Superoxide dismutase 3 dysregulation in a murine model of neonatal lung injury.

Bronchopulmonary dysplasia (BPD), a common chronic respiratory disease that occurs after premature birth, is believed to be secondary to oxidative damage from hyperoxia and inflammation, which leads to impaired alveolar formation and chronic lung dysfunction. We hypothesized that extracellular superoxide dismutase (SOD)3, an antioxidant uniquely targeted to the extracellular matrix (ECM) and alveolar fluid, might have a different response (down-regulation) to hyperoxic injury and recovery in room air (RA), thereby contributing to the persistent airspace injury and inflammation. We used a murine BPD model using postnatal hyperoxia (O2) (4 or 5 d) followed by short-term recovery (14 d) in RA, which mimics the durable effects after injury during alveolar development. This was associated with significantly increased mRNA expression for antioxidant genes mediated by nuclear factor erythroid 2-related factor (Nrf2) in the O2 (n = 4) versus RA group (n = 5). SOD3, an Nrf2-independent antioxidant, was significantly reduced in the O2-exposed mice compared with RA. Immunohistochemistry revealed decreased and disrupted SOD3 deposition in the alveolar ECM of O2-exposed mice. Furthermore, this distinct hyperoxic antioxidant and injury profile was reproducible in murine lung epithelial 12 cells exposed to O2. Overexpression of SOD3 rescued the injury measures in the O2-exposed cells. We establish that reduced SOD3 expression correlates with alveolar injury measures in the recovered neonatal hyperoxic lung, and SOD3 overexpression attenuates hyperoxic injury in an alveolar epithelial cell line. Such findings suggest a candidate mechanism for the pathogenesis of BPD that may lead to targeted interventions.

Hepatocyte growth factor, a determinant of airspace homeostasis in the murine lung.

The alveolar compartment, the fundamental gas exchange unit in the lung, is critical for tissue oxygenation and viability. We explored hepatocyte growth factor (HGF), a pleiotrophic cytokine that promotes epithelial proliferation, morphogenesis, migration, and resistance to apoptosis, as a candidate mediator of alveolar formation and regeneration. Mice deficient in the expression of the HGF receptor Met in lung epithelial cells demonstrated impaired airspace formation marked by a reduction in alveolar epithelial cell abundance and survival, truncation of the pulmonary vascular bed, and enhanced oxidative stress. Administration of recombinant HGF to tight-skin mice, an established genetic emphysema model, attenuated airspace enlargement and reduced oxidative stress. Repair in the TSK/+ mouse was punctuated by enhanced akt and stat3 activation. HGF treatment of an alveolar epithelial cell line not only induced proliferation and scattering of the cells but also conferred protection against staurosporine-induced apoptosis, properties critical for alveolar septation. HGF promoted cell survival was attenuated by akt inhibition. Primary alveolar epithelial cells treated with HGF showed improved survival and enhanced antioxidant production. In conclusion, using both loss-of-function and gain-of-function maneuvers, we show that HGF signaling is necessary for alveolar homeostasis in the developing lung and that augmentation of HGF signaling can improve airspace morphology in murine emphysema. Our studies converge on prosurvival signaling and antioxidant protection as critical pathways in HGF-mediated airspace maintenance or repair. These findings support the exploration of HGF signaling enhancement for diseases of the airspace.

Angiotensin receptor blockade attenuates cigarette smoke-induced lung injury and rescues lung architecture in mice.

Chronic obstructive pulmonary disease (COPD) is a prevalent smoking-related disease for which no disease-altering therapies currently exist. As dysregulated TGF-ß signaling associates with lung pathology in patients with COPD and in animal models of lung injury induced by chronic exposure to cigarette smoke (CS), we postulated that inhibiting TGF-ß signaling would protect against CS-induced lung injury. We first confirmed that TGF-ß signaling was induced in the lungs of mice chronically exposed to CS as well as in COPD patient samples. Importantly, key pathological features of smoking-associated lung disease in patients, e.g., alveolar injury with overt emphysema and airway epithelial hyperplasia with fibrosis, accompanied CS-induced alveolar cell apoptosis caused by enhanced TGF-ß signaling in CS-exposed mice. Systemic administration of a TGF-ß-specific neutralizing antibody normalized TGF-ß signaling and alveolar cell death, conferring improved lung architecture and lung mechanics in CS-exposed mice. Use of losartan, an angiotensin receptor type 1 blocker used widely in the clinic and known to antagonize TGF-ß signaling, also improved oxidative stress, inflammation, metalloprotease activation and elastin remodeling. These data support our hypothesis that inhibition of TGF-ß signaling through angiotensin receptor blockade can attenuate CS-induced lung injury in an established murine model. More importantly, our findings provide a preclinical platform for the development of other TGF-ß-targeted therapies for patients with COPD.

Critical transition in tissue homeostasis accompanies murine lung senescence.

BACKGROUND: Respiratory dysfunction is a major contributor to morbidity and mortality in aged populations. The susceptibility to pulmonary insults is attributed to "low pulmonary reserve", ostensibly reflecting a combination of age-related musculoskeletal, immunologic and intrinsic pulmonary dysfunction. METHODS/PRINCIPAL FINDINGS: Using a murine model of the aging lung, senescent DBA/2 mice, we correlated a longitudinal survey of airspace size and injury measures with a transcriptome from the aging lung at 2, 4, 8, 12, 16 and 20 months of age. Morphometric analysis demonstrated a nonlinear pattern of airspace caliber enlargement with a critical transition occurring between 8 and 12 months of age marked by an initial increase in oxidative stress, cell death and elastase activation which is soon followed by inflammatory cell infiltration, immune complex deposition and the onset of airspace enlargement. The temporally correlative transcriptome showed exuberant induction of immunoglobulin genes coincident with airspace enlargement. Immunohistochemistry, ELISA analysis and flow cytometry demonstrated increased immunoglobulin deposition in the lung associated with a contemporaneous increase in activated B-cells expressing high levels of TLR4 (toll receptor 4) and CD86 and macrophages during midlife. These midlife changes culminate in progressive airspace enlargement during late life stages. CONCLUSION/SIGNIFICANCE: Our findings establish that a tissue-specific aging program is evident during a presenescent interval which involves early oxidative stress, cell death and elastase activation, followed by B lymphocyte and macrophage expansion/activation. This sequence heralds the progression to overt airspace enlargement in the aged lung. These signature events, during middle age, indicate that early stages of the aging immune system may have important correlates in the maintenance of tissue morphology. We further show that time-course analyses of aging models, when informed by structural surveys, can reveal nonintuitive signatures of organ-specific aging pathology.

Folding cooperativity in RNA and DNA is dependent on position in the helix.

Secondary structural motifs play essential roles in the folding and function of RNA and DNA molecules. Previous work from our lab compared the folding of small DNA and RNA hairpin loops containing a sheared GA pair [Moody, E. M., Feerar, J. C., and Bevilacqua, P. C. (2004) Biochemistry 43, 7992-7998]. We found that the small DNA hairpins fold in a highly cooperative manner with indirect coupling, while their RNA counterparts fold in a much less cooperative fashion and display direct coupling. Herein, we extend this study to the double-stranded helix. We carried out double mutant cycles on base pairs having identical nearest-neighbor contexts but located in either external or internal helical registers. In the external register, both RNA and DNA exhibit extensive folding cooperativity between the penultimate and terminal base pair, which is independent of mismatch identity. In contrast, DNA exhibits virtually no folding cooperativity in the center of the helix, while RNA maintains substantial coupling, which is dependent on mismatch identity. Two models account for these non-nearest-neighbor effects: one involves the unfavorable entropy of helix initiation common to DNA and RNA, and the other involves steric and electrostatic strain peculiar to RNA. These data show that RNA can display cooperativity less than, greater than, or equal to that of DNA depending on context and position.

Molecular cloning and tissue expression of chicken AdipoR1 and AdipoR2 complementary deoxyribonucleic acids.

AdipoR1 and AdipoR2 belong to a novel class of transmembrane receptors that mediate the effects of adiponectin. We have cloned the chicken AdipoR1 and AdipoR2 complementary deoxyribonucleic acids (cDNA) and determined their expression in various tissues. We also investigated the effect of feed deprivation on the expression of AdipoR1 or AdipoR2 mRNA in the chicken diencephalon, liver, anterior pituitary gland, and adipose tissue. The chicken AdipoR1 and AdipoR2 cDNA sequences were 76-83% identical to the respective mammalian sequences. A hydrophobicity analysis of the deduced amino acid sequences of chicken AdipoR1/AdipoR2 revealed seven distinct hydrophobic regions representing seven transmembrane domains. By RT-PCR, we detected AdipoR1 and AdipoR2 mRNA in adipose tissue, liver, anterior pituitary gland, diencephalon, skeletal muscle, kidney, spleen, ovary, and blood. AdipoR1 or AdipoR2 mRNA expression in various tissues was quantified by real-time quantitative PCR, and AdipoR1 mRNA expression was the highest in skeletal muscle, adipose tissue and diencephalon, followed by kidney, ovary, liver, anterior pituitary gland, and spleen. AdipoR2 mRNA expression was the highest in adipose tissue followed by skeletal muscle, liver, ovary, diencephalon, anterior pituitary gland, kidney, and spleen. We also found that a 48 h feed deprivation significantly decreased AdipoR1 mRNA quantity in the chicken pituitary gland, while AdipoR2 mRNA quantity was significantly increased in adipose tissue (P<0.05). We conclude that the AdipoR1 and AdipoR2 genes are ubiquitously expressed in chicken tissues and that their expression is altered by feed deprivation in the anterior pituitary gland and adipose tissue.

Adiponectin gene is expressed in multiple tissues in the chicken: food deprivation influences adiponectin messenger ribonucleic acid expression.

Adiponectin is a cytokine hormone originally found to be secreted exclusively by white adipose tissue. However, recent evidences suggest that adiponectin is also produced in brown adipose tissue and skeletal muscle. The present study investigated the expression of adiponectin mRNA in various tissues in the chicken. We also studied the effect of food deprivation on adiponectin gene expression in adipose tissue, liver, anterior pituitary gland, and diencephalon in the chicken. The open reading frame of chicken adiponectin cDNA consists of 735 nucleotides that were 65-68% homologous to various mammalian adiponectin cDNAs. The deduced amino acid sequence of chicken adiponectin contains 22 glycine-X-Y repeats (in which X and Y represent any amino acid) at the N-terminal end as found in the mammalian adiponectin. By RT-PCR and Northern analysis, we detected chicken adiponectin mRNA transcript in adipose tissue, liver, anterior pituitary gland, diencephalon, skeletal muscle, liver, kidney, ovary, and spleen but not in blood. Adiponectin mRNA expression in various tissues was quantitated using real-time quantitative PCR and found to be the highest in adipose tissue, followed by liver, anterior pituitary, diencephalon, kidney, and skeletal muscle. We also found that adiponectin mRNA quantity was significantly decreased after a 48-h food deprivation in adipose tissue, liver, and anterior pituitary gland but not in diencephalon. Our results provide novel evidence that, unlike mammals, adiponectin gene is expressed in several tissues in the chicken and that its expression is influenced by food deprivation.