HO-1 expression in type II pneumocytes after transpulmonary gene delivery

2000 ◽  
Vol 278 (6) ◽  
pp. L1273-L1279 ◽  
Author(s):  
Yi-Hao Weng ◽  
Arthur Tatarov ◽  
Blythe P. Bartos ◽  
Christopher H. Contag ◽  
Phyllis A. Dennery
Keyword(s):  
Oxidative Stress ◽  
Gene Expression ◽  
Gene Transfer ◽  
Expression Patterns ◽  
Alveolar Epithelium ◽  
Luciferase Reporter ◽  
Heme Oxygenase 1 ◽  
Alveolar Type ◽  
Type Ii

Somatic cell gene transfer is a potentially useful strategy to alter lung function. However, achieving efficient transfer to the alveolar epithelium, especially in smaller animals, has not been demonstrated. In this study, the rat heme oxygenase-1 (HO-1) gene was delivered to the lungs of neonatal mice via transpulmonary injection. A bidirectional promoter construct coexpressing both HO-1 and a luciferase reporter gene was used so that in vivo gene expression patterns could be monitored in real time. HO-1 expression levels were also modulated with doxycycline and assessed in vivo with bioluminescent light transmitted through the tissues from the coregulated luciferase reporter. As a model of oxidative stress and HO-1-mediated protection, groups of animals were exposed to hyperoxia. After gene transfer, elevated levels of HO-1 were detected predominantly in alveolar type II cells by immunocytochemistry. With overexpression of HO-1, increased oxidative injury was observed. Furthermore, this model demonstrated a cell-specific effect of lung HO-1 overexpression in oxidative stress. Specific control of expression for therapeutic genes is possible in vivo. The transpulmonary approach may prove useful in targeting gene expression to cells of the alveolar epithelium or to circumscribed areas of the lung.

Inhibition of alveolar type II cell ATP and surfactant synthesis by nitric oxide

1996 ◽  
Vol 270 (6) ◽  
pp. L898-L906 ◽  
Author(s):  
I. Y. Haddad ◽  
S. Zhu ◽  
J. Crow ◽  
E. Barefield ◽  
T. Gadilhe ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Lipid Synthesis ◽  
Alveolar Epithelium ◽  
Alveolar Type ◽  
Type Ii ◽  
Surfactant Synthesis ◽  
Cell Energy ◽  
Type Ii Cell

Alveolar type II (ATII) cells, are often exposed to increased concentration of endogenous and exogenous nitric oxide (.NO). Exposure of freshly isolated rat ATII cells for 2 h to 1-3 microM .NO, generated by S-nitroso-N-penicillamine (SNAP), spermine NONOate, or 3-morpholino-sydnonimine (SIN-1) in the presence of superoxide dismutase, resulted in approximately 60% decrease in the rate of surfactant synthesis, as measured by the rate of incorporation of [methyl-3H]choline into phosphatidylcholine, and 60-80% inhibition of cellular ATP levels, as determined by bioluminescence. Similar results were obtained after incubation of ATII cells with authentic peroxynitrite (0.5 mM) but not SIN-1, a putative generator of peroxynitrite. Addition into the medium of oxyhemoglobin (20 microM), which scavenged .NO, or enhancement of ATII glutathione levels by preincubation with glutathione ester (5 mM) totally prevented the NONOate (100 microM) inhibition of cellular ATP. In contrast to the in vitro findings, normal levels of ATP and lipid synthesis were measured in ATII cells isolated from the lungs of rats that breathed .NO gas (80 ppm) in 21% O2 for 2 h (n = 4). This lack of effect may be due either to the presence of various antioxidants (such as glutathione) in the epithelial lining fluid or to the relatively low concentrations of .NO reaching the alveolar epithelium. We conclude that .NO and peroxynitrite, at concentrations likely to be encountered in vivo during inflammation, decrease ATII cell energy stores and surfactant synthesis, which may lead to derangement of important physiological functions.

Loss of Gadd45a does not modify the pulmonary response to oxidative stress

2005 ◽  
Vol 288 (4) ◽  
pp. L663-L671 ◽  
Author(s):  
Jason M. Roper ◽  
Sean C. Gehen ◽  
Rhonda J. Staversky ◽  
M. Christine Hollander ◽  
Albert J. Fornace ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Dna Damage ◽  
Epithelial Cells ◽  
Oxidative Dna Damage ◽  
Genotoxic Stress ◽  
Clara Cell ◽  
Heme Oxygenase 1 ◽  
Alveolar Type ◽  
Type I ◽  
Type Ii

It is well established that exposure to high levels of oxygen (hyperoxia) injures and kills microvascular endothelial and alveolar type I epithelial cells. In contrast, significant death of airway and type II epithelial cells is not observed at mortality, suggesting that these cell types may express genes that protect against oxidative stress and damage. During a search for genes induced by hyperoxia, we previously reported that airway and alveolar type II epithelial cells uniquely express the growth arrest and DNA damage ( Gadd) 45a gene. Because Gadd45a has been implicated in protection against genotoxic stress, adult Gadd45a (+/+) and Gadd45a (−/−) mice were exposed to hyperoxia to investigate whether it protected epithelial cells against oxidative stress. During hyperoxia, Gadd45a deficiency did not affect loss of airway epithelial expression of Clara cell secretory protein or type II epithelial cell expression of pro-surfactant protein C. Likewise, Gadd45a deficiency did not alter recruitment of inflammatory cells, edema, or overall mortality. Consistent with Gadd45a not affecting the oxidative stress response, p21Cip1/WAF1 and heme oxygenase-1 were comparably induced in Gadd45a (+/+) and Gadd45a (−/−) mice. Additionally, Gadd45a deficiency did not affect oxidative DNA damage or apoptosis as assessed by oxidized guanine and terminal deoxyneucleotidyl transferase-mediated dUTP nick-end labeling staining. Overexpression of Gadd45a in human lung adenocarcinoma cells did not affect viability or survival during exposure, whereas it was protective against UV-radiation. We conclude that increased tolerance of airway and type II epithelial cells to hyperoxia is not attributed solely to expression of Gadd45a.

scholarly journals Mitogenic stimulation accelerates influenza-induced mortality by increasing susceptibility of alveolar type II cells to infection

2017 ◽  
Vol 114 (32) ◽  
pp. E6613-E6622 ◽  
Author(s):  
Nikolaos M. Nikolaidis ◽  
John G. Noel ◽  
Lori B. Pitstick ◽  
Jason C. Gardner ◽  
Yasuaki Uehara ◽  
...  
Keyword(s):  
Viral Infection ◽  
Influenza A ◽  
Ex Vivo ◽  
Keratinocyte Growth Factor ◽  
Alveolar Epithelium ◽  
Host Susceptibility ◽  
Alveolar Type ◽  
Type Ii ◽  
Mitogenic Stimulation

Development of pneumonia is the most lethal consequence of influenza, increasing mortality more than 50-fold compared with uncomplicated infection. The spread of viral infection from conducting airways to the alveolar epithelium is therefore a pivotal event in influenza pathogenesis. We found that mitogenic stimulation with keratinocyte growth factor (KGF) markedly accelerated mortality after infectious challenge with influenza A virus (IAV). Coadministration of KGF with IAV markedly accelerated the spread of viral infection from the airways to alveoli compared with challenge with IAV alone, based on spatial and temporal analyses of viral nucleoprotein staining of lung tissue sections and dissociated lung cells. To better define the temporal relationship between KGF administration and susceptibility to IAV infection in vivo, we administered KGF 120, 48, 24, and 0 h before intrapulmonary IAV challenge and assessed the percentages of proliferating and IAV-infected, alveolar type II (AECII) cells in dispersed lung cell populations. Peak AECII infectivity coincided with the timing of KGF administration that also induced peak AECII proliferation. AECII from mice that were given intrapulmonary KGF before isolation and then infected with IAV ex vivo exhibited the same temporal pattern of proliferation and infectious susceptibility. KGF-induced increases in mortality, AECII proliferation, and enhanced IAV susceptibility were all reversed by pretreatment of the animals with the mTOR inhibitor rapamycin before mitogenic stimulation. Taken together, these data suggest mTOR signaling-dependent, mitogenic conditioning of AECII is a determinant of host susceptibility to infection with IAV.

scholarly journals Improved distribution of small molecules and viral vectors in the murine brain using a hollow fiber catheter

2007 ◽  
Vol 107 (3) ◽  
pp. 568-577 ◽  
Author(s):  
Seunguk OH ◽  
Rick Odland ◽  
Scott R. Wilson ◽  
Kurt M. Kroeger ◽  
Chunyan Liu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Therapy ◽  
Gene Transfer ◽  
Surface Area ◽  
Brain Tissue ◽  
Mouse Brain ◽  
Hollow Fiber ◽  
Luciferase Reporter ◽  
Mammalian Brain

Object A hollow fiber catheter was developed to improve the distribution of drugs administered via direct infusion into the central nervous system (CNS). It is a porous catheter that significantly increases the surface area of brain tissue into which a drug is infused. Methods Dye was infused into the mouse brain through convection-enhanced delivery (CED) using a 28-gauge needle compared with a 3-mm-long hollow fiber catheter. To determine whether a hollow fiber catheter could increase the distribution of gene therapy vectors, a recombinant adenovirus expressing the firefly luciferase reporter was injected into the mouse striatum. Gene expression was monitored using in vivo bioluminescent imaging. To assess the distribution of gene transfer, an adenovirus expressing green fluorescent protein was injected into the striatum using a hollow fiber catheter or a needle. Results Hollow fiber catheter–mediated infusion increased the volume of brain tissue labeled with dye by 2.7 times relative to needle-mediated infusion. In vivo imaging revealed that catheter-mediated infusion of adenovirus resulted in gene expression that was 10 times greater than that mediated by a needle. The catheter appreciably increased the area of brain transduced with adenovirus relative to a needle, affecting a significant portion of the injected hemisphere. Conclusions The miniature hollow fiber catheter used in this study significantly increased the distribution of dye and adenoviral-mediated gene transfer in the mouse brain compared with the levels reached using a 28-gauge needle. Compared with standard single-port clinical catheters, the hollow fiber catheter has the advantage of millions of nanoscale pores to increase surface area and bulk flow in the CNS. Extending the scale of the hollow fiber catheter for the large mammalian brain shows promise in increasing the distribution and efficacy of gene therapy and drug therapy using CED.

scholarly journals Forty-eight hours of unloading and 24 h of reloading lead to changes in global gene expression patterns related to ubiquitination and oxidative stress in humans

2010 ◽  
Vol 109 (5) ◽  
pp. 1404-1415 ◽  
Author(s):  
Kimberly A. Reich ◽  
Yi-Wen Chen ◽  
Paul D. Thompson ◽  
Eric P. Hoffman ◽  
Priscilla M. Clarkson
Keyword(s):  
Oxidative Stress ◽  
Gene Expression ◽  
Stress Response ◽  
Expression Patterns ◽  
Global Gene Expression ◽  
Canonical Pathway ◽  
Gene Expression Patterns ◽  
Heme Oxygenase 1 ◽  
Short Term ◽  
Qrt Pcr

Although short-term disuse does not result in measurable muscle atrophy, studies suggest that molecular changes associated with protein degradation may be initiated within days of the onset of a disuse stimulus. We examined the global gene expression patterns in sedentary men ( n = 7, mean age ± SD = 22.1 ± 3.7 yr) following 48 h unloading (UL) via unilateral lower limb suspension and 24 h reloading (RL). Biopsy samples of the left vastus lateralis muscle were collected at baseline, 48 h UL, and 24 h RL. Expression changes were measured by microarray and gene clustering; identification of enriched functions and canonical pathways were performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) and Ingenuity Pathway Analysis (IPA). Four genes were validated with quantitative RT-PCR (qRT-PCR), and protein levels were measured with Western blot. Of the upregulated genes after UL, the most enriched functional group and highest ranked canonical pathway were related to protein ubiquitination. The oxidative stress response pathway was the second highest ranked canonical pathway. Of the downregulated genes, functions related to mitochondrial metabolism were the most highly enriched. In general, gene expression patterns following UL persisted following RL. qRT-PCR confirmed increases in mRNA for ubiquitin proteasome pathway-related E3 ligase Atrogin1 (but not accompanying increases in protein products) and stress response gene heme oxygenase-1 (HMOX, which showed a trend toward increases in protein products at 48 h UL) as well as extracellular matrix (ECM) component COL4A3. The gene expression patterns were not reversed on RL, suggesting that molecular responses to short-term periods of skeletal muscle inactivity may persist after activity resumes.

cAMP activation of chloride and fluid secretion across the rabbit alveolar epithelium

1998 ◽  
Vol 275 (6) ◽  
pp. L1127-L1133 ◽  
Author(s):  
Vance G. Nielsen ◽  
Michael D. Duvall ◽  
Manuel S. Baird ◽  
Sadis Matalon
Keyword(s):  
Short Circuit ◽  
Alveolar Epithelium ◽  
Short Circuit Current ◽  
Alveolar Epithelial Cells ◽  
Alveolar Type ◽  
Intracellular Camp ◽  
Type Ii ◽  
Alveolar Fluid Clearance ◽  
Ussing Chambers

Active Na+ transport by alveolar epithelial cells has been demonstrated to contribute significantly to alveolar fluid clearance. However, the contribution of transepithelial Cl− movement to the reabsorption of isosmotic fluid across the alveolar epithelium in vivo has not been elucidated. We hypothesized that Cl− transport could be increased across the alveolar epithelium in vivo and across cultured alveolar type II cells by agents that increase intracellular cAMP (e.g., forskolin). In studies where 5% albumin in sodium methanesulfonate (a Cl−-free solution) was administered into the lung, forskolin administration significantly increased intracellular influx of Cl− and fluid into the alveolar space. In vitro studies with cultured rabbit alveolar type II cell monolayers in Ussing chambers demonstrated that elevations in intracellular cAMP increase short-circuit current by increasing both Cl−secretion and Na+ reabsorption. The cystic fibrosis transmembrane conductance regulator channel blocker glibenclamide and the loop diuretic bumetanide partially decreased the forskolin-induced increase in short-circuit current. These data may explain the failure of agonist that stimulated intracellular cAMP to increase alveolar fluid clearance in the rabbit. Moreover, the data suggest that in the event Na+absorptive pathways are damaged, transepithelial Cl− secretion and the consequent intra-alveolar fluid influx may be upregulated.

Exosurf enhances adenovirus-mediated gene transfer to alveolar type II cells

1997 ◽  
Vol 273 (4) ◽  
pp. L741-L748 ◽  
Author(s):  
S. Machelle Manuel ◽  
Yi Guo ◽  
Sadis Matalon
Keyword(s):  
Gene Transfer ◽  
Luciferase Activity ◽  
A549 Cells ◽  
Luciferase Reporter ◽  
Luciferase Expression ◽  
Alveolar Type ◽  
Type Ii ◽  
Luciferase Reporter Gene ◽  
Pulmonary Epithelium

We assessed the role of surfactant replacement mixtures in the enhancement of adenovirus-mediated gene transfer to pulmonary epithelial cells both in vitro and in vivo. A549 cells, a pulmonary epithelium-derived adenocarcinoma cell line, were incubated with either media alone or media containing 10 μg phospholipid/ml Exosurf or Infasurf for 50 min followed by addition of a replication-deficient adenovirus (E1-deleted) expressing the luciferase reporter gene [AdCMV-Luc; 10 plaque-forming units (PFU)/cell] for 4 h. Pretreatment with Exosurf, but not Infasurf, at 37°C, but not at 4°C, enhanced luciferase activity in A549 cells 24 h later by 156% ( P < 0.01). Intratracheal instillation of AdCMV-Luc (2 × 109PFU) into rats resulted in luciferase expression mainly in alveolar macrophages and to a smaller extent in alveolar type II (ATII) cells 24 h later. However, when the AdCMV-Luc instillation was preceded by Exosurf (250 μl; 25 mg/ml), a 10-fold increase in ATII cell luciferase activity was noted. Preincubation of cultured ATII cells with Exosurf also enhanced their transfection by AdCMV-Luc by 515% ( P < 0.001). The results of these studies provide a new strategy for targeting ATII cells for gene delivery.

scholarly journals Angiotensin II Type II Receptor Deficiency Accelerates the Development of Nephropathy in Type I Diabetes via Oxidative Stress and ACE2

2011 ◽  
Vol 2011 ◽  
pp. 1-12 ◽  
Author(s):  
Shiao-Ying Chang ◽  
Yun-Wen Chen ◽  
Isabelle Chenier ◽  
Stella Le Minh Tran ◽  
Shao-Ling Zhang
Keyword(s):  
Oxidative Stress ◽  
Gene Expression ◽  
Angiotensin Ii ◽  
Type I Diabetes ◽  
Ros Generation ◽  
Heme Oxygenase 1 ◽  
Protein Accumulation ◽  
Type I ◽  
Type Ii ◽  
Renal Hypertrophy

Since the functional role(s) of angiotensin II (Ang II) type II receptor (AT2R) in type I diabetes is unknown, we hypothesized that AT2R is involved in decreasing the effects of type I diabetes on the kidneys. We induced diabetes with low-dose streptozotocin (STZ) in both AT2R knockout (AT2RKO) and wild-type (WT) male mice aged 12 weeks and followed them for 4 weeks. Three subgroups nondiabetic, diabetic, and insulin-treated diabetic (Rx insulin implant) were studied. Systolic blood pressure (SBP), physiological parameters, glomerular filtration rate (GFR), renal morphology, gene expression, and apoptosis were assessed. After 4 weeks of diabetes, compared to WT controls, AT2RKO mice clearly developed features of early diabetic nephropathy (DN), such as renal hypertrophy, tubular apoptosis, and progressive extracellular matrix (ECM) protein accumulation as well as increased GFR. AT2RKO mice presented hypertension unaffected by diabetes. Renal oxidative stress (measured as heme oxygenase 1 (HO-1) gene expression and reactive oxygen species (ROS) generation) and intrarenal renin angiotensin system components, such as angiotensinogen (Agt), AT1R, and angiotensin-converting enzyme (ACE) gene expression, were augmented whereas angiotensin-converting enzyme2 (ACE2) gene expression was decreased in renal proximal tubules (RPTs) of AT2RKO mice. The renal changes noted above were significantly enhanced in diabetic AT2RKO mice but partially attenuated in insulin-treated diabetic WT and AT2RKO mice. In conclusion, AT2R deficiency accelerates the development of DN, which appears to be mediated, at least in part, via heightened oxidative stress and ACE/ACE2 ratio in RPTs.

scholarly journals Resveratrol confers endothelial protection via activation of the antioxidant transcription factor Nrf2

2010 ◽  
Vol 299 (1) ◽  
pp. H18-H24 ◽  
Author(s):  
Zoltan Ungvari ◽  
Zsolt Bagi ◽  
Attila Feher ◽  
Fabio A. Recchia ◽  
William E. Sonntag ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Luciferase Reporter ◽  
Erythroid Cell ◽  
Heme Oxygenase 1 ◽  
Related Factor ◽  
Dependent Manner ◽  
Protective Effects ◽  
Resveratrol Treatment ◽  
Gene Assay

Epidemiological studies suggest that Mediterranean diets rich in resveratrol are associated with reduced risk of coronary artery disease. Resveratrol was also shown to confer vasoprotection in animal models of type 2 diabetes and aging. However, the mechanisms by which resveratrol exerts its antioxidative vasculoprotective effects are not completely understood. Using a nuclear factor-E2-related factor-2 (Nrf2)/antioxidant response element-driven luciferase reporter gene assay, we found that in cultured coronary arterial endothelial cells, resveratrol, in a dose-dependent manner, significantly increases transcriptional activity of Nrf2. Accordingly, resveratrol significantly upregulates the expression of the Nrf2 target genes NAD(P)H:quinone oxidoreductase 1, γ-glutamylcysteine synthetase, and heme oxygenase-1. Resveratrol treatment also significantly attenuated high glucose (30 mM)-induced mitochondrial and cellular oxidative stress (assessed by flow cytometry using MitoSox and dihydroethidine staining). The aforementioned effects of resveratrol were significantly attenuated by the small interfering RNA downregulation of Nrf2 or the overexpression of Kelch-like erythroid cell-derived protein 1, which inactivates Nrf2. To test the effects of resveratrol in vivo, we used mice fed a high-fat diet (HFD), which exhibit increased vascular oxidative stress associated with an impaired endothelial function. In HFD-fed Nrf2+/+ mice, resveratrol treatment attenuates oxidative stress (assessed by the Amplex red assay), improves acetylcholine-induced vasodilation, and inhibits apoptosis (assessed by measuring caspase-3 activity and DNA fragmentation) in branches of the femoral artery. In contrast, the aforementioned endothelial protective effects of resveratrol were diminished in HFD-fed Nrf2−/− mice. Taken together, our results indicate that resveratrol both in vitro and in vivo confers endothelial protective effects which are mediated by the activation of Nrf2.

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