The redox state of tissues tends to become progressively more prooxidizing during the aging process. The hypothesis tested in this study was that enhancement of reductive capacity by overexpression of glucose-6-phosphate dehydrogenase (G6PD), a key enzyme for NADPH biosynthesis, could protect against oxidative stress and extend the life span of transgenic Drosophila melanogaster. Overexpression of G6PD was achieved by combining a UAS-G6PD responder transgene at one of four independent loci with either a broad expression (armadillo-GAL4, Tubulin-GAL4, C23-GAL4, and da-GAL4) or a neuronal driver (D42-GAL4 and Appl-GAL4). The mean life spans of G6PD overexpressor flies were extended, in comparison with driver and responder controls, as follows: armadillo-GAL4 (up to 38%), Tubulin-GAL4 (up to 29%), C23-GAL4 (up to 27%), da-GAL4 (up to 24%), D42-GAL4 (up to 18%), and Appl-GAL4 (up to 16%). The G6PD enzymatic activity was increased, as were the levels of NADPH, NADH, and the GSH/GSSG ratio. Resistance to experimental oxidative stress was enhanced. Furthermore, metabolic rates and fertility were essentially the same in G6PD overexpressors and control flies. Collectively, the results demonstrate that enhancement of the NADPH biosynthetic capability can extend the life span of a relatively long-lived strain of flies, which supports the oxidative stress hypothesis of aging.Glucose-6-phosphate dehydrogenase catalyzes the oxidation of glucose-6-phosphate to 6-phosphogluconate and the reduction of NADP ϩ to NADPH, which is the rate-limiting step in the pentose phosphate pathway. This metabolic pathway serves multiple cellular functions, such as the supply of pentose intermediates for nucleotide synthesis, interconversion of 3-7 carbon sugars for various metabolic purposes, and the generation of reducing power in the form of NADPH. The latter metabolite plays an integral role in a broad range of cellular functions, such as (i) acting as a cofactor in reductive biosynthesis, (ii) detoxification of xenobiotics, and (iii) maintenance of the cellular redox state by providing reducing equivalents to antioxidative systems, among others (1).The concept that maintenance of an optimal redox state is essential to cellular survival is now firmly established. Significant amounts of superoxide anion radical and its stoichiometric product, hydrogen peroxide, are generated in mitochondria and peroxisomes under normal physiological conditions (2). Hydrogen peroxide is detoxified by catalase and peroxidases. The activity of the peroxidases is dependent upon the availability of reduced forms of glutathione (GSH) or thioredoxin, and it results in the oxidation of GSH to GSSG and of reduced thioredoxin red (Trx-(SH) 2 ) 2 to oxidized thioredoxin ox (Trx-(S) 2 ). The reducing equivalents for the reductions of GSSG by glutathione reductase and thioredoxin ox by thioredoxin reductase are supplied by NADPH (3). Thus, the regeneration of two major cellular antioxidants, GSH and thioredoxin, is dependent upon the supply of NADPH. In insects, glutathio...

Maternal low protein diet programs offspring to develop hypertension as adults. Transient exposure to angiotensin converting enzyme inhibitors or angiotensin II receptor blockers can result in improvement in hypertension. Male rats whose mothers received a low protein diet during the last half of pregnancy were given either vehicle, continuous enalapril (CE) in their drinking water or were given transient enalapril exposure (TE) after weaning at 21 days of age. The TE group had enalapril in their drinking water for 21 days starting from day 21 of life. All rats were studied at 6 months of age. Vehicle treated rats whose mothers were fed a low protein diet were hypertensive, had albuminuria, and demonstrated upregulation of the intrarenal renin-angiotensin system as evidenced by higher urinary angiotensinogen and urinary angiotensin II levels. In low protein rats both continuous and transient exposure to enalapril normalized blood pressure, urinary angiotensinogen and urinary angiotensin II levels at 6 months of age, but only continuous administration of enalapril decreased urinary albumin excretion. These data support the importance of the intrarenal renin-angiotensin system in mediating hypertension in programmed rats and transient exposure to enalapril can reprogram the hypertension and dysregulation of the intrarenal renin-angiotensin system.

BackgroundDistal alveolar morphogenesis is marked by differentiation of alveolar type (AT)-II to AT-I cells that give rise to the primary site of gas exchange, the alveolar/vascular interface. Endothelial-Monocyte Activating Polypeptide (EMAP) II, an endogenous protein with anti-angiogenic properties, profoundly disrupts distal lung neovascularization and alveolar formation during lung morphogenesis, and is robustly expressed in the dysplastic alveolar regions of infants with Bronchopulmonary dysplasia. Determination as to whether EMAP II has a direct or indirect affect on ATII→ATI trans-differentiation has not been explored.MethodIn a controlled nonvascular environment, an in vitro model of ATII→ATI cell trans-differentiation was utilized to demonstrate the contribution that one vascular mediator has on distal epithelial cell differentiation.ResultsHere, we show that EMAP II significantly blocked ATII→ATI cell transdifferentiation by increasing cellular apoptosis and inhibiting expression of ATI markers. Moreover, EMAP II-treated ATII cells displayed myofibroblast characteristics, including elevated cellular proliferation, increased actin cytoskeleton stress fibers and Rho-GTPase activity, and increased nuclear:cytoplasmic volume. However, EMAP II-treated cells did not express the myofibroblast markers desmin or αSMA.ConclusionOur findings demonstrate that EMAP II interferes with ATII → ATI transdifferentiation resulting in a proliferating non-myofibroblast cell. These data identify the transdifferentiating alveolar cell as a possible target for EMAP II's induction of alveolar dysplasia.

Alveolar growth abnormalities and severe respiratory dysfunction are often fatal. Identifying mechanisms that control epithelial proliferation and enlarged, poorly septated airspaces is essential in developing new therapies for lung disease. The membrane-bound ligand ephrin-B2 is strongly expressed in lung epithelium, and yet in contrast to its known requirement for arteriogenesis, considerably less is known regarding the function of this protein in the epithelium. We hypothesize that the vascular mediator ephrin-B2 governs alveolar growth and mechanics beyond the confines of the endothelium. We used the in vivo manipulation of ephrin-B2 reverse signaling to determine the role of this vascular mediator in the pulmonary epithelium and distal lung mechanics. We determined that the ephrin-B2 gene (EfnB2) is strongly expressed in alveolar Type 2 cells throughout development and into adulthood. The role of ephrin-B2 reverse signaling in the lung was assessed in Efnb2LacZ/6YFDV mutants that coexpress the intracellular truncated ephrin-B2-b-galactosidase fusion and an intracellular point mutant ephrin-B2 protein that is unable to become tyrosine-phosphorylated or to interact with either the SH2 or PDZ domain-containing downstream signaling proteins. In these viable mice, we observed pulmonary hypoplasia and altered pulmonary mechanics, as evidenced by a marked reduction in lung compliance. Associated with the reduction in lung compliance was a significant increase in insoluble fibronectin (FN) basement membrane matrix assembly with FN deposition, and a corresponding increase in the a5 integrin receptor required for FN fibrillogenesis. These experiments indicate that ephrin-B2 reverse signaling mediates distal alveolar formation, fibrillogenesis, and pulmonary compliance.Keywords: arterial; fibronectin; a5b1 integrin; alveoli; pulmonary mechanics Lung development in mammalian species occurs through a series of overlapping stages, distinguished in terms of their histological appearance, and specifically the growth and differentiation of pulmonary epithelial structures. Although the proximal airways are completely formed by the end of gestation, the distal airways and alveoli continue their growth and maturation beyond the time of birth. Concurrent to the development of the epithelial structures, the pulmonary vasculature develops in a sequential fashion, via the coordinated processes of angiogenesis and vasculogenesis (1). Because of the close proximity of the developing airways and vasculature in the lung, vascular mediators have been suggested to influence pulmonary development. For instance, Chen and colleagues (2) and Schwarz and colleagues (3-5) used a mouse fetal lung allograft model to show that endothelial-monocyte activating polypeptide II can not only inhibit neovascularization, but can also significantly impair epithelial morphogenesis. Based on these and similar findings, our laboratory has focused on the role of vascular mediators during pulmonary development.Our studies have focused on the ephrin-B2 (EfnB2)...

To identify cell-intrinsic properties that facilitate interaction between epithelial endodermal and mesenchymal mesodermal cells during lung morphogenesis, we developed a model of lung selfassembly that mimics fetal lung formation in structure, polarity, vasculature, and extracellular matrix expression. Three-dimensional pulmonary bodies (PBs) spontaneously self-assemble from singlecell suspensions and exhibit liquid-like properties that allow measurements of compaction rate and cohesion, and that may help to specify cellular self-organization. We hypothesized that changes in one or more of these parameters could potentially explain the lung hypoplasia associated with abnormal lung development. We examined the impact of endothelial/monocyte-activating polypeptide (EMAP) II in PBs, because EMAPII is highly expressed in lung hypoplasia. EMAPII significantly increased compaction rate and decreased overall cohesion of PBs composed of both epithelial and mesenchymal cells. Moreover, the effects of EMAPII on compaction and cohesion act exclusively through the mesenchymal cell population by interfering with fibronectin matrix assembly. We also show that EMAPII alters epithelial cell polarity and surfactant protein C expression. Our findings demonstrate, for the first time, that PBs possess liquid-like properties that can help to guide the self-assembly of fetal lungs, and that EMAPII expression can influence both mesenchymal and epithelial cells but through different molecular mechanisms.Keywords: cohesion; cell-cell interactions; polarity; extracellular matrix Early lung morphogenesis, initiated by a series of genetic cues, is characterized by the evagination of the foregut epithelium into the underlying mesoderm. Using a stereotypic pattern of reproducible budding and branching events, a tree-like system of epithelial branches gives rise to the mature lung organ. In conjunction with epithelial branching, a complex vascular tree, comprised of vessels arising from angiogenic and vasculogenic mechanisms, establishes an alveolar and vascular interface capable of oxygen exchange. The proximity and interdependence of the distal lung alveoli and vasculature for progression of normal development underscore the importance of an adhesion-based mechanism capable of mediating these cellular interactions. For example, vascular overabundance in lungspecific surfactant protein C (SPC) promoter vascular endothelial growth factor transgenic mice is associated with abnormal branching morphogenesis and inhibition of type I cell differentiation (1). Antiangiogenic proteins also influence distal lung morphogenesis. For instance, endothelial/monocyte-activating polypeptide (EMAP) II has been shown to markedly decrease lung vasculature, induce distal lung hypoplasia, and inhibit distal epithelial cell differentiation in a fetal lung allograft model (2). Conversely, disruption of a gene associated with epithelial structural maturation, such as transforming growth factor-b1, results in vascular malformations in conjunction with the arrest of...