Cyclic stretch stimulates nitric oxide synthase-1-dependent peroxynitrite formation by neonatal rat pulmonary artery smooth muscle.

Peroxynitrite, the reaction product of nitric oxide and superoxide, contributes to the pathogenesis of chronic pulmonary hypertension in immature animals by stimulating proliferation of pulmonary arterial smooth muscle cells (PASMCs). Pulmonary vasoconstriction, secondary to hypoxia and other stimuli, leads to enhanced pulsatile stretch of cells in the vascular wall, particularly in smooth muscle, which we hypothesized would cause increased peroxynitrite generation. Our objectives in this study were to determine whether cyclic mechanical stretch, at supraphysiologic levels, would cause increased production of reactive oxygen species (ROS), nitric oxide, and peroxynitrite in vitro. Early passage neonatal rat PASMCs were seeded and grown to subconfluence on collagen-coated elastomer-bottom plates and subjected to cyclic mechanical stretch (10% ("physiologic") or 20% ("supraphysiologic") at 0.5 Hz) for up to 24 h. Compared to nonstretched controls and to cells subjected to 10% stretch, 20% stretch increased H2O2 (stable marker of ROS) and nitrate/nitrite (stable marker of nitric oxide) in conditioned medium. These effects were accompanied by increased peroxynitrite, as evidenced by increased in situ dihydroethidium fluorescence and immunoreactive nitrotyrosine and by increased expression of nitric oxide synthase (NOS)-1 and NADPH oxidase 4 (NOX4), but not NOS-2. Stretch-induced H2O2 release and increased dihydroethidium fluorescence were prevented by pretreatment with a superoxide scavenger, nonspecific inhibitors of NADPH oxidase or NOS, or a peroxynitrite decomposition catalyst. Short-interfering RNA-mediated knockdown of NOS-1 or NOX4 attenuated increased nitric oxide and H2O2 content, respectively, in stretched-cell-conditioned medium. Knockdown of NOS-1 also attenuated increased immunoreactive nitrotyrosine content and stretch-induced proliferation, whereas knockdown of NOS-2 had no effect. We conclude that increased peroxynitrite generation by neonatal rat PASMCs subjected to supraphysiologic levels of cyclic stretch is NOS-1-dependent and that increased ROS production is predominantly mediated by NADPH oxidase, specifically NOX4.

Lipid hydroperoxide formation regulates postnatal rat lung cell apoptosis and alveologenesis.

An acute increase in oxygen tension after birth imposes an oxidative stress upon the lung. We hypothesized that the resultant increase in reactive oxygen species, specifically lipid hydroperoxides, would trigger postnatal alveologenesis and physiological lung cell apoptosis in the neonatal rat. Neonatal rats were either untreated or treated daily with subcutaneous vehicle or diphenyl phenyl diamine, a scavenger of lipid hydroperoxides and inhibitor of lipid peroxidation, from day 1 to 6 of life. Alveolar formation and physiological lung cell apoptosis were assessed by morphometry, immunohistochemistry, and Western blot analyses on day 7 samples. Substitution experiments were conducted using the prototypic lipid hydroperoxide t-butylhydroperoxide. At a minimum effective dose of 15µg/g body wt, treatment with diphenyl phenyl diamine resulted in a significant increase in tissue fraction and mean linear intercept and significant reductions in small peripheral blood vessels, secondary crest formation, lung and secondary crest cell DNA synthesis, and estimated alveolar number. Decreased numbers of apoptotic type II pneumocytes and mesenchymal cells, and decreased contents of proapoptotic cleaved caspase-3 and -7 and cytoplasmic cytochrome c, and an increase in antiapoptotic Bcl-xL were found in lungs treated with diphenyl phenyl diamine. A prevention of selected changes induced by diphenyl phenyl diamine was observed with concurrent treatment with intraperitoneal t-butylhydroperoxide, at a minimally effective dose of 187µg/g body wt. We conclude that oxidative stress after birth induces lipid hydroperoxide formation, which, in turn, triggers postnatal alveologenesis and physiological lung cell apoptosis in the neonatal rat.

Development of an inflammation-inducible gene expression system using helper-dependent adenoviral vectors.

BACKGROUND: Clinical studies have shown that gene therapy is a promising approach for treating such genetic diseases as the eye disease, Leber's congenital amaurosis. Development of gene therapy approaches for treating chronic inflammatory diseases is, however, more challenging because it requires the production of anti-inflammatory molecules at the diseased tissues only when they are needed. METHODS: We designed such a system by modifying the human interleukin (IL)-6 gene promoter to direct transgene expression and delivered the system into cultured cells as well as mouse lungs using a helper-dependent adenoviral vector. RESULTS: We have demonstrated both in vitro and in vivo that the reporter LacZ or human IL-10 gene can be induced by inflammatory stimuli. CONCLUSIONS: The results obtained indicate that the inflammation inducible gene expression system based on the modified human IL-6 gene promoter has the potential to be used for developing gene therapy for treating inflammatory diseases.