Inhibition of TGF-β signaling and decreased apoptosis in IUGR-associated lung disease in rats

Intrauterine growth restriction is associated with impaired lung function in adulthood. It is unknown whether such impairment of lung function is linked to the transforming growth factor (TGF)-β system in the lung. Therefore, we investigated the effects of IUGR on lung function, expression of extracellular matrix (ECM) components and TGF-β signaling in rats. IUGR was induced in rats by isocaloric protein restriction during gestation. Lung function was assessed with direct plethysmography at postnatal day (P) 70. Pulmonary activity of the TGF-β system was determined at P1 and P70. TGF-β signaling was blocked in vitro using adenovirus-delivered Smad7. At P70, respiratory airway compliance was significantly impaired after IUGR. These changes were accompanied by decreased expression of TGF-β1 at P1 and P70 and a consistently dampened phosphorylation of Smad2 and Smad3. Furthermore, the mRNA expression levels of inhibitors of TGF-β signaling (Smad7 and Smurf2) were reduced, and the expression of TGF-β-regulated ECM components (e.g. collagen I) was decreased in the lungs of IUGR animals at P1; whereas elastin and tenascin N expression was significantly upregulated. In vitro inhibition of TGF-β signaling in NIH/3T3, MLE 12 and endothelial cells by adenovirus-delivered Smad7 demonstrated a direct effect on the expression of ECM components. Taken together, these data demonstrate a significant impact of IUGR on lung development and function and suggest that attenuated TGF-β signaling may contribute to the pathological processes of IUGR-associated lung disease.     

Gene expression of β–catenin is up-regulated in inner dental epithelium and enamel knots during molar tooth morphogenesis in the mouse

Beta–catenin is a multi–functional molecule that is involved in both cell–cell adhesion and signaling. We analyzed changes in β–catenin gene expression during mouse molar tooth development by in situ hybridization. Prominent up–regulation of the expression of this gene was evident exclusively in the enamel knot at the early cap stage. During the cap and bell stages, the enamel knot, inner dental epithelium, and differentiating stratum intermedium expressed the β–catenin gene more strongly than other parts of the enamel organ. During these stages, the strength of the gene expression changed heterogeneously within the inner dental epithelium and stratum intermedium. However, the heterogeneity was not evident at the late bell stage, when the cells in the inner dental epithelium had differentiated into ameloblasts at the cusp tip. No spatiotemporal change in β–catenin gene expression was apparent in the dental papilla except for the cells that differentiated into odontoblasts, which became negative for the expression of the gene after their differentiation. Thus, the up-regulated expression of the β–catenin gene was strongly associated with epithelial morphogenesis. These findings raise the possibility that the up–regulation of the gene expression and the stabilization of the protein by Wnt signaling play a role in the regulation of the activities of β–catenin in tooth morphogenesis.     

Conditional overexpression of TGFβ1 promotes pulmonary inflammation,apoptosis and mortality via TGFβR2 in the developing mouse lung

Background

Earlier studies have reported that transforming growth factor beta 1(TGFβ1) is a critical mediator of hyperoxia-induced acute lung injury (HALI) in developing lungs, leading to impaired alveolarization and a pulmonary phenotype of bronchopulmonary dysplasia (BPD). However, the mechanisms responsible for the TGFβ1-induced inflammatory signals that lead to cell death and abnormal alveolarization are poorly understood. We hypothesized that TGFβ1 signaling via TGFβR2 is necessary for the pathogenesis of the BPD pulmonary phenotype resulting from HALI.

Methods

We utilized lung epithelial cell-specific TGFβ1 overexpressing transgenic and TGFβR2 null mutant mice to evaluate the effects on neonatal mortality as well as pulmonary inflammation and apoptosis in developing lungs. Lung morphometry was performed to determine the impaired alveolarization and multicolor flow cytometry studies were performed to detect inflammatory macrophages and monocytes in lungs. Apoptotic cell death was measured with TUNEL assay, immunohistochemistry and western blotting and protein expression of angiogenic mediators were also analyzed.

Results

Our data reveals that increased TGFβ1 expression in newborn mice lungs leads to increased mortality, macrophage and immature monocyte infiltration, apoptotic cell death specifically in Type II alveolar epithelial cells (AECs), impaired alveolarization, and dysregulated angiogenic molecular markers.

Conclusions

Our study has demonstrated the potential role of inhibition of TGFβ1 signaling via TGFβR2 for improved survival, reduced inflammation and apoptosis that may provide insights for the development of potential therapeutic strategies targeted against HALI and BPD.     

Mechanical ventilation uncouples synthesis and assembly of elastin and increases apoptosis in lungs of newborn mice. Prelude to defective alveolar septation during lung development?

Prolonged mechanical ventilation (MV) with O2-rich gas inhibits lung growth and causes excess, disordered accumulation of lung elastin in preterm infants, often resulting in chronic lung disease (CLD). Using newborn mice, in which alveolarization occurs postnatally, we designed studies to determine how MV with either 40% O2 or air might lead to dysregulated elastin production and impaired lung septation. MV of newborn mice for 8 h with either 40% O2 or air increased lung mRNA for tropoelastin and lysyl oxidase, relative to unventilated controls, without increasing lung expression of genes that regulate elastic fiber assembly (lysyl oxidase-like-1, fibrillin-1, fibrillin-2, fibulin-5, emilin-1). Serine elastase activity in lung increased fourfold after MV with 40% O2, but not with air. We then extended MV with 40% O2 to 24 h and found that lung content of tropoelastin protein doubled, whereas lung content of elastin assembly proteins did not change (lysyl oxidases, fibrillins) or decreased (fibulin-5, emilin-1). Quantitative image analysis of lung sections showed that elastic fiber density increased by 50% after MV for 24 h, with elastin distributed throughout the walls of air spaces, rather than at septal tips, as in control lungs. Dysregulation of elastin was associated with a threefold increase in lung cell apoptosis (TUNEL and caspase-3 assays), which might account for the increased air space size previously reported in this model. Our findings of increased elastin synthesis, coupled with increased elastase activity and reduced lung abundance of proteins that regulate elastic fiber assembly, could explain altered lung elastin deposition, increased apoptosis, and defective septation, as observed in CLD.     

Smooth muscle-specific expression of calcium-independent phospholipase A2β (iPLA2β) participates in the initiation and early progression of vascular inflammation and neointima formation

Whether group VIA phospholipase A(2) (iPLA(2)β) is involved in vascular inflammation and neointima formation is largely unknown. Here, we report that iPLA(2)β expression increases in the vascular tunica media upon carotid artery ligation and that neointima formation is suppressed by genetic deletion of iPLA(2)β or by inhibiting its activity or expression via perivascular delivery of bromoenol lactone or of antisense oligonucleotides, respectively. To investigate whether smooth muscle-specific iPLA(2)β is involved in neointima formation, we generated transgenic mice in which iPLA(2)β is expressed specifically in smooth muscle cells and demonstrate that smooth muscle-specific expression of iPLA(2)β exacerbates ligation-induced neointima formation and enhanced both production of proinflammatory cytokines and vascular infiltration by macrophages. With cultured vascular smooth muscle cell, angiotensin II, arachidonic acid, and TNF-α markedly induce increased expression of IL-6 and TNF-α mRNAs, all of which were suppressed by inhibiting iPLA(2)β activity or expression with bromoenol lactone, antisense oligonucleotides, and genetic deletion, respectively. Similar suppression also results from genetic deletion of 12/15-lipoxygenase or inhibiting its activity with nordihydroguaiaretic acid or luteolin. Expression of iPLA(2)β protein in cultured vascular smooth muscle cells was found to depend on the phenotypic state and to rise upon incubation with TNF-α. Our studies thus illustrate that smooth muscle cell-specific iPLA(2)β participates in the initiation and early progression of vascular inflammation and neointima formation and suggest that iPLA(2)β may represent a novel therapeutic target for preventing cardiovascular diseases.     

The effects of early life Pb exposure on the expression of IL1-β, TNF-α and Aβ in cerebral cortex of mouse pups

ObjectiveTo investigate the effects of maternal lead (Pb) exposure on the learning and memory ability and expression of interleukin1-β (IL1-β), tumor necrosis factor (TNF-α) and beta amyloid protein (Aβ) in cerebral cortex of mice offspring.MethodsPb exposure initiated from beginning of gestation to weaning. Pb acetate administered in drinking solutions was dissolved in distilled deionized water at the concentrations of 0.1%, 0.5% and 1% groups, respectively. On the PND21, the learning and memory ability were tested by water maze test and the Pb levels were also determined by graphite furnace atomic absorption spectrometry. The expression of IL1-β, TNF-α and Aβ in cerebral cortex was measured by immunohistochemistry and western blotting.ResultsThe Pb levels in blood and cerebral cortex of all exposure groups were significantly higher than that of the control group (P < 0.05). In water maze test, the performances of 0.5% and 1% groups were worse than that of the control group (P < 0.05). The expression of IL1-β, TNF-α and Aβ was increased in Pb exposed groups than that of the control group (P < 0.05).ConclusionsThe high expression of IL1-β, TNF-α and Aβ in the cerebral cortex of pups may contribute to the impairment of learning and memory associated with maternal Pb exposure.     

Challenge with 17α-ethinylestradiol (EE2) during early development persistently impairs growth,differentiation, and local expression of IGF-I and IGF-II in immune organs of tilapia

The enormous expansion of world-wide aquaculture has led to increasing interest in the regulation of fish immune system. Estrogen has recently been shown to inhibit the endocrine (liver-derived) and autocrine/paracrine local insulin-like growth factor-I system in fish. In order to address the potential actions of estrogen on the IGF system in immune organs, tilapia were fed with 17α-ethinylestradiol (EE2)-enriched food from 10 to 40 days post fertilization (DPF) to induce functional feminization, an approach commonly used in aquaculture. EE2-treated and control fish were sampled at 75 and 165 DPF. The expression levels of ER-α, IGF-I, IGF-II and growth hormone receptor (GH-R) mRNA in spleen and head kidney were determined by real-time PCR and the expressing sites of IGF-I mRNA identified by in situ hybridisation. Ratios of spleen length and weight to body length and weight were determined. At 165 DPF, the length (4.9% vs. 7.6%) and weight (0.084% vs. 0.132%) ratios were significantly lowered in EE2-treated fish and number and size of the melanomacrophage centres were considerably reduced. At 75 DPF, both in spleen and head kidney of EE2-treated fish the expression levels of IGF-I and IGF-II mRNA were markedly diminished. The suppression was more pronounced for IGF-I (spleen: ?12.071-fold; head kidney: ?8.413-fold) than for IGF-II (spleen: ?4.102-fold; head kidney: ?1.342-fold). In agreement, clearly fewer leucocytes and macrophages in head kidney and spleen of EE2-treated fish contained IGF-I mRNA as shown by in situ hybridisation. ER-α mRNA expression in spleen was increased at 75 DPF but unchanged in head kidney. GH-R gene expression showed a mild upregulation at 165 DPF in both tissues. Thus, exposure to EE2 during early development affected distinctly the IGF system in tilapia immune organs. It led to lasting impairment of spleen growth and differentiation that can be attributed to an interaction of EE2 with IGF-I and, less pronouncedly, IGF-II. Especially, the impairment of spleen and melanomacrophage centres might interfere with the antigen presentation capacity of the immune system and, thus, alter susceptibility to infection.