bluuebunny/crossref_metadata_2025 huggingface dataset:
Albumin transcytosis in mesothelium
Authors: Bodega Francesca , Zocchi Luciano , Agostoni Emilio
Published: March 2015

Apparent permeability to albumin ( P alb) was measured with125I-albumin in specimens of rabbit parietal pericardium from lumen to interstitium (L-I) and from interstitium to lumen (I-L). With albumin concentration (Calb) 0.5%, P alb (× 10−5 cm/s) L-I at 37°C was 0.172 ± 0.019 SE; it decreased to 0.092 ± 0.022 I-L at 37°C, 0.089 ± 0.021 L-I at 12°C, and 0.084 ± 0.018 I-L at 12°C. These findings provide evidence for an active transport L-I, likely transcytosis. With Calb 2.5%, 0.05%, and 0.005%, P alb L-I at 37°C was 0.188 ± 0.023, 0.156 ± 0.021, and 0.090 ± 0.021, respectively; at 12°C it was 0.089 ± 0.017, 0.083 ± 0.019, and 0.087 ± 0.026, respectively. Hence, active albumin transport ceases with Calb 0.005%; P alb values I-L at 12°C and with Calb 0.005% are similar and provide diffusional permeability. With physiological Calb (∼1%), active albumin flux was ∼5 × 10−4μmol · h−1 · cm−2. Apparent permeability to FITC-dextran 70 ( P dx) was also measured. P dx (× 10−5 cm/s) L-I at 37°C with Calb 0.5% was 0.095 ± 0.018; it decreased to 0.026 ± 0.004 I-L (37°C, Calb 0.5%), 0.038 ± 0.007 at 12°C (L-I, Calb 0.5%), 0.030 ± 0.009 with Calb 0.005% (L-I, 37°C), and 0.032 ± 0.011 with nocodazole (L-I, 37°C, Calb 0.5%). These findings provide evidence for transcytosis and confirm conclusions drawn from P alb. Vesicular liquid flow, computed from vesicular dextran flux (fluid-phase only), was ∼3.5 μl · h−1 · cm−2. Transcytosis seems a relevant mechanism, removing protein and liquid from serous cavities.
Δ9-Tetrahydrocannabinol disrupts mitochondrial function and cell energetics
Authors: Sarafian Theodore A. , Kouyoumjian Shaghig , Khoshaghideh Farnaz , Tashkin Donald P. , Roth Michael D.
Published: March 2015

We have observed rapid and extensive depletion of cellular energy stores by Δ9-tetrahydrocannabinol (THC) in the pulmonary transformed cell line A549. ATP levels declined dose dependently with an IC50of 7.5 μg/ml of THC after 24-h exposure. Cell death was observed only at concentrations >10 μg/ml. Studies using JC-1, a fluorescent probe for mitochondrial membrane potential, revealed diminished mitochondrial function at THC concentrations as low as 0.5 μg/ml. At concentrations of 2.5 or 10 μg/ml of THC, a decrease in mitochondrial membrane potential was observed as early as 1 h after THC exposure. Mitochondrial function remained diminished for at least 30 h after THC exposure. Flow cytometry studies on cells exposed to particulate smoke extracts indicate that JC-1 red fluorescence was fivefold lower in cells exposed to marijuana smoke extract relative to cells exposed to tobacco smoke extract. Comparison with a variety of mitochondrial inhibitors demonstrates that THC produced effects similar to that of carbonyl cyanide p-trifluoromethoxyphenylhydrazone, suggesting uncoupling of electron transport. Loss of red JC-1 fluorescence by THC was suppressed by cyclosporin A, suggesting mediation by the mitochondrial permeability transition pore. This disruption of mitochondrial function was sustained for at least 24 h after removal of THC by extensive washing. These results suggest that exposure of the bronchopulmonary epithelium to THC may have important health and physiological consequences.
Expression of the amino acid transporter ATB0+in lung: possible role in luminal protein removal
Authors: Sloan Jennifer L. , Grubb Barbara R. , Mager Sela
Published: March 2015

Normal lung function requires transepithelial clearance of luminal proteins; however, little is known about the molecular mechanisms of protein transport. Protein degradation followed by transport of peptides and amino acids may play an important role in this process. We previously cloned and functionally characterized the neutral and cationic amino acid transporter ATB0+and showed expression in the lung by mRNA analysis. In this study, the tissue distribution, subcellular localization, and function of the transporter in native tissue were investigated. Western blots showed expression of the ATB0+protein in mouse lung, stomach, colon, testis, blastocysts, and human lung. Immunohistochemistry revealed that ATB0+is predominantly expressed on the apical membrane of ciliated epithelial cells throughout mouse airways from trachea to bronchioles and in alveolar type I cells. Electrical measurements from mouse trachea preparations showed Na+- and Cl−-dependent, amino acid-induced short-circuit current consistent with the properties of ATB0+. We hypothesize that, by removing amino acids from the airway lumen, the transporter contributes to protein clearance and, by maintaining a low nutrient environment, plays a role in lung defense.
Distribution of intranasal instillations in mice: effects of volume, time, body position, and anesthesia
Authors: Southam D. S. , Dolovich M. , O'Byrne P. M. , Inman M. D.
Published: March 2015

Intranasal instillation techniques are used to deliver various substances to the upper and lower respiratory tract (URT and LRT) in mice. Here, we quantify the relative distribution achieved with intranasal delivery of a nonabsorbable tracer,99mTc-labeled sulfide-colloid. Relative distribution was determined by killing mice after instillation and quantifying the radioactivity in dissected tissues using gamma scintigraphy. A significant effect of delivery volume on relative distribution was observed when animals were killed 5 min after instillation delivered under gas anesthesia. With a delivery volume of 5 μl, no radiation was detected in the LRT; this increased to a maximum of 55.7 ± 2.5% distribution to the LRT when 50 μl were delivered. The majority of radiation not detected in the LRT was found in the URT. Over the course of the following 1 h, radiation in the LRT remained constant, while that in the URT decreased and appeared in the gastrointestinal tract. Instillation of 25 μl into anesthetized mice resulted in 30.1 ± 6.9% distribution to the LRT, while only 5.3 ± 1.5% ( P < 0.05) of the same volume was detected in the LRT of awake mice. Varying the body position of mice did not affect relative distribution. When using intranasal instillation, the relative distribution between the URT and LRT and the gastrointestinal tract is heavily influenced by delivery volume and level of anesthesia.
Atypical development of the tracheal basement membrane zone of infant rhesus monkeys exposed to ozone and allergen
Authors: Evans Michael J. , Fanucchi Michelle V. , Baker Gregory L. , Van Winkle Laura S. , Pantle Lorraine M. , Nishio Susan J. , Schelegle Edward S. , Gershwin Laurel J. , Miller Lisa A. , Hyde Dallas M. , Sannes Philip L. , Plopper Charles G.
Published: March 2015

Development of the basement membrane zone (BMZ) occurs postnatally in the rhesus monkey. The purpose of this study was to determine whether house dust mite allergen (HDMA) plus ozone altered this process. Rhesus monkeys were exposed to a regimen of HDMA and/or ozone or filtered air for 6 mo. To detect structural changes in the BMZ, we measured immunoreactivity of collagen I. To detect functional changes in the BMZ, we measured perlecan and fibroblast growth factor-2 (FGF-2). We also measured components of the FGF-2 ternary signaling complex [fibroblast growth factor receptor-1 (FGFR-1) and syndecan-4]. The width of the BMZ was irregular in the ozone groups, suggesting atypical development of the BMZ. Perlecan was also absent from the BMZ. In the absence of perlecan, FGF-2 was not bound to the BMZ. However, FGF-2 immunoreactivity was present in basal cells, the lateral intercellular space (LIS), and attenuated fibroblasts. FGFR-1 immunoreactivity was downregulated, and syndecan-4 immunoreactivity was upregulated in the basal cells. This suggests that FGF-2 in basal cells and LIS may be bound to the syndecan-4. We conclude that ozone and HDMA plus ozone effected incorporation of perlecan into the BMZ, resulting in atypical development of the BMZ. These changes are associated with specific alterations in the regulation of FGF-2, FGFR-1, and syndecan-4 in the airway epithelial-mesenchymal trophic unit, which may be associated with the developmental problems of lungs associated with exposure to ozone.
Distal air space epithelial fluid clearance in near-term rat fetuses is fast and requires endogenous catecholamines
Authors: Folkesson Hans G. , Matthay Michael A. , Chapin Cheryl J. , Porta Nicolas F. M. , Kitterman Joseph A.
Published: March 2015

Knowledge about the conversion of the epithelium in the distal air spaces of the lung from secretion to absorption is imperative to the understanding of postnatal lung development; little such information is available in rats. Distal air space fluid clearance was therefore measured in 21- to 22-day gestation rat fetuses and newborn (40 h) rats. Distal air space fluid clearance was measured from the increase in131I-albumin concentration in an isosmolar, physiological solution instilled into the developing lungs. There was no net fluid movement across the distal air space epithelium in the lungs of 21-day gestation fetuses. Twenty-four hours later, distal air space fluid was cleared at a rapid rate in the 22-day gestation fetuses. Within the first 40 h after birth, the rate rapidly declined to adult levels. The high distal air space fluid clearance at 22 days gestation and at 40 h after birth was mediated by β-adrenergic receptors as demonstrated by elevated plasma epinephrine levels and inhibition by propranolol. Interestingly, the elevated distal air space fluid clearance in the 22-day gestation fetuses was only minimally amiloride sensitive; however, amiloride sensitivity increased over the first 40 h after birth. In conclusion, these studies demonstrate that 1) rapid rates of net alveolar fluid clearance occur late in gestation in the rat and 2) this clearance is driven by elevations of endogenous epinephrine.
Transcriptional regulation of CCSP by interferon-γ in vitro and in vivo
Authors: Ramsay P. L. , Luo Z. , Magdaleno S. M. , Whitbourne S. K. , Cao X. , Park M. S. , Welty S. E. , Yu-Lee L.-Y. , DeMayo F. J.
Published: March 2015

Interferon γ (IFN-γ), a potent cytokine inducing a wide range of immunologic activities, is increased in the airway secondary to viral infection or during an inflammatory response. This increase in IFN-γ concentration may alter the expression of specific airway epithelial cell genes that regulate adaptation of airway inflammatory responses. One protein induced by IFN-γ is Clara cell secretory protein (CCSP), which may contribute to the attenuation of airway inflammation. This study was done to investigate the molecular mechanism by which IFN-γ stimulates the expression of the CCSP gene in mouse transformed Clara cells and transgenic mice. Deletion mapping and linker-scanning mutations demonstrated that IFN-γ-induced expression of CCSP was regulated, in part, at the level of transcription. In vitro and in vivo studies verified that the minimal IFN-γ-responsive segment was localized to the proximal 166 bp of the 5′-flanking region. Additionally, IFN-γ-induced expression of CCSP was mediated indirectly through an interferon regulatory factor-1-mediated increase in hepatocyte nuclear factor-3β.
Increased expansion of the lung stimulates calmodulin 2 expression in fetal sheep
Authors: Gillett A. M. , Wallace M. J. , Gillespie M. T. , Hooper S. B.
Published: March 2015

Obstruction of the fetal trachea causes the lungs to expand with accumulated liquid. Although this is a potent stimulus for lung growth, the mechanisms involved are unknown. Our aim was to identify genes that are differentially expressed as a result of increased fetal lung expansion. Using differential display RT-PCR, we isolated a cDNA fragment partially encoding calmodulin 2 (CALM2) and identified the remainder of the coding region by 5′-rapid amplification of cDNA ends. Differential expression of CALM2 was confirmed by Northern blot analysis; CALM2 mRNA levels were increased to 161 ± 5% of control at 2 days of increased lung expansion, induced by tracheal obstruction (TO), and had returned to control levels at days 4 and 10. Using in situ hybridization analysis, we found that the proportion of CALM2-labeled cells increased from 10.3 ± 1.0% to 21.4 ± 6.8% by 2 days of TO. This increase in CALM2 expression was reflected by a tendency for calmodulin protein levels to increase from 122.7 ± 17.3 to 156.5 ± 17.7 at 2 days of TO. Thus increases in fetal lung expansion result in time-dependent changes in CALM2 mRNA levels, which closely parallels the changes in lung DNA synthesis rates. As calmodulin is essential for cell proliferation, increased CALM2 mRNA levels may reflect an important role for calmodulin in expansion-induced fetal lung growth.
Regulation of IL-1β-induced GM-CSF production in human airway smooth muscle cells by carbon monoxide
Authors: Song Ruiping , Ning Wen , Liu Fang , Ameredes Bill T. , Calhoun William J. , Otterbein Leo E. , Choi Augustine M. K.
Published: March 2015

Asthma, a chronic inflammatory disease of the airways, involves the increased expression of inflammatory mediators, including granulocyte-monocyte colony-stimulating factor (GM-CSF). Heme oxygenase-1 (HO-1), a stress-response protein, confers protection against oxidative stress. We hypothesized that carbon monoxide (CO), a byproduct of HO-1-dependent heme catabolism, regulates GM-CSF synthesis in human airway smooth muscle cells (HASMC). IL-1β treatment induced a time-dependent induction of GM-CSF in HASMC. Furthermore, IL-1β stimulated the major MAPK pathways, including ERK1/ERK2, JNK, and p38 MAPK. Exposure of HASMC to CO at low concentration (250 ppm) markedly inhibited IL-1β-induced GM-CSF synthesis (>90%) compared with air-treated controls. CO treatment inhibited IL-1β-induced ERK1/2 activation but did not inhibit JNK and p38 MAPK. Furthermore, CO increased cGMP levels in HASMC. Inhibition of guanylate cyclase by IH-[1,2,4] oxadiazolo[4,3-a]quinoxalin-1–1 (ODQ) abolished the inhibitory effects of CO on GM-CSF synthesis and ERK1/2 activation. Collectively, these data demonstrate that the inhibitory effect of CO on GM-CSF synthesis depends on ERK1/2 MAPK and guanylate cyclase/cGMP-dependent pathways.
Cyclooxygenase is regulated by ET-1 and MAPKs in peripheral lung microvascular smooth muscle cells
Authors: Chen Daohong , Balyakina Elena V. , Lawrence Mayme , Christman Brian W. , Meyrick Barbara
Published: March 2015

We examined the hypothesis that the potent vasoconstrictor endothelin (ET)-1 regulates both its own production and production of the vasodilator prostaglandins PGE2and prostacyclin in sheep peripheral lung vascular smooth muscle cells (PLVSMC). Confluent layers of PLVSMC were exposed to 10 nM ET-1; expression of the prepro (pp)-ET-1, cyclooxygenase (COX)-1, and COX-2 genes was examined by RT-PCR and Western analysis. Intracellular levels of ET-1 were measured by ELISA with and without addition of the protein synthesis inhibitor brefeldin A (50 μg/ml). Prostaglandin levels were measured by gas chromatography-mass spectrometry. Through use of ETAand ETBantagonists (BQ-610 and BQ-788, respectively), the contribution of the ET receptors to COX-1 and -2 expression and ppET-1 gene expression was examined. The contribution of phosphorylated p38 and p44/42 MAPK on COX-1 and COX-2 expression was also examined with MAPK inhibitors (p38, SB-203580 and p44/42, PD-98056). ET-1 resulted in transient increases in ppET-1, COX-1, and COX-2 gene and protein expression and release of 6-keto-PGF1αand PGE2( P < 0.05). Both internalization of ET-1 and synthesis of new peptide contributed to an increase in intracellular ET-1 ( P < 0.05). Although increased ppET-1 was regulated by both ETAand ETB, COX-2 expression was upregulated only by ETA; COX-1 expression was unaffected by either antagonist. ET-1 treatment resulted in transient phosphorylation of p38 and p44/42 MAPK; inhibitors of these MAPKs suppressed expression of COX-2 but not COX-1. Our data indicate that local production of ET-1 regulates COX-2 by activation of the ETAreceptor and phosphorylation of p38 and p44/42 MAPK in PLVSMC.
Neutrophil elastase increases MUC4 expression in normal human bronchial epithelial cells
Authors: Fischer Bernard M. , Cuellar Jacob G. , Diehl Meredith L. , deFreytas Akira M. , Zhang Jin , Carraway Kermit L. , Voynow Judith A.
Published: March 2015

In chronic obstructive pulmonary diseases, the airway epithelium is chronically exposed to neutrophil elastase, an inflammatory protease. The cellular response to neutrophil elastase dictates the balance between epithelial injury and repair. Key regulators of epithelial migration and proliferation are the ErbB receptor tyrosine kinases, including the epidermal growth factor receptor. In this context, we investigated whether neutrophil elastase may regulate expression of MUC4, a membrane-tethered mucin that has recently been identified as a ligand for ErbB2, the major heterodimerization partner of the epidermal growth factor receptor. In normal human bronchial epithelial cells, neutrophil elastase increased MUC4 mRNA levels in both a concentration- and time-dependent manner. RNA stability assays revealed that neutrophil elastase increased MUC4 mRNA levels by prolonging the mRNA half-life from 5 to 21 h. Neutrophil elastase also increased MUC4 glycoprotein levels as determined by Western analysis, using a monoclonal antibody specific for a nontandem repeat MUC4 sequence. Therefore, airway epithelial cells respond to neutrophil elastase exposure by increasing expression of MUC4, a potential activator of epithelial repair mechanisms.
Effect of IL-1β on CRE-dependent gene expression in human airway smooth muscle cells
Authors: Lahiri Thomas , Moore Paul E. , Baraldo Simonetta , Whitehead Timothy R. , McKenna Matthew D. , Panettieri Reynold A. , Shore Stephanie A.
Published: March 2015

IL-1β inhibits isoproterenol (ISO)-induced relaxation of cultured human airway smooth muscle (HASM) cells. The purpose of this study was to determine whether IL-1β can also suppress ISO-induced cAMP response element (CRE)-dependent gene expression. ISO (10 μM) caused a marked increase in CRE-binding protein (CREB) phosphorylation, which was attenuated by IL-1β (2 ng/ml). This effect of IL-1β was abolished by the cyclooxygenase (COX) inhibitor indomethacin. To examine CRE-driven gene expression, we transiently transfected HASM cells with a construct containing CRE upstream of a luciferase reporter gene. ISO (6 h) caused a sixfold increase in luciferase activity. IL-1β (24 h) alone also increased luciferase activity, although to a lesser extent (2-fold). However, the ability of ISO to elicit luciferase expression was markedly reduced in cells treated with IL-1β. Indomethacin, the MEK and p38 inhibitors U-0126 and SB-203580, the protein kinase A inhibitor H-89, and dexamethasone each completely abolished the ability of IL-1β to induce CRE-driven gene expression but only slightly increased the ability of ISO to induce CRE-driven gene expression in IL-1β-treated cells. IL-1β also attenuated dibutyryl cAMP-induced CRE-driven gene expression, but not dibutyryl cAMP-induced CREB phosphorylation. Tumor necrosis factor-α (10 ng/ml) also attenuated ISO-induced CRE-driven gene expression, even though it was without effect on ISO-induced cAMP formation or ISO-induced CREB phosphorylation. The results suggest that IL-1β and tumor necrosis factor-α may attenuate the ability of β-agonists to induce expression of genes with CRE in their regulatory regions at least in part through events downstream of CREB phosphorylation.
Protein transport across the lung epithelial barrier
Authors: Kim Kwang-Jin , Malik Asrar B.
Published: March 2015

Alveolar lining fluid normally contains proteins of important physiological, antioxidant, and mucosal defense functions [such as albumin, immunoglobulin G (IgG), secretory IgA, transferrin, and ceruloplasmin]. Because concentrations of plasma proteins in alveolar fluid can increase in injured lungs (such as with permeability edema and inflammation), understanding how alveolar epithelium handles protein transport is needed to develop therapeutic measures to restore alveolar homeostasis. This review provides an update on recent findings on protein transport across the alveolar epithelial barrier. The use of primary cultured rat alveolar epithelial cell monolayers (that exhibit phenotypic and morphological traits of in vivo alveolar epithelial type I cells) has shown that albumin and IgG are absorbed via saturable processes at rates greater than those predicted by passive diffusional mechanisms. In contrast, secretory component, the extracellular portion of the polymeric immunoglobulin receptor, is secreted into alveolar fluid. Transcytosis involving caveolae and clathrin-coated pits is likely the main route of alveolar epithelial protein transport, although relative contributions of these internalization steps to overall protein handling of alveolar epithelium remain to be determined. The specific pathways and regulatory mechanisms responsible for translocation of proteins across lung alveolar epithelium and regulation of the cognate receptors (e.g., 60-kDa albumin binding protein and IgG binding FcRn) expressed in alveolar epithelium need to be elucidated.
Epinephrine promotes pulmonary angiitis: evidence for a β1-adrenoreceptor-mediated mechanism
Authors: Jain Felipe A. , Zhao Long-hai , Selig Martin K. , Kradin Richard L.
Published: March 2015

Epinephrine (Epi) increases lymphocyte traffic to lung. We investigated whether Epi also modulates pulmonary cell-mediated immune responses in vivo. C57BL/6 mice were immunized with hen-egg lysozyme (HEL) on day 0, challenged with HEL intratracheally at day 12, and killed at day 15. Mice received Epi (0.5 mg/kg) subcutaneously during the sensitization phase, days 1–7 (Epi-SP), or the effector phase, days 12–14 (Epi-EP); controls received saline subcutaneously. Epi-SP mice showed increased airway inflammation ( P < 0.03) and pulmonary angiitis ( P < 0.04) characterized by endothelialitis and subendothelial fibrin deposition. Macrophages and granulocytes were increased in perivascular cuffs in situ ( P < 0.001). CD3+lymphocytes increased in the bronchoalveolar lavage fluid, whereas NK1.1+and CD4+CD25+lymphocytes decreased (all P < 0.05). Atenolol, a selective β1-adrenoreceptor (AR) antagonist, inhibited the increased vascular and airway inflammation and the reduction in CD4+CD25+lymphocytes (all P < 0.05) yielded by Epi, whereas all α/β-AR blockers inhibited airway inflammation. We conclude that Epi-EP selectively promotes vascular inflammation in vivo via a β1-receptor-mediated mechanism.
LPS-induced neutrophilic inflammation and Bcl-2 expression in metaplastic mucous cells
Authors: Foster Jennifer E. , Gott Katherine , Schuyler Mark R. , Kozak Wieslaw , Tesfaigzi Yohannes
Published: March 2015

Our previous studies show that Bcl-2, a regulator of apoptosis, may be involved in the reduction of mucous cell metaplasia (MCM) during recovery from inflammatory responses. The present study was to determine whether neutrophilic inflammation mediates Bcl-2 expression in mucous cells. Rats were intratracheally instilled with 50–1,000 μg of LPS. The number of neutrophils recovered by bronchoalveolar lavage (BAL) increased with the dose of LPS, and the percentage of Bcl-2-expressing cells increased with the numbers of neutrophils in the BAL. Depletion of neutrophils did not reduce MCM, but the percentage of Bcl-2-positive cells increased 1.8-fold in neutrophil-depleted compared with controls. Injection of rats with bezafibrate, an inducer of cytochrome P-450, doubled the number of neutrophils in the BAL, decreased MCM twofold compared with vehicle-injected controls, and reduced Bcl-2 expression. Bcl-2 mRNA levels decreased in a tracheal epithelial cell line treated with bezafibrate. These data demonstrate that Bcl-2 expression is independent of the number of neutrophils in the BAL and that bezafibrate may directly reduce Bcl-2 expression in epithelial cells.
Inhaled nitric oxide increases surfactant protein gene expression in the intact lamb
Authors: Stuart Regan B. , Ovadia Boaz , Suzara Vincent V. , Ross Patrick A. , Thelitz Stephan , Fineman Jeffrey R. , Gutierrez Jorge A.
Published: March 2015

Inhaled nitric oxide (iNO) is used to treat a number of disease processes. Although in vitro data suggest that nitric oxide (NO) alters surfactant protein gene expression, the effects in vivo have not been studied. The objective of this study was to evaluate the effects of iNO on surfactant protein (SP)-A, -B, and -C gene expression in the intact lamb. Thirteen 4-wk-old lambs were mechanically ventilated with 21% oxygen and received iNO at 40 ppm ( n = 7) or vehicle gas ( n = 6) for 24 h. Peripheral lung biopsies were obtained at 0, 12, and 24 h and analyzed for surfactant mRNA, protein, and total DNA content. Inhaled NO increased SP-A and SP-B mRNA content by 80% from 0 to 12 h and by 78 and 71%, respectively, from 0 to 24 h. There was an increase in SP-A and SP-B protein content by 45% from 0 to 12 h, and a decrease by 70 and 65%, respectively, from 0 to 24 h. DNA content was unchanged. The mechanisms and physiological effects of these findings warrant further investigation.
Oxygen regulates mitogen-stimulated proliferation of fetal human airway smooth muscle cells
Authors: Pandya Hitesh C. , Snetkov Vladimir A. , Twort Charles H. C. , Ward Jeremy P. T. , Hirst Stuart J.
Published: March 2015

Increased airway smooth muscle (ASM) content is characteristic of infants with chronic lung disease of prematurity/bronchopulmonary dysplasia. Oxygen therapy, reactive oxygen species (ROS), and immature antioxidant defenses are major risk factors in chronic lung disease of prematurity/bronchopulmonary dysplasia, but their interrelationship is unclear. The direct effects of raised Po 2 and modulation of ROS were examined on proliferation of cultured fetal human ASM cells. A bell-shaped relationship was found between Po 2 and DNA synthesis induced by fetal bovine serum, platelet-derived growth factor, and basic fibroblastic growth factor, with peak responses occurring at 10-kPa Po 2. Changes in DNA synthesis by Po 2 did not occur in the absence of mitogen. ROS generation, estimated by dichlorodihydrofluorescein oxidation, was increased by mitogens but was unaffected by nonmitogens (bradykinin, histamine). There was an inverse relationship between ROS generation and Po 2, and mitogen-induced ROS generation was substantially potentiated as the Po 2 fell. H2O2 mimicked the effect of Po 2 on fetal bovine serum-stimulated proliferation, whereas treatment with antioxidants (GSH, N-acetylcysteine) reduced it. These data demonstrate that increases in Po 2 above levels found in utero modulate proliferation of fetal ASM cells but only in the presence of growth factors. They also strongly suggest that, under these conditions, proliferation is mediated in part by generation of ROS.
An enhancer region determines hSP-B gene expression in bronchiolar and ATII epithelial cells in transgenic mice
Authors: Yang Li , Naltner Angela , Kreiner Allison , Yan Dong , Cowen Angelynn , Du Hong , Yan Cong
Published: March 2015

Regulation of the surfactant protein B gene (SP-B) is developmentally controlled and highly tissue specific. To elucidate the SP-B gene temporal/spatial expression pattern in lung development at the transcriptional level, a transgenic mouse model line carrying the human SP-B (hSP-B) 1.5-kb 5′-flanking regulatory region and the lacZ gene was established. Expression of hSP-B 1.5-kb lacZ gene started at the onset of lung formation [embryonic day 9 (E9)] and was restricted to epithelial cells throughout prenatal and postnatal lung development. In the adult lung, hSP-B 1.5-kb lacZ gene expression was restricted to bronchiolar and alveolar type II epithelial cells. In lung explant culturing studies, the hSP-B 1.5-kb lacZ gene was highly expressed in newly formed epithelial tubules during the respiratory branching process. In a second transgenic mouse line, an enhancer region, which binds to thyroid transcription factor-1, retinoic acid receptor, signal transducers and activators of transcription 3, and nuclear receptor coactivators (SRC-1, ACTR, TIF2, and CBP/p300), was deleted from the hSP-B 1.5-kb lacZ gene. The deletion abolished hSP-B lacZ gene expression in bronchiolar epithelial cells and significantly reduced its expression level in alveolar type II epithelial cells in transgenic mice.
Tracheal occlusion stimulates cell cycle progression and type I cell differentiation in lungs of fetal rats
Authors: Yoshizawa Jyoji , Chapin Cheryl J. , Sbragia Lourenço , Ertsey Robert , Gutierrez Jorge A. , Albanese Craig T. , Kitterman Joseph A.
Published: March 2015

Fetal tracheal occlusion (TO) has been reported to stimulate lung growth but decreases number and maturation of type II cells, effects that vary with gestational age and duration of TO. We examined effects of a novel method of TO (unipolar microcautery to seal the trachea) produced at 19.5–20 days (d) of gestation in fetal rats; fetuses were delivered at term, 22 d. Controls were sham operated and unoperated littermates. TO increased wet lung weight but not dry lung weight or lung DNA and protein. To evaluate further the effects of TO, we examined the cell cycle regulators, cyclins D1 and A, in fetal lungs. Cyclin D1 increased with TO ( P < 0.005). TO also increased expression of the type I epithelial cell marker RTI40 (mRNA and protein). TO decreased mRNA for surfactant proteins (SP)-A and -C but did not affect protein levels of SP-A and -B and of RTII70, a type II epithelial cell marker. We conclude that TO by microcautery, even of short duration, has diverse pulmonary effects including stimulating increased levels of cyclin D1 with probable cell cycle progression, type I cell differentiation, and possibly inhibiting type II cell function.
Time-dependent reversal of sepsis-induced PMN uptake and lung vascular injury by expression of CD18 antagonist
Authors: Xu Ning , Gao Xiao-Pei , Minshall Richard D. , Rahman Arshad , Malik Asrar B.
Published: March 2015

We determined the time-dependent effects of conditional expression of neutrophil inhibitory factor (NIF), a specific 41-kDa CD18 integrin antagonist, on the time course of NIF expression and lung PMN (polymorphonuclear leukocyte) infiltration and vascular injury in a model of Escherichia coli-induced sepsis in mice. Studies were made in mice transduced with the E-selectin (ES) promoter-NIF construct (using liposomes) in which the NIF cDNA was driven by the inflammation- and endothelial cell-specific ES promoter. We observed time-dependent expression of NIF in pulmonary vascular endothelium that paralleled the ES expression. Expression of both was evident at 1 h after E. coli challenge, peaked at 3–6 h, and returned to basal level within 48 h. We observed that increases in PMN uptake and transalveolar PMN migration induced by E. coli challenge were reversed in a time-dependent manner following NIF expression in mice. NIF expression also prevented the progression of lung vascular injury and edema formation following E. coli challenge. Thus the conditional expression of NIF using the ES promoter can reverse, in a time-dependent manner, lung PMN infiltration and vascular injury induced by gram-negative sepsis. The results support the model that initial engagement of CD18 integrins enables the further recruitment of additional PMN into lung tissues such that PMN continue to sequester and migrate after E. coli challenge.
Role of anti-L-selectin antibody in burn and smoke inhalation injury in sheep
Authors: Katahira Jiro , Murakami Kazunori , Schmalstieg Frank C. , Cox Robert , Hawkins Hal , Traber Lillian D. , Traber Daniel L.
Published: March 2015

We hypothesized that the antibody neutralization of L-selectin would decrease the pulmonary abnormalities characteristic of burn and smoke inhalation injury. Three groups of sheep ( n = 18) were prepared and randomized: the LAM-(1–3) group ( n = 6) was injected intravenously with 1 mg/kg of leukocyte adhesion molecule (LAM)-(1-3) (mouse monoclonal antibody against L-selectin) 1 h after the injury, the control group ( n = 6) was not injured or treated, and the nontreatment group ( n = 6) was injured but not treated. All animals were mechanically ventilated during the 48-h experimental period. The ratio of arterial Po 2 to inspired O2 fraction decreased in the LAM-(1–3) and nontreatment groups. Lung lymph flow and pulmonary microvascular permeability were elevated after injury. This elevation was significantly reduced when LAM-(1–3) was administered 1 h after injury. Nitrate/nitrite (NO x ) amounts in plasma and lung lymph increased significantly after the combined injury. These changes were attenuated by posttreatment with LAM-(1–3). These results suggest that the changes in pulmonary transvascular fluid flux result from injury of lung endothelium by polymorphonuclear leukocytes. In conclusion, posttreatment with the antibody for L-selectin improved lung lymph flow and permeability index. L-selectin appears to be principally involved in the increased pulmonary transvascular fluid flux observed with burn/smoke insult. L-selectin may be a useful target in the treatment of acute lung injury after burn and smoke inhalation.
Surfactant strengthens the inhibitory effect of C-reactive protein on human lung macrophage cytokine release
Authors: Casals Cristina , Arias-Díaz Javier , Valiño Fernando , Sáenz Alejandra , García Cruz , Balibrea José L. , Vara Elena
Published: March 2015

In this study we investigated the effect of acute-phase levels of C-reactive protein (CRP) on cytokine production by pulmonary macrophages in the presence or absence of pulmonary surfactant. Both human alveolar and interstitial macrophages as well as human surfactant were obtained from multiple organ donor lungs. Precultured macrophages were stimulated with LPS alone or together with IFN-γ in the presence or absence of CRP, surfactant, and combinations. Releases of TNF-α and of IL-1β to the medium were determined. We found that CRP could modulate lung inflammation in humans by decreasing the production of proinflammatory cytokines by both alveolar and interstitial macrophages stimulated with LPS alone or together with IFN-γ. The potential interaction between CRP and surfactant phospholipids did not overcome the effect of either CRP or surfactant on TNF-α and IL-1β release by lung macrophages. On the contrary, CRP and pulmonary surfactant together had a greater inhibitory effect than either alone on the release of proinflammatory cytokines by lung macrophages.
Mitochondrial ROS initiate phosphorylation of p38 MAP kinase during hypoxia in cardiomyocytes
Authors: Kulisz Andre , Chen Ningfang , Chandel Navdeep S. , Shao Zuohui , Schumacker Paul T.
Published: March 2015

The p38 mitogen-activated protein kinase (MAPK) is phosphorylated in response to oxidative stress. Mitochondria in cardiomyocytes increase their generation of reactive oxygen species (ROS) during hypoxia (1–5% O2). These ROS participate in signal transduction pathways involved in adaptive responses, including ischemic preconditioning and gene transcription. The present study therefore tested the hypothesis that hypoxia induces p38 MAPK phosphorylation by augmenting mitochondrial ROS generation. In cardiomyocytes, phosphorylation of p38 was observed in a Po 2-dependent manner during hypoxia. This response was inhibited by rotenone, thenoyltrifluoroacetone, and myxothiazol, inhibitors of mitochondrial complexes I, II, and III, respectively. A similar inhibition was observed in the cells pretreated with anion channel inhibitor DIDS, which may block ROS release from mitochondria. During normoxia, increases in mitochondrial ROS elicited by azide (1–2 mM) or by the mitochondrial inhibitor antimycin A caused increased phosphorylation of p38. Brief treatment with exogenous H2O2 during normoxia also induced phosphorylation of p38 as hypoxia, but this effect was not abolished by myxothiazol or DIDS. The antioxidant N-acetyl-cysteine abolished the p38 response to hypoxia, presumably by scavenging H2O2, but the mitogen extracellular receptor kinase inhibitor PD-98059 did not inhibit p38 phosphorylation during hypoxia. Thus physiological hypoxia leads to p38 phosphorylation through a mechanism that requires electron flux in the proximal region of the mitochondrial electron transport chain, which suggests that either H2O2 or superoxide participates in activating that process.
Eotaxin/CCL11 is involved in acute, but not chronic, allergic airway responses toAspergillus fumigatus
Authors: Schuh Jane M. , Blease Kate , Kunkel Steven L. , Hogaboam Cory M.
Published: March 2015

Eotaxin/CCL11 is a major chemoattractant for eosinophils and Th2 cells. As such, it represents an attractive target in the treatment of allergic disease. The present study addresses the role of eotaxin/CCL11 during acute and chronic allergic airway responses to the fungus Aspergillus fumigatus. Mice lacking the eotaxin gene (Eo−/−) and wild-type mice (Eo+/+) were sensitized to A. fumigatus and received either an intratracheal challenge with soluble A. fumigatusantigens (acute model) or an intratracheal challenge with live A. fumigatus spores or conidia (chronic model). Airway hyperresponsiveness and eosinophil, but not T cell, recruitment were significantly decreased at 24 h after the soluble allergen in A. fumigatus-sensitized Eo−/− mice compared with similarly sensitized Eo+/+ mice. In contrast, the development of chronic allergic airway disease due to A. fumigatus conidia was not altered by the lack of eotaxin. Together, these data suggest that eotaxin initiates allergic airway disease due to A. fumigatus, but this chemokine did not appear to contribute to the maintenance of A. fumigatus-induced allergic airway disease.
Contributions of nitric oxide synthase isozymes to exhaled nitric oxide and hypoxic pulmonary vasoconstriction in rabbit lungs
Authors: Vaughan David J. , Brogan Thomas V. , Kerr Mark E. , Deem Steven , Luchtel Daniel L. , Swenson Erik R.
Published: March 2015

We investigated the source(s) for exhaled nitric oxide (NO) in isolated, perfused rabbits lungs by using isozyme-specific nitric oxide synthase (NOS) inhibitors and antibodies. Each inhibitor was studied under normoxia and hypoxia. Only nitro-l-arginine methyl ester (l-NAME, a nonselective NOS inhibitor) reduced exhaled NO and increased hypoxic pulmonary vasoconstriction (HPV), in contrast to 1400W, an inhibitor of inducible NOS (iNOS), and 7-nitroindazole, an inhibitor of neuronal NOS (nNOS). Acetylcholine-mediated stimulation of vascular endothelial NOS (eNOS) increased exhaled NO and could only be inhibited by l-NAME. Selective inhibition of airway and alveolar epithelial NO production by nebulized l-NAME decreased exhaled NO and increased hypoxic pulmonary artery pressure. Immunohistochemistry demonstrated extensive staining for eNOS in the epithelia, vasculature, and lymphatic tissue. There was no staining for iNOS but moderate staining for nNOS in the ciliated cells of the epithelia, lymphoid tissue, and cartilage cells. Our findings show virtually all exhaled NO in the rabbit lung is produced by eNOS, which is present throughout the airways, alveoli, and vessels. Both vascular and epithelial-derived NO modulate HPV.
Effects of diazepam and stress on lung inflammatory response in OVA-sensitized rats
Authors: De Paula Portela Carlos , Tibério Iolanda De F. L. C. , Leick-Maldonado Edna A. , Martins Milton A. , Palermo-Neto João
Published: March 2015

The influence of stress and diazepam treatment on airway inflammation was investigated in ovalbumin (OVA)-sensitized rats. Animals were injected with OVA plus aluminum hydroxide intraperitoneally ( day 0) and boosted with OVA subcutaneously ( day 7). From the first to 13th day after sensitization, rats were treated with diazepam, and 1 h later they were placed in a shuttle box where they received 50 mild escapable foot shocks/day preceded by a sound signal (S). Response during the warning (S) canceled shock delivery and terminated the S. On day 14, rats were submitted to a single session of 50 inescapable foot shocks preceded by S and then were challenged with OVA. High levels of stress were detected in shocked animals, manifested as ultrasonic vocalizations. Morphometric analysis of stressed animals revealed a significant increase in both edema and lymphomononucleated cells in airways compared with controls. Diazepam treatment reduced edema in stressed and nonstressed rats. No differences were found in polymorphonucleated cell infiltration. Diazepam treatment reduced lymphomononucleated cell infiltration in stressed animals. These data suggest that stress and diazepam treatment play relevant roles in edema and lymphomononucleated airway inflammation in OVA-sensitized rats.
Identification of protein disulfide isomerase as an endothelial hypoxic stress protein
Authors: Graven Krista K. , Molvar Christopher , Roncarati Jill S. , Klahn Brian D. , Lowrey Shawna , Farber Harrison W.
Published: March 2015

Endothelial cells (EC) exposed to hypoxia upregulate a unique set of five stress proteins. These proteins are upregulated in human and bovine aortic and pulmonary artery EC and are distinct from heat shock or glucose-regulated proteins. We previously identified two of these proteins as the glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase and enolase and postulated that the remaining proteins were also glycolytic enzymes. Using SDS-PAGE, tryptic digestion, and NH2-terminal amino acid sequencing, we report here the identification of the 56-kDa protein as protein disulfide isomerase (PDI). PDI is upregulated by hypoxia at the mRNA level and follows a time course similar to that of the protein, with maximal upregulation detected after exposure to 18 h of 0% O2. Neither smooth muscle cells nor fibroblasts upregulate PDI to the same extent as EC, which correlates with their decreased hypoxia tolerance. Upregulation of PDI specifically in EC may contribute to their ability to tolerate hypoxia and may occur through PDI's functions as a prolyl hydroxylase subunit, protein folding catalyst, or molecular chaperone.
Phorbol esters increase MLC phosphorylation and actin remodeling in bovine lung endothelium without increased contraction
Authors: Bogatcheva Natalia V. , Verin Alexander D. , Wang Peiyi , Birukova Anna A. , Birukov Konstantin G. , Mirzopoyazova Tamara , Adyshev Djanybek M. , Chiang Eddie T. , Crow Michael T. , Garcia Joe G. N.
Published: March 2015

Direct protein kinase C (PKC) activation with phorbol myristate acetate (PMA) results in the loss of endothelial monolayer integrity in bovine lung endothelial cells (EC) but produces barrier enhancement in human lung endothelium. To extend these findings, we studied EC contractile events and observed a 40% increase in myosin light chain (MLC) phosphorylation in bovine endothelium following PMA challenge. The increase in PMA-mediated MLC phosphorylation occurred at sites distinct from Ser19/Thr18, sites catalyzed by MLC kinase (MLCK), and immunoblotting with antibodies specific to phosphorylated Ser19/Thr18demonstrated profound time-dependent Ser19/Thr18dephosphorylation. These events occurred in conjunction with rearrangement of stress fibers into a grid-like network, but without an increase in cellular contraction as measured by silicone membrane wrinkling assay. The PMA-induced MLC dephosphorylation was not due to kinase inhibition but, rather, correlated with rapid increases in myosin-associated phosphatase 1 (PPase 1) activity. These data suggest that PMA-mediated EC barrier regulation may involve dual mechanisms that alter MLC phosphorylation. The increase in bovine MLC phosphorylation likely occurs via direct PKC-dependent MLC phosphorylation in conjunction with decreases in Ser19/Thr18phosphorylation catalyzed by MLCK due to PMA-induced increases in PPase 1 activity. Together, these events result in stress fiber destabilization and profound actin rearrangement in bovine endothelium, which may result in the physiological alterations observed in these models.
Modulation of cGMP by human HO-1 retrovirus gene transfer in pulmonary microvessel endothelial cells
Authors: Abraham Nader G. , Quan Shuo , Mieyal Paul A. , Yang Liming , Burke-Wolin Theresa , Mingone Christopher J. , Goodman Alvin I. , Nasjletti Alberto , Wolin Michael S.
Published: March 2015

Carbon monoxide (CO) stimulates guanylate cyclase (GC) and increases guanosine 3′,5′-cyclic monophosphate (cGMP) levels. We transfected rat-lung pulmonary endothelial cells with a retrovirus-mediated human heme oxygenase (hHO)-1 gene. Pulmonary cells that expressed hHO-1 exhibited a fourfold increase in HO activity associated with decreases in the steady-state levels of heme and cGMP without changes in soluble GC (sGC) and endothelial nitric oxide synthase (NOS) proteins or basal nitrite production. Heme elicited significant increases in CO production and intracellular cGMP levels in both pulmonary endothelial and pulmonary hHO-1-expressing cells. Nω-nitro-l-arginine methyl ester (l-NAME), an inhibitor of NOS, significantly decreased cGMP levels in heme-treated pulmonary endothelial cells but not heme-treated hHO-1-expressing cells. In the presence of exogenous heme, CO and cGMP levels in hHO-1-expressing cells exceeded the corresponding levels in pulmonary endothelial cells. Acute exposure of endothelial cells to SnCl2, which is an inducer of HO-1, increased cGMP levels, whereas chronic exposure decreased heme and cGMP levels. These results indicate that prolonged overexpression of HO-1 ultimately decreases sGC activity by limiting the availability of cellular heme. Heme activates sGC and enhances cGMP levels via a mechanism that is largely insensitive to NOS inhibition.
Microtubule-active agents modify nitric oxide production in pulmonary artery endothelial cells
Authors: Su Yunchao , Zharikov Sergei I. , Block Edward R.
Published: March 2015

The effects of specific microtubule-active agents on nitric oxide (NO) production were examined in pulmonary artery endothelial cells (PAEC). PAEC were incubated with taxol, which stabilizes microtubules, or nocodazole, which disrupts microtubules, or both for 2–4 h. We then examined NO production, endothelial NO synthase (eNOS) activity, and eNOS association with heat shock protein (HSP) 90. Incubation of PAEC with taxol (15 μM) for 2–4 h resulted in an increase in NO production, eNOS activity, and the amount of HSP90 binding to eNOS. Incubation of PAEC with nocodazole (50 μM) for 2–4 h induced a decrease in NO production, eNOS activity, and the amount of HSP90 binding to eNOS. The presence of taxol in the culture medium prevented the effects of nocodazole on NO production and eNOS activity in PAEC. Geldanamycin, a HSP90 inhibitor, prevented the taxol-induced increase in eNOS activity. Taxol and nocodazole did not affect eNOS, HSP90, and tubulin protein contents in PAEC, as detected using Western blot analysis. These results indicate that the polymerization state of the microtubule cytoskeleton regulates NO production and eNOS activity in PAEC. The changes in eNOS activity induced by modification of microtubules are due, at least in part, to the altered binding of HSP90 to eNOS protein.