Detection of epithelial to mesenchymal transition in airways of a bleomycin induced pulmonary fibrosis model derived from an α-smooth muscle actin-Cre transgenic mouse

BACKGROUND: Epithelial to mesenchymal transition (EMT) in alveolar epithelial cells (AECs) has been widely observed in patients suffering interstitial pulmonary fibrosis. In vitro studies have also demonstrated that AECs could convert into myofibroblasts following exposure to TGF-β1. In this study, we examined whether EMT occurs in bleomycin (BLM) induced pulmonary fibrosis, and the involvement of bronchial epithelial cells (BECs) in the EMT. Using an α-smooth muscle actin-Cre transgenic mouse (α-SMA-Cre/R26R) strain, we labelled myofibroblasts in vivo. We also performed a phenotypic analysis of human BEC lines during TGF-β1 stimulation in vitro. METHODS: We generated the α-SMA-Cre mouse strain by pronuclear microinjection with a Cre recombinase cDNA driven by the mouse α-smooth muscle actin (α-SMA) promoter. α-SMA-Cre mice were crossed with the Cre-dependent LacZ expressing strain R26R to produce the double transgenic strain α-SMA-Cre/R26R. β-galactosidase (βgal) staining, α-SMA and smooth muscle myosin heavy chains immunostaining were carried out simultaneously to confirm the specificity of expression of the transgenic reporter within smooth muscle cells (SMCs) under physiological conditions. BLM-induced peribronchial fibrosis in α-SMA-Cre/R26R mice was examined by pulmonary βgal staining and α-SMA immunofluorescence staining. To confirm in vivo observations of BECs undergoing EMT, we stimulated human BEC line 16HBE with TGF-β1 and examined the localization of the myofibroblast markers α-SMA and F-actin, and the epithelial marker E-cadherin by immunofluorescence. RESULTS: βgal staining in organs of healthy α-SMA-Cre/R26R mice corresponded with the distribution of SMCs, as confirmed by α-SMA and SM-MHC immunostaining. BLM-treated mice showed significantly enhanced βgal staining in subepithelial areas in bronchi, terminal bronchioles and walls of pulmonary vessels. Some AECs in certain peribronchial areas or even a small subset of BECs were also positively stained, as confirmed by α-SMA immunostaining. In vitro, addition of TGF-β1 to 16HBE cells could also stimulate the expression of α-SMA and F-actin, while E-cadherin was decreased, consistent with an EMT. CONCLUSION: We observed airway EMT in BLM-induced peribronchial fibrosis mice. BECs, like AECs, have the capacity to undergo EMT and to contribute to mesenchymal expansion in pulmonary fibrosis

Genetic Determinants of Circulating Sphingolipid Concentrations in European Populations

Sphingolipids have essential roles as structural components of cell membranes and in cell signalling, and disruption of their metabolism causes several diseases, with diverse neurological, psychiatric, and metabolic consequences. Increasingly, variants within a few of the genes that encode enzymes involved in sphingolipid metabolism are being associated with complex disease phenotypes. Direct experimental evidence supports a role of specific sphingolipid species in several common complex chronic disease processes including atherosclerotic plaque formation, myocardial infarction (MI), cardiomyopathy, pancreatic beta-cell failure, insulin resistance, and type 2 diabetes mellitus. Therefore, sphingolipids represent novel and important intermediate phenotypes for genetic analysis, yet little is known about the major genetic variants that influence their circulating levels in the general population. We performed a genome-wide association study (GWAS) between 318,237 single-nucleotide polymorphisms (SNPs) and levels of circulating sphingomyelin (SM), dihydrosphingomyelin (Dih-SM), ceramide (Cer), and glucosylceramide (GluCer) single lipid species (33 traits); and 43 matched metabolite ratios measured in 4,400 subjects from five diverse European populations. Associated variants (32) in five genomic regions were identified with genome-wide significant corrected p-values ranging down to 9.08 x 10(-66). The strongest associations were observed in or near 7 genes functionally involved in ceramide biosynthesis and trafficking: SPTLC3, LASS4, SGPP1, ATP10D, and FADS1-3. Variants in 3 loci (ATP10D, FADS3, and SPTLC3) associate with MI in a series of three German MI studies. An additional 70 variants across 23 candidate genes involved in sphingolipid-metabolizing pathways also demonstrate association (p = 10(-4) or less). Circulating concentrations of several key components in sphingolipid metabolism are thus under strong genetic control, and variants in these loci can be tested for a role in the development of common cardiovascular, metabolic, neurological, and psychiatric diseases

Sulforaphane restores cellular glutathione levels and reduces chronic periodontitis neutrophil hyperactivity in vitro

The production of high levels of reactive oxygen species by neutrophils is associated with the local and systemic destructive phenotype found in the chronic inflammatory disease periodontitis. In the present study, we investigated the ability of sulforaphane (SFN) to restore cellular glutathione levels and reduce the hyperactivity of circulating neutrophils associated with chronic periodontitis. Using differentiated HL60 cells as a neutrophil model, here we show that generation of extracellular O2 . - by the nicotinamide adenine dinucleotide (NADPH) oxidase complex is increased by intracellular glutathione depletion. This may be attributed to the upregulation of thiol regulated acid sphingomyelinase driven lipid raft formation. Intracellular glutathione was also lower in primary neutrophils from periodontitis patients and, consistent with our previous findings, patients neutrophils were hyper-reactive to stimuli. The activity of nuclear factor erythroid-2-related factor 2 (Nrf2), a master regulator of the antioxidant response, is impaired in circulating neutrophils from chronic periodontitis patients. Although patients' neutrophils exhibit a low reduced glutathione (GSH)/oxidised glutathione (GSSG) ratio and a higher total Nrf2 level, the DNA-binding activity of nuclear Nrf2 remained unchanged relative to healthy controls and had reduced expression of glutamate cysteine ligase catalytic (GCLC), and modifier (GCLM) subunit mRNAs, compared to periodontally healthy subjects neutrophils. Pre-treatment with SFN increased expression of GCLC and GCM, improved intracellular GSH/GSSG ratios and reduced agonist-activated extracellular O2 . - production in both dHL60 and primary neutrophils from patients with periodontitis and controls. These findings suggest that a deficiency in Nrf2-dependent pathways may underpin susceptibility to hyper-reactivity in circulating primary neutrophils during chronic periodontitis. © 2013 Dias et al

Functional Implications of Novel Human Acid Sphingomyelinase Splice Variants

BACKGROUND: Acid sphingomyelinase (ASM) hydrolyses sphingomyelin and generates the lipid messenger ceramide, which mediates a variety of stress-related cellular processes. The pathological effects of dysregulated ASM activity are evident in several human diseases and indicate an important functional role for ASM regulation. We investigated alternative splicing as a possible mechanism for regulating cellular ASM activity. METHODOLOGY/PRINCIPAL FINDINGS: We identified three novel ASM splice variants in human cells, termed ASM-5, -6 and -7, which lack portions of the catalytic- and/or carboxy-terminal domains in comparison to full-length ASM-1. Differential expression patterns in primary blood cells indicated that ASM splicing might be subject to regulatory processes. The newly identified ASM splice variants were catalytically inactive in biochemical in vitro assays, but they decreased the relative cellular ceramide content in overexpression studies and exerted a dominant-negative effect on ASM activity in physiological cell models. CONCLUSIONS/SIGNIFICANCE: These findings indicate that alternative splicing of ASM is of functional significance for the cellular stress response, possibly representing a mechanism for maintaining constant levels of cellular ASM enzyme activity