Rat lung peroxiredoxins I and II are differentially regulated during development and by hyperoxia
Authors: Kim Han-Suk , Kang Sang Won , Rhee Sue Goo , Clerch Linda Biadasz
Published: December 2017

Peroxiredoxin I (Prx I) and peroxiredoxin II (Prx II) are found in abundance in the cytoplasm of cells and catalyze the reduction of hydrogen peroxide with the use of electrons provided by thioredoxin. Here we examined Prx I and Prx II expression in rat lung during perinatal development and in response to hyperoxia. Prx I protein increased during late gestation and after birth fell to adult levels; conversely, Prx I mRNA increased after birth. Prx II protein concentration was unchanged in the perinatal period, but Prx II mRNA increased after birth. In response to hyperoxia begun on postnatal day 4, there was no change in Prx II expression; however, Prx I mRNA, protein, and enzymatic activity increased significantly. These data show that 1) Prx I and Prx II are developmentally regulated at the level of translational efficiency and 2) Prx I, but not Prx II, is inducible and is upregulated during the late-gestational preparation for the oxidative stress experienced by the lung at birth and during exposure to hyperoxia in the neonatal period.
Possible involvement of Rho kinase in Ca2+sensitization and mobilization by MCh in tracheal smooth muscle
Authors: Ito Satoru , Kume Hiroaki , Honjo Haruo , Katoh Hideki , Kodama Itsuo , Yamaki Kenichi , Hayashi Hideharu
Published: December 2017

We examined the effects of Rho kinase on contraction and intracellular Ca2+concentration ([Ca2+]i) in guinea pig trachealis by measuring isometric force and the fura 2 signal [340- to 380-nm fluorescence ratio (F340/F380)]. A Rho kinase inhibitor, Y-27632 (1–1,000 μM), inhibited methacholine (MCh)-induced contraction, with a reduction in F340/F380 in a concentration-dependent manner. The values of EC50for contraction and F340/F380 induced by 1 μM MCh with Y-27632 were 27.3 ± 5.1 and 524.1 ± 31.0 μM, respectively. With 0.1 μM MCh, the values for these parameters were decreased to 1.0 ± 0.1 and 98.2 ± 6.2 μM, respectively. Tension-F340/F380 curves for MCh indicated that Y-27632 caused an ∼50% inhibition of MCh-induced contraction, without a reduction in F340/F380. These effects of Y-27632 were not inhibited by a protein kinase C inhibitor, GF-109203X. Our results indicate that inhibition of Rho kinase attenuates both Ca2+sensitization and [Ca2+]i.
Interleukin-6 family cytokines: signaling and effects in human airway smooth muscle cells
Authors: Lahiri Thomas , Laporte Johanne D. , Moore Paul E. , Panettieri Reynold A. , Shore Stephanie A.
Published: December 2017

Interleukin (IL)-1β induces cyclooxygenase (COX)-2 expression and prostanoid formation in cultured human airway smooth muscle (HASM) cells. In other cell types, IL-6 family cytokines induce COX-2 or augment IL-1β-induced COX-2 expression. The purpose of this study was to determine whether IL-6 family cytokines were involved in COX-2 expression in HASM cells. RT-PCR was used to demonstrate that the necessary receptor components for IL-6-type cytokine binding are expressed in HASM cells. IL-6 and oncostatin M (OSM) each caused a dose-dependent phosphorylation of signal transducer and activator of transcription-3, whereas IL-11 did not. IL-6, IL-11, and OSM alone had no effect on COX-2 expression. However, OSM caused dose-dependent augmentation of COX-2 expression and prostaglandin (PG) E2release induced by IL-1β. In contrast, IL-6 and IL-11 did not alter IL-1β-induced COX-2 expression. IL-6 did increase IL-1β-induced PGE2formation in unstimulated cells but not in cells stimulated with arachidonic acid (AA; 10−5M), suggesting that IL-6 effects were mediated at the level of AA release. Our results indicate that IL-6 and OSM are capable of inducing signaling in HASM cells. In addition, OSM and IL-1β synergistically cause COX-2 expression and PGE2release.