bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of NO synthase inhibition on myocardial metabolism during moderate ischemia
Authors: Martin Claus , Schulz Rainer , Post Heiner , Gres Petra , Heusch Gerd
Published: March 2015

Nitric oxide (NO) is involved in the control of myocardial metabolism. In normoperfused myocardium, NO synthase inhibition shifts myocardial metabolism from free fatty acid (FFA) toward carbohydrate utilization. Ischemic myocardium is characterized by a similar shift toward preferential carbohydrate utilization, although NO synthesis is increased. The importance of NO for myocardial metabolism during ischemia has not been analyzed in detail. We therefore assessed the influence of NO synthase inhibition with NG-nitro-l-arginine (l-NNA) on myocardial metabolism during moderate ischemia in anesthetized pigs. In control animals, the increase in left ventricular pressure with l-NNA was mimicked by aortic constriction. Before ischemia, l-NNA decreased myocardial FFA consumption (MV˙FFA; P < 0.05), while consumption of carbohydrate and O2(MV˙o2) remained constant. ATP equivalents [calculated with the assumption of complete oxidative substrate decomposition (ATPeq)] decreased withl-NNA ( P < 0.05), associated with a decrease of regional myocardial function ( P < 0.05). In contrast, aortic constriction had no effect on MV˙FFA, while MV˙o2increased ( P < 0.05) and ATPeqand regional myocardial function remained constant. During ischemia, alterations in myocardial metabolism were similar in control and l-NNA-treated animals: MV˙FFAdecreased ( P < 0.05) and net lactate consumption was reversed to net lactate production ( P < 0.05). Regional myocardial function was decreased ( P < 0.05), although more markedly in animals receiving l-NNA ( P < 0.05). We conclude that the efficiency of oxidative metabolism was impaired by l-NNA per se, paralleled by impaired regional myocardial function. During ischemia, l-NNA had no effect on myocardial substrate consumption, indicating that NO synthases were no longer effectively involved in the control of myocardial metabolism.
Evaluation of a new fiber-optic pressure recording system for cardiovascular measurements in mice
Authors: Woldbaek Per Reidar , Strømme Tævje Andreas , Sande Jørn Bodvar , Christensen Geir , Tønnessen Theis , Ilebekk Arnfinn
Published: March 2015

We have tested a new fiber-optic pressure recording system, Samba, with a thin fiber [outer diameter (OD) = 0.25 mm] and a pressure sensor (length and OD = 0.42 mm) attached to the end. The accuracy of the system tested in vitro was good, with a coefficient of variation of 2.54% at 100 mmHg. The drift was <0.45 mmHg/h, and the temperature sensitivity was ∼0.07 mmHg/1°C between 22 and 37°C. The frequency response characteristics were similar to a 1.4-Fr Millar catheter (0–200 Hz). Introduction of the Samba sensor from the right carotid artery into the left ventricle in six mice caused no drop in mean aortic pressure, whereas introduction of a 1.4-Fr Millar catheter (OD = 0.47 mm; n = 6) caused a pressure drop from 91.6 ± 9.2 to 65.1 ± 6.2 mmHg; P < 0.05. Thus the Samba sensor system may represent a new alternative to assess hemodynamic variables in the murine cardiovascular system.
Kallikrein gene delivery attenuates cardiac remodeling and promotes neovascularization in spontaneously hypertensive rats
Authors: Bledsoe Grant , Chao Lee , Chao Julie
Published: March 2015

Hypertension that results in left ventricular (LV) hypertrophy and/or fibrosis can lead to cardiac dysfunction. Spontaneously hypertensive rats (SHR) develop high blood pressure and LV hypertrophy at an early age and are a popular model of human essential hypertension. To investigate the role of the tissue kallikrein-kinin system in cardiac remodeling, an adenovirus containing the human tissue kallikrein gene was injected intravenously into adult SHR and normotensive Wistar-Kyoto (WKY) rats. The blood pressure of WKY rats remained unchanged throughout the experiment. Alternatively, kallikrein gene transfer reduced blood pressure in SHR for the first 2 wk, but had no effect from 3 to 5 wk. Five weeks after kallikrein gene delivery, SHR showed significant reductions in LV-to-heart weight ratio, LV long axis, and cardiomyocyte size; however, these parameters were unaffected in WKY rats. Interestingly, cardiac collagen density was decreased in both SHR and WKY rats receiving the kallikrein gene. Kallikrein gene transfer also increased cardiac capillary density in SHR, but not in WKY rats. The morphological changes after kallikrein gene transfer were associated with decreases in JNK activation as well as transforming growth factor (TGF)-β1 and plasminogen activator inhibitor-1 levels in the heart. In addition, kallikrein gene delivery elevated LV nitric oxide and cGMP levels in both rat strains. These results indicate that kallikrein-kinin attenuates cardiac hypertrophy and fibrosis and enhances capillary growth in SHR through the suppression of JNK, TGF-β1, and plasminogen activator inhibitor-1 via the nitric oxide-cGMP pathway.
Differential pre- and postsynaptic effects of desipramine on cardiac sympathetic nerve terminals in RHF
Authors: Liang Chang-Seng , Himura Yoshihiro , Kashiki Michihiro , Stevens Suzanne Y.
Published: March 2015

Right heart failure (RHF) is characterized by chamber-specific reductions of myocardial norepinephrine (NE) reuptake, β-receptor density, and profiles of cardiac sympathetic nerve ending neurotransmitters. To study the functional linkage between NE uptake and the pre- and postsynaptic changes, we administered desipramine (225 mg/day), a NE uptake inhibitor, to dogs with RHF produced by tricuspid avulsion and progressive pulmonary constriction or sham-operated dogs for 6 wk. Animals receiving no desipramine were studied as controls. We measured myocardial NE uptake activity using [3H]NE, β-receptor density by [125I]iodocyanopindolol, inotropic responses to dobutamine, and noradrenergic terminal neurotransmitter profiles by glyoxylic acid-induced histofluorescence for catecholamines, and immunocytochemical staining for tyrosine hydroxylase and neuropeptide Y. Desipramine decreased myocardial NE uptake activity and had no effect on the resting hemodynamics in both RHF and sham animals but decreased myocardial β-adrenoceptor density and β-adrenergic inotropic responses in both ventricles of the RHF animals. However, desipramine treatment prevented the reduction of sympathetic neurotransmitter profiles in the failing heart. Our results indicate that NE uptake inhibition facilitates the reduction of myocardial β-adrenoceptor density and β-adrenergic subsensitivity in RHF, probably by increasing interstitial NE concentrations, but protects the cardiac noradrenergic nerve endings from damage, probably via blockade of NE-derived neurotoxic metabolites into the nerve endings.
Oxidative capacity in failing hearts
Authors: Gong Guangrong , Liu Jingbo , Liang Peihua , Guo Tao , Hu Qingsong , Ochiai Ko , Hou Mingxiao , Ye Yun , Wu Xiaoyun , Mansoor Abdul , From Arthur H. L. , Ugurbil Kamil , Bache Robert J. , Zhang Jianyi
Published: March 2015

Although high-energy phosphate metabolism is abnormal in failing hearts [congestive heart failure (CHF)], it is unclear whether oxidative capacity is impaired. This study used the mitochondrial uncoupling agent 2,4-dinitrophenol (DNP) to determine whether reserve oxidative capacity exists during the high workload produced by catecholamine infusion in hypertrophied and failing hearts. Left ventricular hypertrophy (LVH) was produced by ascending aortic banding in 21 swine; 9 animals developed CHF. Basal myocardial phosphocreatine (PCr)/ATP measured with31P NMR spectroscopy was decreased in both LVH and CHF hearts (corresponding to an increase in free [ADP]), whereas ATP was decreased in hearts with CHF. Infusion of dobutamine and dopamine (each 20 μg · kg–1· min–1iv) caused an approximate doubling of myocardial oxygen consumption ([Formula: see text]) in all groups and decreased PCr/ATP in the normal and LVH groups. During continuing catecholamine infusion, DNP (2–8 mg/kg iv) caused further increases of [Formula: see text] in normal and LVH hearts with no change in PCr/ATP. In contrast, DNP caused no increase in [Formula: see text] in the failing hearts; the associated decrease of PCr/ATP suggests that DNP decreased the mitochondrial proton gradient, thereby causing ADP to increase to maintain adequate ATP synthesis.
Systemic and regional hemodynamic effects of angiotensin-(1–7) in rats
Authors: Sampaio Walkyria O. , Nascimento Antônio A. S. , Santos Robson A. S.
Published: March 2015

The systemic and regional hemodynamics effects of ANG-(1–7) were examined in urethane-anesthetized rats. The blood flow distribution (kidneys, skin, mesentery, lungs, spleen, brain, muscle, and adrenals), cardiac output, and total peripheral resistance were investigated by using fluorescent microspheres. Blood pressure and heart rate were recorded from the brachial artery. ANG-(1–7) infusion (110 fmol · min−1 · 10 min−1 iv) significantly increased blood flow to the kidney (5.10 ± 1.07 to 8.30 ± 0.97 ml · min−1 · g−1), mesentery (0.73 ± 0.16 to 1.17 ± 0.49 ml · min−1 · g−1), brain (1.32 ± 0.44 to 2.18 ± 0.85 ml · min−1 · g−1), and skin (0.07 ± 0.02 to 0.18 ± 0.07 ml · min−1 · g−1) and the vascular conductance in these organs. ANG-(1–7) also produced a significant increase in cardiac index (30%) and a decrease in total peripheral resistance (2.90 ± 0.55 to 2.15 ± 0.28 mmHg · ml−1 · min · 100 g). Blood flow to the spleen, muscle, lungs, and adrenals, as well as the blood pressure and heart rate, were not altered by the ANG-(1–7) infusion. The selective ANG-(1–7) antagonist A-779 reduced the blood flow in renal, cerebral, mesenteric, and cutaneous beds and blocked the ANG-(1–7)-induced vasodilatation in the kidney, mesentery, and skin, suggesting a significant role of endogenous ANG-(1–7) in these territories. The effects of ANG-(1–7) on the cerebral blood flow, cardiac index, systolic volume, and total peripheral resistance were partially attenuated by A-779. A high dose of ANG-(1–7) (11 pmol · min−1 · 10 min−1) caused an opposite effect of that produced by the low dose. Our results show for the first time that ANG-(1–7) has a previously unsuspected potent effect in the blood flow distribution and systemic hemodynamics.
Decreased renal NO excretion and reduced glomerular tuft area in mice lacking the bradykinin B2receptor
Authors: Schanstra Joost P. , Duchene Johan , Praddaude Françoise , Bruneval Patrick , Tack Ivan , Chevalier Jacques , Girolami Jean-Pierre , Bascands Jean-Loup
Published: March 2015

Bradykinin B2receptor knockout mice (B2−/−) have been useful to study the role of bradykinin under pathological conditions. With the use of these mice, it was shown that bradykinin plays an important role in angiogenesis, heart failure, salt-induced hypertension, and kidney fibrosis. Data on the role of the bradykinin B2receptor under physiological conditions using these mice are controversial and scarce, because these mice have no typical phenotype. For this reason, we have studied, under physiological conditions, renal hemodynamics as well as a number of morphometric glomerular parameters of B2−/−mice on a homogenized genetic background and on mice bred in a pathogen-free environment. Backcrossed B2−/−mice had normal blood pressure and normal apparent renal hemodynamics and morphology. However, reduced renal nitrite excretion and glomerular cGMP content were found, which was associated with a reduced glomerular capillary surface area. These differences had, however, no detectable effects on renal hemodynamics. These differences between B2−/−and wild-type mice might become important under pathological conditions as shown by a number of studies using these bradykinin B2receptor knockout mice.
An in vitro airway wall model of remodeling
Authors: Choe Melanie M. , Sporn Peter H. S. , Swartz Melody A.
Published: March 2015

Recent studies have shown that mechanical forces on airway epithelial cells can induce upregulation of genes involved in airway remodeling in diseases such as asthma. However, the relevance of these responses to airway wall remodeling is still unclear since 1) mechanotransduction is highly dependent on environment (e.g., matrix and other cell types) and 2) inflammatory mediators, which strongly affect remodeling, are also present in asthma. To assess the effects of mechanical forces on the airway wall in a relevant three-dimensional inflammatory context, we have established a tissue culture model of the human airway wall that can be induced to undergo matrix remodeling. Our model contains differentiated human bronchial epithelial cells characterized by tight junctions, cilia formation, and mucus secretion atop a collagen gel embedded with human lung fibroblasts. We found that addition of activated eosinophils and the application of 50% strain to the same system increased the epithelial thickness compared with either condition alone, suggesting that mechanical strain affects airway wall remodeling synergistically with inflammation. This integrated model more closely mimics airway wall remodeling than single-cell, conditioned media, or even two-dimensional coculture systems and is relevant for examining the importance of mechanical strain on airway wall remodeling in an inflammatory environment, which may be crucial for understanding and treating pathologies such as asthma.
Protein nitration in rat lungs during hyperoxia exposure: a possible role of myeloperoxidase
Authors: Narasaraju Telugu A. , Jin Nili , Narendranath Chintagari R. , Chen Zhongming , Gou Deming , Liu Lin
Published: March 2015

Several studies have suggested that exposure to hyperoxia causes lung injury through increased generation of reactive oxygen and nitrogen species. The present study was aimed to investigate the effects of hyperoxia exposure on protein nitration in lungs. Rats were exposed to hyperoxia (>95%) for 48, 60, and 72 h. Histopathological analysis showed a dramatic change in the severity of lung injury in terms of edema and hemorrhage between 48- and 60-h exposure times. Western blot for nitrotyrosine showed that several proteins with molecular masses of 29-66 kDa were nitrated in hyperoxic lung tissues. Immunohistochemical analyses indicate nitrotyrosine staining of alveolar epithelial and interstitial regions. Furthermore, immunoprecipitation followed by Western blot revealed the nitration of surfactant protein A and t1α, proteins specific for alveolar epithelial type II and type I cells, respectively. The increased myeloperoxidase (MPO) activity and total nitrite levels in bronchoalveolar lavage and lung tissue homogenates were observed in hyperoxic lungs. Neutrophils and macrophages isolated from the hyperoxia-exposed rats, when cocultured with a rat lung epithelial L2 cell line, caused a significant protein nitration in L2 cells. Inclusion of nitrite further increased the protein nitration. These studies suggest that protein nitration during hyperoxia may be mediated in part by MPO generated from activated phagocytic cells, and such protein modifications may contribute to hyperoxia-mediated lung injury.
Involvement of CD40-CD40L signaling in postischemic lung injury
Authors: Moore Timothy M. , Shirah W. Bradley , Khimenko Pavel L. , Paisley Peyton , Lausch Robert N. , Taylor Aubrey E.
Published: March 2015

Our studies show that ischemia-reperfusion (I/R) in the isolated rat lung causes retention of lymphocytes, which is associated with increased microvascular permeability, as determined by quantitative measurement of the microvascular filtration coefficient ( K f,c). Immunoneutralization of either CD40 or CD40L, cell surface proteins important in lymphocyte-endothelial cell proinflammatory events, results in significantly lower postischemic K f,c values. Antagonism of CD40-CD40L signaling also results in attenuation of I/R-elicited macrophage inflammatory protein-2 production. Rat lymphocytes activated ex vivo with phorbol 12-myristate, 13-acetate increased K f,c in isolated lungs independently of I/R, and this increase was prevented by pretreating lungs with anti-CD40. In addition to lymphocyte involvement via CD40-CD40L interactions, our studies also show that I/R injury is potentiated by antagonism of IL-10 produced locally within the postischemic lung, whereas exogenous, rat recombinant IL-10 provided protection against I/R-induced microvascular damage. Thus acute lymphocyte involvement in lung I/R injury involves CD40-CD40L signaling mechanisms, and these events may be influenced by local IL-10 generation.
Maternal loading with very low-density lipoproteins stimulates fetal surfactant synthesis
Authors: Ryan Alan J. , Medh Jheem D. , McCoy Diann M. , Salome Ronald G. , Mallampalli Rama K.
Published: March 2015

We examined whether administration of very low-density lipoproteins (VLDL) to pregnant rats increases surfactant phosphatidylcholine (PtdCho) content in fetal pre-type II alveolar epithelial cells. VLDL-triglycerides are hydrolyzed to fatty acids by lipoprotein lipase (LPL), an enzyme activated by heparin. Fatty acids released by LPL can incorporate into the PtdCho molecule or activate the key biosynthetic enzyme cytidylyltransferase (CCT). Dams were given BSA, heparin, VLDL, or VLDL with heparin intravenously. Radiolabeled VLDL given to the pregnant rat crossed the placenta and was distributed systemically in the fetus and incorporated into disaturated PtdCho (DSPtdCho) in pre-type II cells. Maternal administration of VLDL with heparin increased DSPtdCho content in cells by 45% compared with control ( P < 0.05). VLDL produced a dose-dependent, saturable, and selective increase in CCT activity. VLDL did not significantly alter immunoreactive CCT content but increased palmitic, stearic, and oleic acids in pre-type II cells. Furthermore, hypertriglyceridemic apolipoprotein E knockout mice contained significantly greater levels of DSPtdCho content in alveolar lavage and CCT activity compared with either LDL receptor knockout mice or wild-type controls that have normal serum triglycerides. Thus the nutritional or genetic modulation of serum VLDL-triglycerides provides specific fatty acids that stimulate PtdCho synthesis and CCT activity thereby increasing surfactant content.
Aberrant lung structure, composition, and function in a murine model of Hermansky-Pudlak syndrome
Authors: Lyerla Timothy A. , Rusiniak Michael E. , Borchers Michael , Jahreis Gerald , Tan Jian , Ohtake Patricia , Novak Edward K. , Swank Richard T.
Published: March 2015

Hermansky-Pudlak syndrome (HPS) is a genetically heterogeneous inherited disease causing hypopigmentation and prolonged bleeding times. An additional serious clinical problem of HPS is the development of lung pathology, which may lead to severe lung disease and premature death. No cure for the disease exists, and previously, no animal model for the HPS lung abnormalities has been reported. A mouse model of HPS, which is homozygously recessive for both the Hps1 (pale ear) and Hps2 (pearl) genes, exhibits striking abnormalities of lung type II cells. Type II cells and lamellar bodies of this mutant are greatly enlarged, and the lamellar bodies are engorged with surfactant. Mutant lungs accumulate excessive autofluorescent pigment. The air spaces of mutant lungs contain age-related elevations of inflammatory cells and foamy macrophages. In vivo measurement of lung hysteresivity demonstrated aberrant lung function in mutant mice. All these features are similar to the lung pathology described in HPS patients. Morphometry of mutant lungs indicates a significant emphysema. These mutant mice provide a model to further investigate the lung pathology and therapy of HPS. We hypothesize that abnormal type II cell lamellar body structure/function may predict future lung pathology in HPS.
A549 subclones demonstrate heterogeneity in toxicological sensitivity and antioxidant profile
Authors: Watanabe Nobuo , Dickinson Dale A. , Krzywanski David M. , Iles Karen E. , Zhang Hongqiao , Venglarik Charles J. , Forman Henry Jay
Published: March 2015

In A549 cell culture, significant variability was found in sensitivity to actinomycin D. Using limiting dilution, actinomycin D-susceptible (G4S) and -resistant (D3R) subclones were isolated. G4S cells were also susceptible to protein synthesis inhibitors, a redox cycling quinone, and an electrophile with concomitant activation of caspases 3 and 9. D3R cells were resistant to these agents without caspase activation. Antioxidant profiles revealed that D3R cells had significantly higher glutathione and glutathione reductase activity but markedly lower catalase, glutathione peroxidase, and aldehyde reductase activities than G4S cells. Thus A549 cells contain at least two distinct subpopulations with respect to predisposition to cell death and antioxidant profile. Because sensitivities to agents and the antioxidant profile were inconsistent, mechanisms independent of antioxidants, including the apparent inability to activate caspases in D3R cells, may play an important role. Regardless, the results suggest that antioxidant profiles of asymmetrical cell populations cannot predict sensitivity to oxidants and warn that the use of single subclones is advisable for mechanistic studies using A549 or other unstable cell lines.
Asbestos induces mitochondrial DNA damage and dysfunction linked to the development of apoptosis
Authors: Shukla Arti , Jung Michael , Stern Maria , Fukagawa Naomi K. , Taatjes Douglas J. , Sawyer Dennis , Van Houten Bennett , Mossman Brooke T.
Published: March 2015

To test the hypothesis that asbestos-mediated cell injury is mediated through an oxidant-dependent mitochondrial pathway, isolated mesothelial cells were examined for mitochondrial DNA damage as determined by quantitative PCR. Mitochondrial DNA damage occurred at fourfold lower concentrations of crocidolite asbestos compared with concentrations required for nuclear DNA damage. DNA damage by asbestos was preceded by oxidant stress as shown by confocal scanning laser microscopy using MitoTracker Green FM and the oxidant probe Redox Sensor Red CC-1. These events were associated with dose-related decreases in steady-state mRNA levels of cytochrome c oxidase, subunit 3 (COIII), and NADH dehydrogenase 5. Subsequently, dose-dependent decreases in formazan production, an indication of mitochondrial dysfunction, increased mRNA expression of pro- and antiapoptotic genes, and increased numbers of apoptotic cells were observed in asbestos-exposed mesothelial cells. The possible contribution of mitochondrial-derived pathways to asbestos-induced apoptosis was confirmed by its significant reduction after pretreatment of cells with a caspase-9 inhibitor. Apoptosis was decreased in the presence of catalase. Last, use of HeLa cells transfected with a mitochondrial transport sequence targeting the human DNA repair enzyme 8-oxoguanine DNA glycosylase to mitochondria demonstrated that asbestos-induced apoptosis was ameliorated with increased cell survival. Studies collectively indicate that mitochondria are initial targets of asbestos-induced DNA damage and apoptosis via an oxidant-related mechanism.
Negative impact of DEP exposure on human airway epithelial cell adhesion, stiffness, and repair
Authors: Doornaert Blandine , Leblond Valerie , Galiacy Stephane , Gras Gabriel , Planus Emmanuelle , Laurent Valerie , Isabey Daniel , Lafuma Chantal
Published: March 2015

Epidemiological and experimental studies suggest that diesel exhaust particles (DEPs) may be associated with increased respiratory mortality and morbidity. Several recent studies have also shown that DEPs increase the production of inflammatory cytokines by human bronchial epithelium (HBE) cells in vitro. The present study investigates the effects of DEPs on the interaction of l-HBE cells (16HBE14o-) with the cell and matrix microenvironment based on evaluation of integrin-type cell/matrix ligand expression, cytoskeleton (CSK) stiffness, and matrix remodeling via matrix metalloproteinase (MMP)-1, MMP-2, and MMP-9 expression. The results showed that DEP exposure induced: 1) a net dose-dependent decrease in CSK stiffness through actin fibers, 2) a concomitant specific reduction of both α3- and β1-integrin subunits extensively expressed on the HBE cell surface, 3) a decrease in the level of CD44, which is a major HBE cell-cell and HBE cell-matrix adhesion molecule; and 4) an isolated decrease in MMP-1 expression without any change in tissue inhibitor of matrix metalloproteinase (TIMP)-1 or TIMP-2 tissue inhibitors. Restrictive modulation of cell-matrix interaction, cell-cell connection, CSK stiffness, and fibrillary collagen remodeling results in a decreased wound closure capacity and an increased deadhesion capacity. In conclusion, on the basis of these results, we can propose that, in addition to their ability to increase the production of inflammatory cytokines, DEPs could also alter the links between actin CSK and the extracellular matrix, suggesting that they might facilitate HBE cell detachment in vivo.
Inducible lung-specific urokinase expression reduces fibrosis and mortality after lung injury in mice
Authors: Sisson Thomas H. , Hanson Kerstin E. , Subbotina Natalya , Patwardhan Anjali , Hattori Noboru , Simon Richard H.
Published: March 2015

Plasminogen activator inhibitor-1 (PAI-1)-deficient transgenic mice have improved survival and less fibrosis after intratracheal bleomycin instillation. We hypothesize that PAI-1 deficiency limits scarring through unopposed plasminogen activation. If this is indeed true, then we would expect increased urokinase-type plasminogen activator (uPA) expression to result in a similar reduction in scarring and improvement in mortality. To test our hypothesis, using the tetracycline gene regulatory system, we have generated a transgenic mouse model with the features of inducible, lung-specific uPA production. After doxycycline administration, these transgenic animals expressed increased levels of uPA in their bronchoalveolar lavage (BAL) fluid that accelerated intrapulmonary fibrin clearance. Importantly, this increased plasminogen activator production led to a reduction in both lung collagen accumulation and mortality after bleomycin-induced injury. These results suggest that PAI-1 deficiency does protect against the effects of bleomycin-induced lung injury through unopposed plasmin generation. By allowing the manipulation of plasminogen activation at different phases of the fibrotic process, this model will serve as a powerful tool in further investigations into the pathogenesis of pulmonary fibrosis.
Glutathione prevents inhibition of fibroblast-mediated collagen gel contraction by cigarette smoke
Authors: Kim Hui Jung , Liu Xiangde , Wang Hangjun , Kohyama Tadashi , Kobayashi Tetsu , Wen Fu-Qiang , Romberger Debra J. , Abe Shinji , MacNee William , Rahman Irfan , Rennard Stephen I.
Published: March 2015

Cigarette smoke, the major risk factor for the development of emphysema, contains over 4,700 chemical compounds, including free radicals and other oxidants (1014/puff). An imbalance between oxidants and antioxidants has been proposed in the pathogenesis of chronic obstructive pulmonary disease. Inhibition of repair processes has been suggested to be one pathway contributing to the development of emphysema. We hypothesized that cigarette smoke inhibition of repair might result from a shift of the oxidant/antioxidant balance in favor of oxidants. To evaluate this hypothesis, N-acetyl-l-cysteine (NAC), which serves as a substrate for glutathione (GSH) production, and buthionine sulfoximine (BSO), which inhibits GSH production, were incubated in the presence and absence of cigarette smoke extract (CSE) with fibroblasts in three-dimensional collagen gels. Neither agent alone altered gel contraction. CSE inhibition of gel contraction, however, was mitigated by NAC and potentiated by BSO. Parallel effects were observed on cigarette smoke inhibition of fibronectin production and mRNA expression as well as by changes in intracellular GSH content. Pretreatment of fibroblasts with NAC or BSO resulted in similar effects, suggesting that neither agent was acting directly on smoke but, rather, was altering cellular response to smoke. In conclusion, smoke inhibition of fibroblast repair, as reflected by collagen gel contraction and fibronectin production, may be modulated by intracellular GSH levels.
SPRR1B overexpression enhances entry of cells into the G0 phase of the cell cycle
Authors: Tesfaigzi Yohannes , Wright Paul S. , Belinsky Steven A.
Published: March 2015

Many studies have established the role of SPRR1B during squamous differentiation of skin and respiratory epithelial cells. However, its role in nonsquamous cells is largely unknown. We reported that expression of SPRR1B in Chinese hamster ovary (CHO) cells is increased as they enter the G0 phase of the cell cycle. The purpose of this study was to further investigate the SPRR1B expression pattern in nonsquamous tumors and to study its role in these cells. Expression of SPRR1B was detected by Northern blotting in a higher percentage of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced compared with beryllium metal-induced rat lung adenocarcinomas. In situ hybridizations confirmed that SPRR1B is expressed in individual or clusters of cells of nonsquamous cells from mouse, rat, and human adenocarcinomas. The same pattern of expression was observed in adenocarcinomas formed in nude mice from cell lines established from adenocarcinomas. SPRR1B expression was downregulated in the cell lines derived from adenocarcinoma when cells were enriched in G0 at low confluence. Tetraploidy was induced in CHO, mouse, and human tumor cell lines by stably overexpressing SPRR1B, whereas control cells showed no change in ploidy. Inducible expression of this protein for shorter periods using the ecdyson system did not affect growth rate or the ploidy of CHO cells but accelerated entry into G0/G1 compared with controls. These findings indicate that SPRR1B is likely coupled primarily to signals responsible for withdrawal from the proliferative state rather than the final stages of cellular quiescence and that its overexpression for prolonged periods may disrupt the normal progression of mitosis.
Sharing signals: connecting lung epithelial cells with gap junction channels
Authors: Koval Michael
Published: March 2015

Gap junction channels enable the direct flow of signaling molecules and metabolites between cells. Alveolar epithelial cells show great variability in the expression of gap junction proteins (connexins) as a function of cell phenotype and cell state. Differential connexin expression and control by alveolar epithelial cells have the potential to enable these cells to regulate the extent of intercellular coupling in response to cell stress and to regulate surfactant secretion. However, defining the precise signals transmitted through gap junction channels and the cross talk between gap junctions and other signaling pathways has proven difficult. Insights from what is known about roles for gap junctions in other systems in the context of the connexin expression pattern by lung cells can be used to predict potential roles for gap junctional communication between alveolar epithelial cells.
Dopamine increases lung liquid clearance during mechanical ventilation
Authors: Saldías F. J. , Comellas A. P. , Pesce L. , Lecuona E. , Sznajder J. I.
Published: March 2015

Short-term mechanical ventilation with high tidal volume (HVT) causes mild to moderate lung injury and impairs active Na+transport and lung liquid clearance in rats. Dopamine (DA) enhances active Na+transport in normal rat lungs by increasing Na+-K+-ATPase activity in the alveolar epithelium. We examined whether DA would increase alveolar fluid reabsorption in rats ventilated with HVT for 40 min compared with those ventilated with low tidal volume (LVT) and with nonventilated rats. Similar to previous reports, HVT ventilation decreased alveolar fluid reabsorption by ∼50% ( P < 0.001). DA increased alveolar fluid reabsorption in nonventilated control rats (by ∼60%), LVT ventilated rats (by ∼55%), and HVT ventilated rats (by ∼200%). In parallel studies, DA increased Na+-K+-ATPase activity in cultured rat alveolar epithelial type II cells (ATII). Depolymerization of cellular microtubules by colchicine inhibited the effect of DA on HVT ventilated rats as well as on Na+-K+-ATPase activity in ATII cells. Neither DA nor colchicine affected the short-term Na+-K+-ATPase α1- and β1-subunit mRNA steady-state levels or total α1- and β1-subunit protein abundance in ATII cells. Thus we reason that DA improved alveolar fluid reabsorption in rats ventilated with HVT by upregulating the Na+-K+-ATPase function in alveolar epithelial cells.
Intra-amniotic injection of IL-1 induces inflammation and maturation in fetal sheep lung
Authors: Willet Karen E. , Kramer Boris W. , Kallapur Suhas G. , Ikegami Machiko , Newnham John P. , Moss Timothy J. , Sly Peter D. , Jobe Alan H.
Published: March 2015

Antenatal inflammation may be an important triggering event in the pathogenesis of bronchopulmonary dysplasia but may also accelerate fetal lung maturation. We examined the effects of intra-amniotic (IA) interleukin (IL)-1α and IL-1β on maturation of the fetal sheep lung. These cytokine effects were compared with IA endotoxin, a potent proinflammatory stimulus that accelerated lung maturation. Date-bred ewes received 15 or 150 μg recombinant ovine IL-1α or IL-1β or 10 mg Escherichia coli endotoxin by IA injection at 118 days gestation (term = 150 days), and fetuses were delivered at 125 days. IL-1α and IL-1β improved lung function and increased alveolar saturated phosphatidylcholine (Sat PC) and surfactant protein mRNA expression at the higher dose. The maturation response to IL-1α was greater than that to IL-1β, which was similar to endotoxin response. Inflammation was also more pronounced after IL-1α treatment. Only endotoxin animals had residual inflammation of the fetal membranes at 7 days. Lung compliance, lung volume, and alveolar Sat PC were positively correlated with residual alveolar wash leukocyte numbers 7 days after IL-1 treatment, suggesting a link between lung inflammation and maturation.
Haptoglobin reduces lung injury associated with exposure to blood
Authors: Yang Funmei , Haile David J. , Berger Franklin G. , Herbert Damon C. , Van Beveren Emily , Ghio Andrew J.
Published: March 2015

The biological functions of the acute- phase protein haptoglobin (Hp) may be related to its ability to bind hemoglobin (Hb) or to modulate immune response. Hp is expressed at a high level in lung cells, yet its protective role(s) in the lung is not known. With the use of transgenic mice overexpressing Hp in alveolar macrophages, we demonstrated that Hp diminished Hb-induced lung injury when the lung was exposed to whole blood. In transgenic mouse lungs, Hb was more efficiently removed, and the induction of stress- responsive heme oxygenase-1 gene was significantly lower when compared with wild-type mice. At 24 h after blood treatment, the ferritin level that serves as an index for intracellular iron content was also lower in alveolar macrophages in transgenic mice than in wild-type mice. We propose that an Hp-mediated Hb catabolism process exists in alveolar macrophages. This process is likely coupled to an iron mobilization pathway and may be an efficient mechanism to reduce oxidative damage associated with hemolysis.
Ventilation-induced lung injury and mechanotransduction: stretching it too far?
Authors: Uhlig Stefan
Published: March 2015

10.1152/ajplung.00124. 2001. The Acute Respiratory Distress Syndrome Network clinical trial on ventilation of critically ill patients has drawn attention to the potential side effects of mechanical ventilation. Both clinical and basic research have demonstrated that injurious ventilation strategies can initiate or perpetuate local and systemic inflammatory responses. There are four principal mechanisms that can produce such a response. 1) Ventilation, especially with high ventilation pressures and zero positive end-expiratory pressure, can cause stress failure of the plasma membrane and of epithelial and endothelial barriers. Stress failure of the plasma membrane causes necrosis, which leads to liberation of both preformed inflammatory mediators and agents that stimulate other cells that are still intact to produce such mediators. 2) Stress failure of the barriers causes loss of compartmentalization with spread of mediators and bacteria throughout the body as a consequence. 3) Less injurious ventilation strategies that do not cause tissue destruction can elicit release of mediators by more specific mechanisms, presumably through activation of stretch-activated signaling cascades (mechanotransduction). 4) Ventilation with increasing positive pressures raises the pressure in the pulmonary circulation and thus vascular shear stress, both of which are known stimuli for endothelial cells. These different mechanisms should be taken into account in the design and the interpretation of studies on molecular mechanisms of ventilation-induced lung injury.
Differentiation of human pulmonary type II cells in vitro by glucocorticoid plus cAMP
Authors: Gonzales Linda W. , Guttentag Susan H. , Wade Kelly C. , Postle Anthony D. , Ballard Philip L.
Published: March 2015

Mature alveolar type II cells that produce pulmonary surfactant are essential for adaptation to extrauterine life and prevention of infant respiratory distress syndrome. We have developed a new in vitro model to further investigate regulation of type II cell differentiation. Epithelial cells isolated from human fetal lung were cultured in serum-free medium on plastic. Cells treated with dexamethasone + cAMP analog and isobutylmethylxanthine for 4 days exhibited increased phosphatidylcholine synthesis and content of disaturated phosphatidylcholine species, manyfold increases in all surfactant proteins with processing to mature forms, and abundant lamellar bodies. DNA microarray analysis identified ∼3,100 expressed genes, including subsets of genes induced 2- to >100-fold (∼2.5%) or repressed 2- to 18-fold (∼1.2%) by hormone treatment. Of the highly regulated genes, most were coregulated in an additive or synergistic manner by dexamethasone and cAMP agents. Approximately 90% of the regulated genes identified by this initial microarray analysis have not been previously recognized as hormone responsive. One newly identified hormone-induced gene is Nkx2.1 (thyroid transcription factor-1), which has a critical role in surfactant protein gene expression. Our findings indicate that glucocorticoid + cAMP is sufficient and necessary for precocious induction of functional type II cells in this in vitro system and that these hormones act primarily in combination to regulate expression of a subset of specific genes.
Na transport proteins are expressed by rat alveolar epithelial type I cells
Authors: Borok Zea , Liebler Janice M. , Lubman Richard L. , Foster Martha J. , Zhou Beiyun , Li Xian , Zabski Stephanie M. , Kim Kwang-Jin , Crandall Edward D.
Published: March 2015

Despite a presumptive role for type I (AT1) cells in alveolar epithelial transport, specific Na transporters have not previously been localized to these cells. To evaluate expression of Na transporters in AT1 cells, double labeling immunofluorescence microscopy was utilized in whole lung and in cytocentrifuged preparations of partially purified alveolar epithelial cells (AEC). Expression of Na pump subunit isoforms and the α-subunit of the rat (r) epithelial Na channel (α-ENaC) was evaluated in isolated AT1 cells identified by their immunoreactivity with AT1 cell-specific antibody markers (VIIIB2 and/or anti-aquaporin-5) and lack of reactivity with antibodies specific for AT2 cells (anti-surfactant protein A) or leukocytes (anti-leukocyte common antigen). Expression of the Na pump α1-subunit in AEC was assessed in situ. Na pump subunit isoform and α-rENaC expression was also evaluated by RT-PCR in highly purified (∼95%) AT1 cell preparations. Labeling of isolated AT1 cells with anti-α1and anti-β1Na pump subunit and anti-α-rENaC antibodies was detected, while reactivity with anti-α2Na pump subunit antibody was absent. AT1 cells in situ were reactive with anti-α1Na pump subunit antibody. Na pump α1- and β1- (but not α2-) subunits and α-rENaC were detected in highly purified AT1 cells by RT-PCR. These data demonstrate that AT1 cells express Na pump and Na channel proteins, supporting a role for AT1 cells in active transalveolar epithelial Na transport.
Smad3 deficiency attenuates bleomycin-induced pulmonary fibrosis in mice
Authors: Zhao Jingsong , Shi Wei , Wang Yan-Ling , Chen Hui , Bringas Pablo , Datto Michael B. , Frederick Joshua P. , Wang Xiao-Fan , Warburton David
Published: March 2015

Transforming growth factor-β (TGF-β) signaling plays an important regulatory role during lung fibrogenesis. Smad3 was identified in the pathway for transducing TGF-β signals from the cell membrane to the nucleus. Using mice without Smad3 gene expression, we investigated whether Smad3 could regulate bleomycin-induced pulmonary fibrosis in vivo. Mice deficient in Smad3 demonstrated suppressed type I procollagen mRNA expression and reduced hydroxyproline content in the lungs compared with wild-type mice treated with bleomycin. Furthermore, loss of Smad3 greatly attenuated morphological fibrotic responses to bleomycin in the mouse lungs, suggesting that Smad3 is implicated in the pathogenesis of pulmonary fibrosis. These results show that Smad3 contributes to bleomycin-induced lung injury and that Smad3 may serve as a novel target for potential therapeutic treatment of lung fibrosis.
Detection of chondroitin sulfates and decorin in developing fetal and neonatal rat lung
Authors: Wang Yiqiong , Sakamoto Kaori , Khosla Jody , Sannes Philip L.
Published: March 2015

Chondroitin sulfates and their related proteoglycans are components of extracellular matrix that act as key determinants of growth and differentiation characteristics of developing lungs. Changes in their immunohistochemical distribution during progressive organ maturation were examined with monospecific antibodies to chondroitin sulfate, a nonbasement membrane chondroitin sulfate proteoglycan, and the specific chondroitin sulfate-containing proteoglycan decorin in whole fetuses and lungs from newborn and adult rats. Alveolar and airway extracellular matrix immunostained heavily in the prenatal rat for both chondroitin sulfate and chondroitin sulfate proteoglycan, whereas decorin was confined to developing airways and vessels. These sites retained their respective levels of reactivity with all antibodies through 1–10 days postnatal but thereafter became progressively more diminished and focal in alveolar regions. The heavy staining seen early in development was interpreted to reflect a significant and wide distribution of chondroitin sulfates, chondroitin sulfate proteoglycans, and decorin in rapidly growing tissues, whereas the reduced and more focal reactivity observed at later time points coincided with known focal patterns of localization of fibrillar elements of the extracellular matrix and a more differentiated state.
FGF-10 prevents mechanical stretch-induced alveolar epithelial cell DNA damage via MAPK activation
Authors: Upadhyay Daya , Correa-Meyer Eduardo , Sznajder Jacob I. , Kamp David W.
Published: March 2015

Cyclic stretch of alveolar epithelial cells (AEC) can alter normal lung barrier function. Fibroblast growth factor-10 (FGF-10), an alveolar type II cell mitogen that is critical for lung development, may have a role in promoting AEC repair. We studied whether cyclic stretch induces AEC DNA damage and whether FGF-10 would be protective. Cyclic stretch (30 min of 30% strain amplitude and 30 cycles/min) caused AEC DNA strand break formation, as assessed by alkaline unwinding technique and DNA nucleosomal fragmentation. Pretreatment of AEC with FGF-10 (10 ng/ml) blocked stretch-induced DNA strand break formation and DNA fragmentation. FGF-10 activated AEC mitogen-activated protein kinase (MAPK), and MAPK inhibitors prevented FGF-10-induced AEC MAPK activation and abolished the protective effects of FGF-10 against stretch-induced DNA damage. In addition, a Grb2-SOS inhibitor (SH3b-p peptide), a RAS inhibitor (farnesyl transferase inhibitor 277), and a RAF-1 inhibitor (forskolin) each prevented FGF-10-induced extracellular signal-regulated kinase (ERK) 1/2 phosphorylation in AEC. Moreover, N17-A549 cells that express a RAS dominant/negative protein prevented the FGF-10-induced ERK1/2 phosphorylation and RAS activation in AEC. We conclude that cyclic stretch causes AEC DNA damage and that FGF-10 attenuates these effects by mechanisms involving MAPK activation via the Grb2-SOS/Ras/RAF-1/ERK1/2 pathway.
Alveolar epithelial cell inhibition of fibroblast proliferation is regulated by MCP-1/CCR2 and mediated by PGE2
Authors: Moore Bethany B. , Peters-Golden Marc , Christensen Paul J. , Lama Vibha , Kuziel William A. , Paine Robert , Toews Galen B.
Published: March 2015

CC chemokine receptor 2 (CCR2) −/− mice are protected from experimental pulmonary fibrosis, a disease increasingly recognized as being mediated by dysfunctional interactions between epithelial cells and fibroblasts. We have sought to investigate the interactions between alveolar epithelial cells (AECs) and fibroblasts in these fibrosis-resistant (CCR2 −/−) and fibrosis-sensitive (CCR2 +/+) mice. AECs from CCR2 −/− mice suppress fibroblast proliferation more than AECs from CCR2 +/+ mice (77 vs. 43%). Exogenous administration of the CCR2 ligand monocyte chemoattractant protein-1 (MCP-1) to the fibroblast-AEC cocultures reverses the suppression mediated by CCR2 +/+ AECs but has no effect with CCR2 −/− AECs. MCP-1 regulates AEC function but not fibroblast function. AEC inhibition of fibroblast proliferation was mediated by a soluble, aspirin-sensitive factor. Accordingly, AECs from CCR2 −/− mice produce greater quantities of PGE2than do AECs from CCR2 +/+ mice, and MCP-1 inhibits AEC-derived PGE2synthesis. Diminished PGE2production by AECs results in enhanced fibroproliferation. Thus an important profibrotic mechanism of MCP-1/CCR2 interactions is to limit PGE2production in AECs after injury, thus promoting fibrogenesis.
Ceramide accumulation precedes caspase-3 activation during apoptosis of A549 human lung adenocarcinoma cells
Authors: Ravid Tommer , Tsaba Adili , Gee Peter , Rasooly Reuven , Medina Edward A. , Goldkorn Tzipora
Published: March 2015

Ceramide, the basic structural unit of sphingolipids, controls the balance between cell growth and death by inducing apoptosis. We have previously shown that accumulation of ceramide, triggered by hydrogen peroxide (H2O2) or by short-chain ceramide analogs, induces apoptosis of lung epithelial cells. Here we elucidate the link between caspase-3 activation, at the execution phase, and ceramide accumulation, at the commitment phase of apoptosis in A549 human lung adenocarcinoma cells. The induction of ceramide accumulation by various triggers of ceramide generation, such as H2O2, C6-ceramide, or UDP-glucose-ceramide glucosyltransferase inhibitordl-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol, triggered the activation of caspase-3. This ceramide elevation also induced the cleavage of the death substrate poly(ADP-ribose) polymerase and was followed by apoptotic cell death. Ceramide-mediated apoptosis was blocked by a general caspase inhibitor, Boc-d-fluoromethylketone, and by overexpression of the antiapoptotic protein Bcl-2. Notably, overexpression of Bcl-2 reduced the basal cellular levels of ceramide and prevented the induction of ceramide generation by C6-ceramide, which implies ceramide generation as a possible target for the antiapoptotic effects of Bcl-2.