AJP: Lung Cellular and Molecular Physiology current issue

Interactions of nanoparticles with pulmonary structures and cellular responses

2008-05-01

Combustion-derived and synthetic nano-sized particles (NSP) have gained considerable interest among pulmonary researchers and clinicians for two main reasons. 1) Inhalation exposure to combustion-derived NSP was associated with increased pulmonary and cardiovascular morbidity and mortality as suggested by epidemiological studies. Experimental evidence has provided a mechanistic picture of the adverse health effects associated with inhalation of combustion-derived and synthetic NSP. 2) The toxicological potential of NSP contrasts with the potential application of synthetic NSP in technological as well as medicinal settings, with the latter including the use of NSP as diagnostics or therapeutics. To shed light on this paradox, this article aims to highlight recent findings about the interaction of inhaled NSP with the structures of the respiratory tract including surfactant, alveolar macrophages, and epithelial cells. Cellular responses to NSP exposure include the generation of reactive oxygen species and the induction of an inflammatory response. Furthermore, this review places special emphasis on methodological differences between experimental studies and the caveats associated with the dose metrics and points out ways to overcome inherent methodological problems.

Xanthine oxidoreductase in respiratory and cardiovascular disorders

Boueiz, A., Damarla, M., Hassoun, P. M. — 2008-05-01

In addition to its critical role in purine metabolism, xanthine oxidoreductase (XOR) has been implicated in the development of tissue oxidative damage in a wide variety of respiratory and cardiovascular disorders such as acute lung injury, ischemia-reperfusion injury, atherosclerosis, heart failure, and arterial hypertension. Although much remains to be clarified about the regulation and signaling pathways of this enzyme, it is quite evident from abundant investigation in animal models and some human trials that XOR inhibition can favorably alter critical disease processes and impact outcomes. From promising bench-to-bedside data, a better understanding of this enigmatic enzyme is emerging. However, the positive findings related to XOR inhibition need to be confirmed in large-scale, well-designed clinical trials. This will hopefully provide new opportunities for therapeutic intervention. This article reviews the available evidence involving XOR in oxidative states with specific emphasis on respiratory and cardiovascular diseases.

Caveolin-1 stops profibrogenic signaling?

Kim, H. P., Choi, A. M. K. — 2008-05-01

Antifibrotic properties of caveolin-1 scaffolding domain in vitro and in vivo

2008-05-01

Lung fibrosis involves the overexpression of ECM proteins, primarily collagen, by -smooth muscle actin (ASMA)-positive cells. Caveolin-1 is a master regulator of collagen expression by cultured lung fibroblasts and of lung fibrosis in vivo. A peptide equivalent to the caveolin-1 scaffolding domain (CSD peptide) inhibits collagen and tenascin-C expression by normal lung fibroblasts (NLF) and fibroblasts from the fibrotic lungs of scleroderma patients (SLF). CSD peptide inhibits ASMA expression in SLF but not NLF. Similar inhibition of collagen, tenascin-C, and ASMA expression was also observed when caveolin-1 expression was upregulated using adenovirus. These observations suggest that the low caveolin-1 levels in SLF cause their overexpression of collagen, tenascin-C, and ASMA. In mechanistic studies, MEK, ERK, JNK, and Akt were hyperactivated in SLF, and CSD peptide inhibited their activation and altered their subcellular localization. These studies and experiments using kinase inhibitors suggest many differences between NLF and SLF in signaling cascades. To validate these data, we determined that the alterations in signaling molecule activation observed in SLF also occur in fibrotic lung tissue from scleroderma patients and in mice with bleomycin-induced lung fibrosis. Finally, we demonstrated that systemic administration of CSD peptide to bleomycin-treated mice blocks epithelial cell apoptosis, inflammatory cell infiltration, and changes in tissue morphology as well as signaling molecule activation and collagen, tenascin-C, and ASMA expression associated with lung fibrosis. CSD peptide may be a prototype for novel treatments for human lung fibrosis that act, in part, by inhibiting the expression of ASMA and ECM proteins.

Caveolin-1: a critical regulator of pulmonary vascular architecture and nitric oxide bioavailability in pulmonary hypertension

Ryter, S. W., Choi, A. M. K. — 2008-05-01

Increased pulmonary vascular resistance and defective pulmonary artery filling in caveolin-1-/- mice

2008-05-01

Caveolin-1, the structural and signaling protein of caveolae, is an important negative regulator of endothelial nitric oxide synthase (eNOS). We observed that mice lacking caveolin-1 (Cav1^–/–) had twofold increased plasma NO levels but developed pulmonary hypertension. We measured pulmonary vascular resistance (PVR) and assessed alterations in small pulmonary arteries to determine the basis of the hypertension. PVR was 46% greater in Cav1^–/– mice than wild-type (WT), and increased PVR in Cav1^–/– mice was attributed to precapillary sites. Treatment with N^G-nitro-l-arginine methyl ester (l-NAME) to inhibit NOS activity raised PVR by 42% in WT but 82% in Cav1^–/– mice, indicating greater NO-mediated pulmonary vasodilation in Cav1^–/– mice compared with WT. Pulmonary vasculature of Cav1^–/– mice was also less reactive to the vasoconstrictor thromboxane A₂ mimetic (U-46619) compared with WT. We observed redistribution of type I collagen and expression of smooth muscle -actin in lung parenchyma of Cav1^–/– mice compared with WT suggestive of vascular remodeling. Fluorescent agarose casting also showed markedly decreased density of pulmonary arteries and artery filling defects in Cav1^–/– mice. Scanning electron microscopy showed severely distorted and tortuous pulmonary precapillary vessels. Thus caveolin-1 null mice have elevated PVR that is attributed to remodeling of pulmonary precapillary vessels. The elevated basal plasma NO level in Cav1^–/– mice compensates partly for the vascular structural abnormalities by promoting pulmonary vasodilation.

Intra-alveolar tissue factor pathway inhibitor is not sufficient to block tissue factor procoagulant activity

2008-05-01

The alveolar compartment in acute lung injury contains high levels of tissue factor (TF) procoagulant activity favoring fibrin deposition. We previously reported that the alveolar epithelium can release TF procoagulant activity in response to a proinflammatory stimulus. To test the hypothesis that the alveolar epithelium further modulates intra-alveolar fibrin deposition through secretion of an endogenous inhibitor to TF, tissue factor pathway inhibitor (TFPI), we measured TFPI levels in edema fluid (EF) from patients with acute respiratory distress syndrome. To determine whether the alveolar epithelium can release TFPI, both full-length TFPI and truncated TFPI were measured (ELISA) in pulmonary edema fluid from patients with acute respiratory distress syndrome (ARDS) and a control group of patients with hydrostatic pulmonary edema (HYDRO). TFPI protein was also measured in conditioned media (CM) and cell lysates (CL) from human alveolar epithelial cells (A549) after exposure to cytomix (TNF-, IL-1β, IFN-). TFPI protein levels were higher in pulmonary edema fluid from patients with ARDS vs. HYDRO. TFPI protein was increased in CM and did not change in CL after cytomix treatment; TFPI mRNA levels (RT-PCR) did not change. Despite the high levels of TFPI, both the EF and CM retained significant TF procoagulant activity as measured by plasma recalcification time. The majority of intra-alveolar TFPI was in a truncated, inactive form, whereas the majority of TFPI released from cells was full length, suggesting different mechanisms of inactivation. In summary, the alveolar epithelium releases TFPI in response to an inflammatory stimulus but does not increase TFPI gene transcription or protein production. Levels of intra-alveolar TFPI in ARDS are not sufficient to block intra-alveolar TF procoagulant activity due to truncation and inactivation of intra-alveolar TFPI.

Reversal of elastase-induced pulmonary emphysema and promotion of alveolar epithelial cell proliferation by simvastatin in mice

2008-05-01

Besides lowering cholesterol, statins exert multiple effects, such as anti-inflammatory activity and improvement of endothelial cell function. We examined whether simvastatin (SS) protects against the development of elastase-induced pulmonary emphysema in mice by using mean linear intercepts of alveoli (Lm) as a morphometric parameter of emphysema. After injection of intratracheal elastase on day 0, C57BL/6 mice were treated daily with SS (SS+ group) or PBS (SS– group) for 2 wk. A 21% decrease in Lm on day 7 was observed in the SS+ group vs. the SS– group. Anti-inflammatory effects of SS were observed as a decrease in percentage of neutrophils up to day 3, and in hydroxyproline concentration on day 3, in bronchoalveolar lavage fluid (BALF). SS also increased the number of proliferating cell nuclear antigen (PCNA)-positive alveolar epithelial cells between days 3 and 14. To confirm the role of statins in promoting proliferation of alveolar cells, mice were treated with SS (SS+) vs. PBS (SS–) for 12 days, starting 3 wk after elastase administration. After SS treatment, Lm decreased by 52% and PCNA-positive alveolar epithelial cells increased compared with the SS– group. Concentrations of vascular endothelial growth factor in BALF and endothelial nitric oxide synthase protein expression in pulmonary vessels tended to be higher in the SS+ group vs. the SS– group in this protocol. In conclusion, SS inhibited the development of elastase-induced pulmonary emphysema in mice. This therapeutic effect was due not only to anti-inflammation but also to the promotion of alveolar epithelial cell regeneration, partly mediated by restoring endothelial cell functions.

PPAR-{gamma} agonists inhibit profibrotic phenotypes in human lung fibroblasts and bleomycin-induced pulmonary fibrosis

2008-05-01

Pulmonary fibrosis is characterized by alterations in fibroblast phenotypes resulting in excessive extracellular matrix accumulation and anatomic remodeling. Current therapies for this condition are largely ineffective. Peroxisome proliferator-activated receptor- (PPAR-) is a member of the nuclear hormone receptor superfamily, the activation of which produces a number of biological effects, including alterations in metabolic and inflammatory responses. The role of PPAR- as a potential therapeutic target for fibrotic lung diseases remains undefined. In the present study, we show expression of PPAR- in fibroblasts obtained from normal human lungs and lungs of patients with idiopathic interstitial pneumonias. Treatment of lung fibroblasts and myofibroblasts with PPAR- agonists results in inhibition of proliferative responses and induces cell cycle arrest. In addition, PPAR- agonists, including a constitutively active PPAR- construct (VP16-PPAR-), inhibit the ability of transforming growth factor-β1 to induce myofibroblast differentiation and collagen secretion. PPAR- agonists also inhibit fibrosis in a murine model, even when administration is delayed until after the initial inflammation has largely resolved. These observations indicate that PPAR- is an important regulator of fibroblast/myofibroblast activation and suggest a role for PPAR- ligands as novel therapeutic agents for fibrotic lung diseases.

20-HETE increases superoxide production and activates NAPDH oxidase in pulmonary artery endothelial cells

2008-05-01

Reactive oxygen species (ROS) signal vital physiological processes including cell growth, angiogenesis, contraction, and relaxation of vascular smooth muscle. Because cytochrome P-450 family 4 (CYP4)/20-hydroxyeicosatetraenoic acid (20-HETE) has been reported to enhance angiogenesis, pulmonary vascular tone, and endothelial nitric oxide synthase function, we explored the potential of this system to stimulate bovine pulmonary artery endothelial cell (BPAEC) ROS production. Our data are the first to demonstrate that 20-HETE increases ROS in BPAECs in a time- and concentration-dependent manner as detected by enhanced fluorescence of oxidation products of dihydroethidium (DHE) and dichlorofluorescein diacetate. An analog of 20-HETE elicits no increase in ROS and blocks 20-HETE-evoked increments in DHE fluorescence, supporting its function as an antagonist. Endothelial cells derived from bovine aortas exhibit enhanced ROS production to 20-HETE quantitatively similar to that of BPAECs. 20-HETE-induced ROS production in BPAECs is blunted by pretreatment with polyethylene-glycolated SOD, apocynin, inhibition of Rac1, and a peptide-based inhibitor of NADPH oxidase subunit p47^phox association with gp91. These data support 20-HETE-stimulated, NADPH oxidase-derived, and Rac1/2-dependent ROS production in BPAECs. 20-HETE promotes translocation of p47^phox and tyrosine phosphorylation of p47^phox in a time-dependent manner as well as increased activated Rac1/2, providing at least three mechanisms through which 20-HETE activates NADPH oxidase. These observations suggest that 20-HETE stimulates ROS production in BPAECs at least in part through activation of NADPH oxidase within minutes of application of the lipid.

Substance P acts via the neurokinin receptor 1 to elicit bronchoconstriction, oxidative stress, and upregulated ICAM-1 expression after oil smoke exposure

Li, P.-C., Chen, W.-C., Chang, L.-C., Lin, S.-C. — 2008-05-01

This study aimed to 1) assess whether substance P (SP) acts via neurokinin (NK)-1 and NK-2 receptors to stimulate neurogenic inflammation (indicated by formation of ICAM-1 expression and oxidative stress) following oil smoke exposure (OSE) in rats; and 2) determine if pretreatment with antioxidants ameliorates the deleterious effects of OSE. Rats were pretreated with NK-1 receptor antagonist CP-96345, NK-2 receptor antagonist SR-48968, vitamin C, or catechins. OSE was for 30–120 min. Rats were killed 0–8 h later. Total lung resistance (R_L), airway smooth muscle activity (ASMA), lung ICAM-1 expression, neurogenic plasma extravasation (via India ink and Evans blue dye), bronchoalveolar lavage fluid SP concentrations, and reactive oxygen species formation [via lucigenin- and luminal-amplified chemiluminescence (CL)] were assessed. Lung histology was performed. SP concentrations increased significantly in nonpretreated rats following OSE in a dose-dependent manner. R_L and total ASMA increased over time after OSE. Vitamin C and catechin pretreatments were associated with significantly reduced lucigenin CL 2 and 4 h after OSE. Pretreatment with catechins significantly reduced luminal CL counts 4 and 8 h after OSE. Evans blue levels were significantly reduced following 60 and 120 min of OSE in catechin- and CP-96345-pretreated rats. ICAM-1 protein expression was significantly decreased in all pretreatment groups after OSE. Thickening of the alveolar capillary membrane, focal hemorrhaging, interstitial pneumonitis, and peribronchiolar inflammation were apparent in OSE lungs. These findings suggest that SP acts via the NK-1 receptor to provoke neurogenic inflammation, oxidative stress, and ICAM-1 expression after OSE in rats.

TRX-ASK1-JNK signaling regulation of cell density-dependent cytotoxicity in cigarette smoke-exposed human bronchial epithelial cells

2008-05-01

Cigarette smoke is a major environmental air pollutant that injures airway epithelium and incites subsequent diseases including chronic obstructive pulmonary disease. The lesion that smoke induces in airway epithelium is still incompletely understood. Using a LIVE/DEAD cytotoxicity assay, we observed that subconfluent cultures of bronchial epithelial cells derived from both human and monkey airway tissues and an immortalized normal human bronchial epithelial cell line (HBE1) were more susceptible to injury by cigarette smoke extract (CSE) and by direct cigarette smoke exposure than cells in confluent cultures. Scraping confluent cultures also caused an enhanced cell injury predominately in the leading edge of the scraped confluent cultures by CSE. Cellular ATP levels in both subconfluent and confluent cultures were drastically reduced after CSE exposure. In contrast, GSH levels were significantly reduced only in subconfluent cultures exposed to smoke and not in confluent cultures. Western blot analysis demonstrated ERK activation in both confluent and subconfluent cultures after CSE. However, activation of apoptosis signal-regulating kinase 1 (ASK1), JNK, and p38 were demonstrated only in subconfluent cultures and not in confluent cultures after CSE. Using short interfering RNA (siRNA) to JNK1 and JNK2 and a JNK inhibitor, we attenuated CSE-mediated cell death in subconfluent cultures but not with an inhibitor of the p38 pathway. Using the tetracycline (Tet)-on inducible approach, overexpression of thioredoxin (TRX) attenuated CSE-mediated cell death and JNK activation in subconfluent cultures. These results suggest that the TRX-ASK1-JNK pathway may play a critical role in mediating cell density-dependent CSE cytotoxicity.

Prostasin expression is regulated by airway surface liquid volume and is increased in cystic fibrosis

2008-05-01

Airway surface liquid (ASL) absorption is initiated by Na⁺ entry via epithelial Na⁺ channels (ENaC), which establishes an osmotic gradient that drives fluid from the luminal to serosal airway surface. We and others have recently reported that a protease/anti-protease balance regulates ENaC in human airway epithelial cells (HAEC) and provides a mechanism for autoregulation of ASL volume. In cystic fibrosis (CF), this balance is disturbed, leading to constitutive proteolytic activation of ENaC and the pathological Na⁺ hyperabsorption characteristic of this airway disease. Prostasin is a glycosylphosphatidylinositol-anchored serine protease that activates ENaC and is expressed on the surface epithelium lining the airway. In this report we present evidence that prostasin expression is regulated by the ASL volume, allowing for increased proteolytic activation of ENaC when the ASL volume is high. Prostasin activity is further regulated by the cognate serpin protease nexin-1 (PN-1), which is expressed in HAEC and inhibits Na⁺ absorption by forming an inactive complex with prostasin and preventing the proteolytic processing of prostasin. Whereas these mechanisms regulate prostasin expression in response to ASL volume in non-CF epithelia, HAEC cultured from CF patients express >50% more prostasin on the epithelial surface. These findings suggest that a proteolytic cascade involving prostasin, an upstream prostasin-activating protease, and PN-1 regulate Na⁺ absorption in the airway and that abnormal prostasin expression contributes to excessive proteolytic activation of ENaC in CF patients.

The regulation of selective and nonselective Na+ conductances in H441 human airway epithelial cells

Brown, S. G., Gallacher, M., Olver, R. E., Wilson, S. M. — 2008-05-01

Analysis of membrane currents recorded from hormone-deprived H441 cells showed that the membrane potential (V_m) in single cells (approximately –80 mV) was unaffected by lowering [Na⁺]_o or [Cl^–]_o, indicating that cellular Na⁺ and Cl^– conductances (G_Na and G_Cl, respectively) are negligible. Although insulin (20 nM, ~24 h) and dexamethasone (0.2 µM, ~24 h) both depolarized V_m by ~20 mV, the response to insulin reflected a rise in G_Cl mediated via phosphatidylinositol 3-kinase (PI3K) whereas dexamethasone acted by inducing a serum- and glucocorticoid-regulated kinase 1 (SGK1)-dependent rise in G_Na. Although insulin stimulation/PI3K-P110 expression did not directly increase G_Na, these maneuvers augmented the dexamethasone-induced conductance. The glucocorticoid/SGK1-induced G_Na in single cells discriminated poorly between Na⁺ and K⁺ (P_Na/P_K ~0.6), was insensitive to amiloride (1 mM), but was partially blocked by LaCl₃ (La³⁺; 1 mM, ~80%), pimozide (0.1 mM, ~40%), and dichlorobenzamil (15 µM, ~15%). Cells growing as small groups, on the other hand, expressed an amiloride-sensitive (10 µM), selective G_Na that displayed the same pattern of hormonal regulation as the nonselective conductance in single cells. These data therefore 1) confirm that H441 cells can express selective or nonselective G_Na (14, 48), 2) show that these conductances are both induced by glucocorticoids/SGK1 and subject to PI3K-dependent regulation, and 3) establish that cell-cell contact is vitally important to the development of Na⁺ selectivity and amiloride sensitivity.

Regulation of angiopoietin expression by bacterial lipopolysaccharide

Mofarrahi, M., Nouh, T., Qureshi, S., Guillot, L., Mayaki, D., Hussain, S. N. A. — 2008-05-01

Angiopoietins are ligands for Tie-2 receptors and play important roles in angiogenesis and inflammation. While angiopoietin-1 (Ang-1) inhibits inflammatory responses, angiopoietin-2 (Ang-2) promotes cytokine production and vascular leakage. In this study, we evaluated in vivo and in vitro effects of Escherichia coli lipopolysaccharides (LPS) on angiopoietin expression. Wild-type C57/BL6 mice were injected with saline (control) or E. coli LPS (20 mg/ml ip) and killed 6, 12, and 24 h later. The diaphragm, lung, and liver were excised and assayed for mRNA and protein expression of Ang-1, Ang-2, and Tie-2 protein and tyrosine phosphorylation. LPS injection elicited a severalfold rise in Ang-2 mRNA and protein levels in the three organs. By comparison, both Ang-1 and Tie-2 levels in the diaphragm, liver, and lung were significantly attenuated by LPS administration. In addition, Tie-2 tyrosine phosphorylation in the lung was significantly reduced in response to LPS injection. In vitro exposure to E. coli LPS elicited cell-specific changes in Ang-1 expression, with significant induction in Ang-1 expression being observed in cultured human epithelial cells, whereas significant attenuation of Ang-1 expression was observed in response to E. coli LPS exposure in primary human skeletal myoblasts. In both cell types, E. coli LPS elicited substantial induction of Ang-2 mRNA, a response that was mediated in part through NF-B. We conclude that in vivo endotoxemia triggers functional inhibition of the Ang-1/Tie-2 receptor pathway by reducing Ang-1 and Tie-2 expression and inducing Ang-2 levels and that this response may contribute to enhanced vascular leakage in sepsis.

Reduced spontaneous relaxation in immature guinea pig airway smooth muscle is associated with increased prostanoid release

Wang, L., Pozzato, V., Turato, G., Madamanchi, A., Murphy, T. M., Chitano, P. — 2008-05-01

Airway smooth muscle (ASM) from infant guinea pigs has less spontaneous relaxation during stimulation than ASM from adults. Inhibition of cyclooxygenase (COX), which catalyzes the production of prostanoids, increases this relaxation in infant ASM and abolishes age differences, thus suggesting that prostanoids reduce relaxation in infant ASM. In this study, we investigated whether leukotrienes are also involved in reducing spontaneous relaxation; whether the two COX isoforms, COX-1 and COX-2, differentially regulate spontaneous relaxation; and whether prostanoid release is developmentally regulated in guinea pig ASM. In different age groups, we measured relaxation during and after electrical stimulation in tracheal strips as well as prostanoid release from tracheal segments. Relaxation was studied in the absence and in the presence of a lipoxygenase inhibitor, a cysteinyl leukotriene receptor-1 antagonist, a COX-1 inhibitor, or a COX-2 inhibitor. We found that inhibition of lipoxygenase or cysteinyl leukotriene receptor-1 antagonism did not increase spontaneous relaxation at any age, thus excluding a role for leukotrienes in this phenomenon. Inhibition of COX-2, but not COX-1, promoted spontaneous relaxation. The basal release of prostanoids was more abundant in tissue from infant animals and decreased significantly with age. Thromboxane B₂ was the most abundant metabolite released at all ages. Electrical stimulation and epithelium removal did not affect the age difference in prostanoid release. We conclude that increased basal prostanoid release contributes to the reduced spontaneous relaxation in immature guinea pig ASM compared with older animals. By regulating ASM relaxation, prostanoids may play a role in the airway hyperresponsiveness at a young age.

Alterations to surfactant precede physiological deterioration during high tidal volume ventilation

2008-05-01

Lung injury due to mechanical ventilation is associated with an impairment of endogenous surfactant. It is unknown whether this impairment is a consequence of or an active contributor to the development and progression of lung injury. To investigate this issue, the present study addressed three questions: Do alterations to surfactant precede physiological lung dysfunction during mechanical ventilation? Which components are responsible for surfactant's biophysical dysfunction? Does exogenous surfactant supplementation offer a physiological benefit in ventilation-induced lung injury? Adult rats were exposed to either a low-stretch [tidal volume (Vt) = 8 ml/kg, positive end-expiratory pressure (PEEP) = 5 cmH₂O, respiratory rate (RR) = 54–56 breaths/min (bpm), fractional inspired oxygen (Fi_O₂) = 1.0] or high-stretch (Vt = 30 ml/kg, PEEP = 0 cmH₂O, RR = 14–16 bpm, Fi_O₂ = 1.0) ventilation strategy and monitored for either 1 or 2 h. Subsequently, animals were lavaged and the composition and function of surfactant was analyzed. Separate groups of animals received exogenous surfactant after 1 h of high-stretch ventilation and were monitored for an additional 2 h. High stretch induced a significant decrease in blood oxygenation after 2 h of ventilation. Alterations in surfactant pool sizes and activity were observed at 1 h of high-stretch ventilation and progressed over time. The functional impairment of surfactant appeared to be caused by alterations to the hydrophobic components of surfactant. Exogenous surfactant treatment after a period of high-stretch ventilation mitigated subsequent physiological lung dysfunction. Together, these results suggest that alterations of surfactant are a consequence of the ventilation strategy that impair the biophysical activity of this material and thereby contribute directly to lung dysfunction over time.

Contributions of nitric oxide synthase isoforms to pulmonary oxygen toxicity, local vs. mediated effects

2008-05-01

Reactive species of oxygen and nitrogen have been collectively implicated in pulmonary oxygen toxicity, but the contributions of specific molecules are unknown. Therefore, we assessed the roles of several reactive species, particularly nitric oxide, in pulmonary injury by exposing wild-type mice and seven groups of genetically altered mice to >98% O₂ at 1, 3, or 4 atmospheres absolute. Genetically altered animals included knockouts lacking either neuronal nitric oxide synthase (nNOS^–/–), endothelial nitric oxide synthase (eNOS^–/–), inducible nitric oxide synthase (iNOS^–/–), extracellular superoxide dismutase (SOD3^–/–), or glutathione peroxidase 1 (GPx1^–/–), as well as two transgenic variants (S1179A and S1179D) having altered eNOS activities. We confirmed our earlier finding that normobaric hyperoxia (NBO₂) and hyperbaric hyperoxia (HBO₂) result in at least two distinct but overlapping patterns of pulmonary injury. Our new findings are that the role of nitric oxide in the pulmonary pathophysiology of hyperoxia depends both on the specific NOS isozyme that is its source and on the level of hyperoxia. Thus, iNOS predominates in the etiology of lung injury in NBO₂, and SOD3 provides an important defense. But in HBO₂, nNOS is a major contributor to pulmonary injury, whereas eNOS is protective. In addition, we demonstrated that nitric oxide derived from nNOS is involved in a neurogenic mechanism of HBO₂-induced lung injury that is linked to central nervous system oxygen toxicity through adrenergic/cholinergic pathways.

Apoetm1Unc mice have impaired alveologenesis, low lung function, and rapid loss of lung function

Massaro, D., Massaro, G. D. — 2008-05-01

Diminished lung function, indicated by a low forced expiratory volume in one second (FEV₁), and short physical stature, predict early mortality from all causes, including cardiovascular, among smokers and never smokers. The basis for these associations is unclear, and, it is not known if there is a pulmonary morphological component to the relationship between low FEV₁ and early death in a general population. Some apolipoprotein E genotypes also predict atherosclerosis and early mortality. These considerations led us to examine the Apoe^tm1Unc (Apoe) mouse, in which the apolipoprotein E gene is deleted, and that develops dyslipidemia, atherosclerosis at an early age, and has a shorter life span than the founder wild-type (wt) strain. We asked if Apoe mice have a morphological or functional pulmonary phenotype. We measured the size, number, and surface area of pulmonary gas-exchange units (alveoli) and mechanical properties of the lung. Compared with wt mice, Apoe mice had: 1) diminished developmental alveologenesis, 2) increased airway resistance in early adulthood, 3) high lung volume and high dynamic and static compliance in later adulthood, 4) more rapid loss of lung recoil with age, and 5) were less long than wt mice. These findings in mice indicate the association of a low FEV₁ with early death in humans may have developmental, and accelerated ageing, related pulmonary components, and that dietary, genetic, or dietary and genetic influences, on lipid metabolism may be an upstream cause of inflammation and oxidative stress, currently considered to be major risk factors for COPD.

Differential roles of ATR and ATM in p53, Chk1, and histone H2AX phosphorylation in response to hyperoxia: ATR-dependent ATM activation

Kulkarni, A., Das, K. C. — 2008-05-01

Elevated level of oxygen (hyperoxia) is widely used in critical care units and in respiratory insufficiencies. In addition, hyperoxia has been implicated in many diseases such as bronchopulmonary dysplasia or acute respiratory distress syndrome. Although hyperoxia is known to cause DNA base modifications and strand breaks, the DNA damage response has not been adequately investigated. We have investigated the effect of hyperoxia on DNA damage signaling and show that hyperoxia is a unique stress that activates the ataxia telangiectasia mutant (ATM)- and Rad3-related protein kinase (ATR)-dependent p53 phosphorylations (Ser6, -15, -37, and -392), phosphorylation of histone H2AX (Ser139), and phosphorylation of checkpoint kinase 1 (Chk1). In addition, we show that phosphorylation of p53 (Ser6) and histone H2AX (Ser139) depend on both ATM and ATR. We demonstrate that ATR activation precedes ATM activation in hyperoxia. Finally, we show that ATR is required for ATM activation in hyperoxia. Taken together, we report that ATR is the major DNA damage signal transducer in hyperoxia that activates ATM.

Differential effects of intravenous anesthetics on capacitative calcium entry in human pulmonary artery smooth muscle cells

Yang, M., Ding, X., Murray, P. A. — 2008-05-01

We assessed the roles of the protein kinase C (PKC) and the tyrosine kinase (TK) signaling pathways in regulating capacitative calcium entry (CCE) in human pulmonary artery smooth muscle cells (PASMCs) and investigated the effects of intravenous anesthetics (midazolam, propofol, thiopental, ketamine, etomidate, morphine, and fentanyl) on CCE in human PASMCs. Fura-2-loaded human PASMCs were placed in a dish (37°C) on an inverted fluorescence microscope. Intracellular Ca²⁺ concentration ([Ca²⁺]_i) was measured as the 340/380 fluorescence ratio in individual PASMCs. Thapsigargin, a sarcoplasmic reticulum Ca²⁺-adenosine triphosphatase inhibitor, was used to deplete intracellular Ca²⁺ stores after removing extracellular Ca²⁺. CCE was then activated by restoring extracellular Ca²⁺ (2.2 mM). The effects of PKC activation and inhibition, TK inhibition, and the intravenous anesthetics on CCE were assessed. Thapsigargin caused a transient increase in [Ca²⁺]_i. Restoring extracellular Ca²⁺ caused a rapid peak increase in [Ca²⁺]_i, followed by a sustained increase in [Ca²⁺]_i; i.e., CCE was stimulated in human PASMCs. PKC activation attenuated (P < 0.05), whereas PKC inhibition potentiated (P < 0.05), both peak and sustained CCE. TK inhibition attenuated (P < 0.05) both peak and sustained CCE. Midazolam, propofol, and thiopental each attenuated (P < 0.05) both peak and sustained CCE, whereas ketamine, etomidate, morphine, and fentanyl had no effect on CCE. Our results suggest that CCE in human PASMCs is influenced by both the TK and PKC signaling pathways. Midazolam, propofol, and thiopental each attenuated CCE, whereas ketamine, etomidate, morphine, and fentanyl had no effect on CCE.

Effect of obesity on pulmonary inflammation induced by acute ozone exposure: role of interleukin-6

Lang, J. E., Williams, E. S., Mizgerd, J. P., Shore, S. A. — 2008-05-01

To determine the role of interleukin (IL)-6 in the increased ozone (O₃)-induced inflammation and injury observed in obese vs. lean mice, lean wild-type and leptin-deficient obese (ob/ob) mice were injected with anti-IL-6 antibody (Ab) or isotype control Ab 24 h before exposure to either O₃ (2 ppm for 3 h) or room air. Four or 24 h after O₃ exposure, bronchoalveolar lavage (BAL) was performed, and the lungs were harvested for Western blotting. Anti-IL-6 Ab caused substantial reductions in O₃-induced increases in BAL IL-6 in mice of both genotypes. Four hours following O₃, ob/ob mice had increased BAL neutrophils compared with controls, and anti-IL-6-Ab virtually abolished this difference. At 24 h, O₃-induced increases in BAL protein and BAL serum albumin were augmented in ob/ob vs. wild-type mice, and anti-IL-6 Ab ablated these obesity-related differences in epithelial barrier injury. O₃ increased tyrosine phosphorylation of STAT-3 and STAT-1. There was no effect of obesity on STAT-3 phosphorylation, whereas obesity decreased STAT-1 expression, resulting in reduced STAT-1 phosphorylation. IL-6 neutralization did not alter STAT-3 or STAT-1 phosphorylation in ob/ob or wild-type mice. O₃ increased BAL leukemia inhibitory factor (LIF) to a greater extent in obese than in lean mice, and LIF may account for effects on STAT phosphorylation. Our results suggest that IL-6 plays a complex role in pulmonary responses to O₃, a role that differs between wild-type and ob/ob mice. Moreover, obesity-related differences in activation of STAT proteins may contribute to some of the differences in the response of obese vs. lean mice.