Application of ToxTracker for the toxicological assessment of tobacco and nicotine delivery products.

Consumer demands and innovation have led to an increasingly diverse range of nicotine delivery systems, driven by a desire to reduce risk associated with traditional combustible cigarettes. This speed of change provides a mandate for rapid new product assessment. We have used the validated technology ToxTracker®, to assess biomarkers of DNA damage, protein misfolding, oxidative and cellular stress, across the categories of cigarette (1R6F), tobacco heating product (THP 1.4) and electronic cigarette (ePen 3). In addition, we compared the commonly used test matrices for tobacco and nicotine products; whole aerosol aqueous extracts (AqE) and gas vapour phase (GVP), determining their suitability across the product categories. We demonstrated a significant reduction in oxidative stress and cytotoxicity for THP 1.4 over cigarette, further reduced for ePen 3, when assessed by both dilution and nicotine dosimetry. We also identified that while the extraction matrices AqE and GVP from combustible products were equivalent in the induced responses, this was not true of the other category examples, moreover THP 1.4 GVP demonstrates a >50 % reduction in both toxicity and cytotoxicity endpoints over AqE. This indicates that unlike cigarette, the active components or toxicants for THP and electronic cigarette are associated with the aerosol fraction of these categories.

Engineering multi-layered tissue constructs using acoustic levitation.

Engineering tissue structures that mimic those found in vivo remains a challenge for modern biology. We demonstrate a new technique for engineering composite structures of cells comprising layers of heterogeneous cell types. An acoustofluidic bioreactor is used to assemble epithelial cells into a sheet-like structure. On transferring these cell sheets to a confluent layer of fibroblasts, the epithelial cells cover the fibroblast surface by collective migration maintaining distinct epithelial and fibroblast cell layers. The collective behaviour of the epithelium is dependent on the formation of cell-cell junctions during levitation and contrasts with the behaviour of mono-dispersed epithelial cells where cell-matrix interactions dominate and hinder formation of discrete cell layers. The multilayered tissue model is shown to form a polarised epithelial barrier and respond to apical challenge. The method is useful for engineering a wide range of layered tissue types and mechanistic studies on collective cell migration.

Hydration and nanomechanical changes in collagen fibrils bearing advanced glycation end-products.

Accumulation of advanced glycation end-products (AGEs) in biological tissues occurs as a consequence of normal ageing and pathology. Most biological tissues are composed of considerable amounts of collagen, with collagen fibrils being the most abundant form. Collagen fibrils are the smallest discernible structural elements of load-bearing tissues and as such, they are of high biomechanical importance. The low turnover of collagen cause AGEs to accumulate within the collagen fibrils with normal ageing as well as in pathologies. We hypothesized that collagen fibrils bearing AGEs have altered hydration and mechanical properties. To this end, we employed atomic force and Brillouin light scattering microscopy to measure the extent of hydration as well as the transverse elastic properties of collagen fibrils treated with ribose. We find that hydration is different in collagen fibrils bearing AGEs and this is directly related to their mechanical properties. Collagen fibrils treated with ribose showed increased hydration levels and decreased transverse stiffness compared to controlled samples. Our results show that BLS and AFM yield complementary evidence on the effect of hydration on the nanomechanical properties of collagen fibrils.

Nanoscale dysregulation of collagen structure-function disrupts mechano-homeostasis and mediates pulmonary fibrosis.

Matrix stiffening with downstream activation of mechanosensitive pathways is strongly implicated in progressive fibrosis; however, pathologic changes in extracellular matrix (ECM) that initiate mechano-homeostasis dysregulation are not defined in human disease. By integrated multiscale biomechanical and biological analyses of idiopathic pulmonary fibrosis lung tissue, we identify that increased tissue stiffness is a function of dysregulated post-translational collagen cross-linking rather than any collagen concentration increase whilst at the nanometre-scale collagen fibrils are structurally and functionally abnormal with increased stiffness, reduced swelling ratio, and reduced diameter. In ex vivo and animal models of lung fibrosis, dual inhibition of lysyl oxidase-like (LOXL) 2 and LOXL3 was sufficient to normalise collagen fibrillogenesis, reduce tissue stiffness, and improve lung function in vivo. Thus, in human fibrosis, altered collagen architecture is a key determinant of abnormal ECM structure-function, and inhibition of pyridinoline cross-linking can maintain mechano-homeostasis to limit the self-sustaining effects of ECM on progressive fibrosis.

The histone deacetylase inhibitor, romidepsin, as a potential treatment for pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive disease that usually affects elderly people. It has a poor prognosis and there are limited therapies. Since epigenetic alterations are associated with IPF, histone deacetylase (HDAC) inhibitors offer a novel therapeutic strategy to address the unmet medical need. This study investigated the potential of romidepsin, an FDA-approved HDAC inhibitor, as an anti-fibrotic treatment and evaluated biomarkers of target engagement that may have utility in future clinical trials. The anti-fibrotic effects of romidepsin were evaluated both in vitro and in vivo together with any harmful effect on alveolar type II cells (ATII). Bronchoalveolar lavage fluid (BALF) from IPF or control donors was analyzed for the presence of lysyl oxidase (LOX). In parallel with an increase in histone acetylation, romidepsin potently inhibited fibroblast proliferation, myofibroblast differentiation and LOX expression. ATII cell numbers and their lamellar bodies were unaffected. In vivo, romidepsin inhibited bleomycin-induced pulmonary fibrosis in association with suppression of LOX expression. LOX was significantly elevated in BALF of IPF patients compared to controls. These data show the anti-fibrotic effects of romidepsin, supporting its potential use as novel treatment for IPF with LOX as a companion biomarker for evaluation of early on-target effects.

Mechanical Strain Causes Adaptive Change in Bronchial Fibroblasts Enhancing Profibrotic and Inflammatory Responses.

Asthma is characterized by periodic episodes of bronchoconstriction and reversible airway obstruction; these symptoms are attributable to a number of factors including increased mass and reactivity of bronchial smooth muscle and extracellular matrix (ECM) in asthmatic airways. Literature has suggested changes in cell responses and signaling can be elicited via modulation of mechanical stress acting upon them, potentially affecting the microenvironment of the cell. In this study, we hypothesized that mechanical strain directly affects the (myo)fibroblast phenotype in asthma. Therefore, we characterized responses of bronchial fibroblasts, from 6 normal and 11 asthmatic non-smoking volunteers, exposed to cyclical mechanical strain using flexible silastic membranes. Samples were analyzed for proteoglycans, α-smooth muscle actin (αSMA), collagens I and III, matrix metalloproteinase (MMP) 2 & 9 and interleukin-8 (IL-8) by qRT-PCR, Western blot, zymography and ELISA. Mechanical strain caused a decrease in αSMA mRNA but no change in either αSMA protein or proteoglycan expression. In contrast the inflammatory mediator IL-8, MMPs and interstitial collagens were increased at both the transcriptional and protein level. The results demonstrate an adaptive response of bronchial fibroblasts to mechanical strain, irrespective of donor. The adaptation involves cytoskeletal rearrangement, matrix remodelling and inflammatory cytokine release. These results suggest that mechanical strain could contribute to disease progression in asthma by promoting inflammation and remodelling responses.

A genome-wide association study identifies CDHR3 as a susceptibility locus for early childhood asthma with severe exacerbations.

Asthma exacerbations are among the most frequent causes of hospitalization during childhood, but the underlying mechanisms are poorly understood. We performed a genome-wide association study of a specific asthma phenotype characterized by recurrent, severe exacerbations occurring between 2 and 6 years of age in a total of 1,173 cases and 2,522 controls. Cases were identified from national health registries of hospitalization, and DNA was obtained from the Danish Neonatal Screening Biobank. We identified five loci with genome-wide significant association. Four of these, GSDMB, IL33, RAD50 and IL1RL1, were previously reported as asthma susceptibility loci, but the effect sizes for these loci in our cohort were considerably larger than in the previous genome-wide association studies of asthma. We also obtained strong evidence for a new susceptibility gene, CDHR3 (encoding cadherin-related family member 3), which is highly expressed in airway epithelium. These results demonstrate the strength of applying specific phenotyping in the search for asthma susceptibility genes.

Improving RDS treatment with current drugs.

Currently attributed to a lack of foetal lung development, respiratory distress syndrome is the eighth largest cause of infant mortality (USA). Corticosteroids have proved successful but are not infallible in this indication having both a 24-hour latency and little effect on surfactant production. In vivo evidence shows a triggering event in vaginal delivery leads to a rapid final preparation of the lungs, accelerating fluid re-adsorption and surfactant production. It may be possible to reproduce accelerated adaptation synergistically with natural and steroidal maturation; however this would require looking again at ß-agonists. Vulnerable pregnancies may be better served by corticosteroids, oxytocin tocolytics. A single dose of a ß-agonist immediately before delivery may maximise adaptation while avoiding previous failings of this therapy. Trends in premature birth and caesarean section, make prevention of this syndrome increasingly challenging, however room for improvement may be possible with current therapies.

JunD is a profibrogenic transcription factor regulated by Jun N-terminal kinase-independent phosphorylation.

JunD is implicated in the regulation of hepatic stellate cell (HSC) activation and liver fibrosis via its transcriptional regulation of the tissue inhibitor of metalloproteinases-1 (TIMP-1) gene. In the present study we found in vivo evidence of a role for JunD in fibrogenesis. Expression of JunD was demonstrated in alpha-SMA-positive activated HSCs of fibrotic rodents and human livers. The junD-/- mice were protected from carbon tetrachloride-induced fibrosis. The livers of injured junD-/- mice displayed significantly reduced formation of fibrotic crosslinked collagen and a smaller number of alpha-SMA-positive HSCs compared with those of wild-type (wt) mice. Hepatic TIMP-1 mRNA expression in injured junD-/- mice was 78% lower and in culture activated junD-/- HSCs was 50%-80% lower than that in wt mice. In examining the signal transduction mechanisms that regulate JunD-dependent TIMP-1 expression, we found a role for phosphorylation of the Ser100 residue of JunD but ruled out JNK as a mediator of this event, suggesting ERK1/2 is utilized. In conclusion, a signaling pathway for the development of fibrosis involves the regulation of TIMP-1 expression by phosphorylated JunD.

Nuclear factor-kappaB1 (p50) limits the inflammatory and fibrogenic responses to chronic injury.

In this study we addressed the role of the nuclear factor (NF)-kappaB1/p50 subunit in chronic injury of the liver by determining the inflammatory and fibrotic responses of nfkappab1-null mice in an experimental model that mimics chronic liver disease. Mice received repeated hepatic injuries throughout 12 weeks by intraperitoneal injection of the hepatotoxin carbon tetrachloride. In response nfkappab1(-/-) mice developed more severe neutrophilic inflammation and fibrosis compared to nfkappab1(+/+) mice. This phenotype was associated with elevated hepatic expression of tumor necrosis factor (TNF)-alpha, which was localized to regions of the liver associated with inflammation and fibrosis. Hepatic stellate cells are important regulators of hepatic inflammatory and fibrogenic events but normally do not express TNF-alpha. Hepatic stellate cells derived from nfkappab1(-/-) mice expressed TNF-alpha promoter activity, mRNA, and protein. By contrast the expression of other NF-kappaB-responsive genes (ICAM1 and interleukin-6) was similar between nfkappab1(-/-) and nfkappab1(+/+) cells. We provide experimental evidence that the inappropriate expression of TNF-alpha by nfkappab1(-/-) cells is because of lack of a p50-dependent histone deacetylase 1 (HDAC1)-mediated repression of TNF-alpha gene transcription. Taken together these data indicate that the p50 NF-kappaB subunit plays a critical protective role in the injured liver by limiting the expression of TNF-alpha and its recruitment of inflammatory cells.

Induction of myofibroblast MMP-9 transcription in three-dimensional collagen I gel cultures: regulation by NF-kappaB, AP-1 and Sp1.

Chronic liver injury leads to a progressive wound healing response that eventually results in hepatic fibrosis characterised by net deposition of fibrillar extracellular matrix (ECM) and a qualitative shift from type IV to type I/III collagen. The pivotal cellular event underlying this response is hepatic stellate cell (HSC) activation towards a myofibroblast-like phenotype. Activated HSC contribute to ECM remodelling via secretion of type I/III collagens and matrix metalloproteinases (MMPs). Previous studies showed that three-dimensional (3D) contact of activated HSC with type I collagen further stimulates the ECM remodelling properties of HSC by inducing the type IV gelatinase, MMP-9. The aim of the current study was to confirm transcriptional activation of the MMP-9 gene and identify transcription factors regulating this response. Gelatin zymography and Northern blotting were used to confirm induction of MMP-9 protein and mRNA expression in primary rat HSC cultured in a three-dimensional collagen I gel lattice. MMP-9 promoter studies in transfected HSC and electrophoretic mobility shift assay (EMSA) were employed to study transcriptional events. Both NF-kappaB and AP-1 DNA were induced in HSC cultured in 3D collagen I gels and binding sites for these factors in the MMP-9 promoter were crucial for induction of transcription. By contrast removal of an Sp1 site in the promoter enhanced transcription, while over-expression of either Sp1 or Sp3 repressed transcription. It is concluded that 3D contact of activated HSC with collagen I stimulates MMP-9 expression by elevating NF-kappaB and AP-1 activities which are able to overcome the repressive influence of Sp1/Sp3 on MMP-9 gene transcription.

High efficiency gene transfer into cultured primary rat and human hepatic stellate cells using baculovirus vectors.

BACKGROUND/AIMS: Gene transfer into hepatic stellate cells (HSC) is inefficient when using plasmid-based transfection methods; viral-based systems are therefore being developed. A baculovirus system has recently been shown to be useful for expressing genes in mammalian cells. The aim of this study was to determine if baculovirus vectors can infect and express target genes in rat and human HSC and to assess potential cytotoxic and modulatory effects of infection. METHODS: A recombinant baculovirus vector (AcCALacZ) carrying the LacZ gene was used to infect HSC. beta-Galactosidase assays and electron microscopy were used to determine efficiency of infection and gene expression. Counting of trypan blue negative cells was used to assess cytotoxic/cytostatic effects of infection. Measurement of protein content of cells and alpha-smooth muscle actin expression were performed to assess the effects of baculovirus on cell function/phenotype. RESULTS: Baculovirus infection of activated HSC was highly efficient (> 90%) and provided long-term LacZ gene expression (15 days) in the absence of cytotoxic, cytostatic or modulatory effects. Infection of freshly isolated cells was also observed but at lower levels (20%). CONCLUSIONS: Baculovirus vectors can therefore be used to deliver target genes to cultured rat and human HSC with high efficiency and longevity in the absence of detrimental effects on cell function.