Cutaneous wound repair requires the de novo induction of a specialized form of fibroblast, the α-smooth muscle actin (α-SMA)-expressing myofibroblast, which migrates into the wound where it adheres to and contracts extracellular matrix (ECM), resulting in wound closure. Persistence of the myofibroblast results in scarring and fibrotic disease. In this report, we show that, compared with wild-type littermates, PKCϵ-/- mice display delayed impaired cutaneous wound closure and a reduction in myofibroblasts. Moreover, both in the presence and absence of TGFβ, dermal fibroblasts from PKCϵ-/- mice cultured on fibronectin show impaired abilities to form `supermature' focal adhesions and α-SMA stress fibers, and reduced pro-fibrotic gene expression. Smad3 phosphorylation in response to TGFβ1 was impaired in PKCϵ-/- fibroblasts. PKCϵ-/- fibroblasts show reduced FAK and Rac activation, and adhesive, contractile and migratory abilities. Overexpressing constitutively active Rac1 rescues the defective FAK phosphorylation, cell migration, adhesion and stress fiber formation of these PKCϵ-/- fibroblasts, indicating that Rac1 operates downstream of PKCϵ, yet upstream of FAK. These results suggest that loss of PKCϵ severely impairs myofibroblast formation and function, and that targeting PKCϵ may be beneficial in selectively modulating wound healing and fibrotic responses in vivo.
Rodent models produce data which underpin biomedical research and non-clinical drug trials, but translation from rodents into successful clinical outcomes is often lacking. There is a growing body of evidence showing that improving experimental design is key to improving the predictive nature of rodent studies and reducing the number of animals used in research. Age, one important factor in experimental design, is often poorly reported and can be overlooked. The authors conducted a survey to assess the age used for a range of models, and the reasoning for age choice. From 297 respondents providing 611 responses, researchers reported using rodents most often in the 6-20 week age range regardless of the biology being studied. The age referred to as 'adult' by respondents varied between six and 20 weeks. Practical reasons for the choice of rodent age were frequently given, with increased cost associated with using older animals and maintenance of historical data comparability being two important limiting factors. These results highlight that choice of age is inconsistent across the research community and often not based on the development or cellular ageing of the system being studied. This could potentially result in decreased scientific validity and increased experimental variability. In some cases the use of older animals may be beneficial. Increased scientific rigour in the choice of the age of rodent may increase the translation of rodent models to humans.
Objectives. Nitric oxide (•NO) is an important physiological signalling molecule and a potent vasodilator. We have previously demonstrated abnormal • NO metabolism in the plasma of patients with systemic sclerosis (SSc; scleroderma), a disease that features vascular dysfunction as well as collagen overproduction and fibrosis. The aim of the present study was to examine nitric oxide synthase (NOS) expression and activity and assess the potential role of antioxidants in the scleroderma-like syndrome of the tight-skin 1 (TSK-1/+) mouse, an experimental animal model for fibrosis.
Journal Article Pseudohyperaldosteronism induced by habitual ingestion of liquorice Get access A M Holmes, B.Sc., M.B., M.R.C.P, A M Holmes, B.Sc., M.B., M.R.C.P Division of Metabolism, Department of Medicine, The Royal Infirmary, Manchester, M13 9 WL Search for other works by this author on: Oxford Academic Google Scholar J Young, J Young Technical Officer Division of Metabolism, Department of Medicine, The Royal Infirmary, Manchester, M13 9 WL Present address: M.R.C. Blood Pressure Unit, Western Infirmary, Glasgow. Search for other works by this author on: Oxford Academic Google Scholar P K Marrott, M.B., B.S., M.R.C.P, P K Marrott, M.B., B.S., M.R.C.P Registrar Division of Metabolism, Department of Medicine, The Royal Infirmary, Manchester, M13 9 WL Present address: Registrar, Birch Hill Hospital, Rochdale. Search for other works by this author on: Oxford Academic Google Scholar E Prentice E Prentice Junior Technical Officer Division of Metabolism, Department of Medicine, The Royal Infirmary, Manchester, M13 9 WL Present address: M.R.C. Blood Pressure Unit, Western Infirmary, Glasgow. Search for other works by this author on: Oxford Academic Google Scholar Postgraduate Medical Journal, Volume 46, Issue 540, June 1970, Pages 625–629, https://doi.org/10.1136/pgmj.46.540.625 Published: 01 October 1970
Malignant mesothelioma (MM) has no biomarker driven therapies in routine clinical use. We used a drug screen of molecularly characterised MM lines to identify novel genomic biomarker driven therapy. This led to the discovery of a subset of MMs, defined by loss of function (LOF) of the nuclear deubiquitinase BRCA associated protein 1 (BAP1), that demonstrate heightened sensitivity to tumour necrosis factor related apoptosis inducing ligand (TRAIL). We then validated this association across in vitro, in vivo and ex vivo models and delineated the underlying mechanism.
Methods
15 MM lines were characterised for mutations in five MM tumour driver genes and screened for response to 95 compounds. The identified BAP1-TRAIL association was validated in an extended panel of MM lines with apoptosis and cell viability assays. 25 early passage MM cultures, mouse xenograft and human tumour explant models were used to further validate the association. Knock-in and knock-out models in BAP1 mutant and wild type lines confirmed the effect of loss of BAP1 expression on TRAIL sensitivity. 6 mutant BAP1 constructs were generated and identified which functional BAP1 sites modulate TRAIL sensitivity. The effect of BAP1 function on the downstream death receptor/apoptosis pathway components was determined using microarray and immunoblot analysis.
Results
BAP1 LOF significantly correlated with TRAIL sensitivity in established MM lines (p=0.015) and primary MM cultures (p<0.0064). This association was confirmed in mouse xenograft (p<0.05) and tumour explant models. Mutagenesis of BAP1 confirmed deubiquitinase activity and its ability to bind to ASXL proteins to form the polycomb repressor deubiquitinase complex (PR-DUB) as determinants of TRAIL sensitivity, implicating modulation of transcriptional programmes as an underlying mechanism. Consistent with this, knockdown of ASXL1 also increased TRAIL sensitivity in MM lines and loss of BAP1 deubiquitinase and ASXL binding activity altered gene, and protein expression of components of the apoptotic machinery favouring apoptosis upon activation of death receptors.
Conclusions
We identify loss of BAP1 as a novel biomarker for TRAIL sensitivity in MM. BAP1 LOF is observed in up to 67% of MM tumours and BAP1 immunohistochemistry is in use as a diagnostic tool; hence this approach is validated and ready for immediate and actionable clinical use for this disease.
Connective tissue growth factor (CTGF, CCN2) is a matricellular protein which plays key roles in normal mammalian development and in tissue homeostasis and repair. In pathological conditions, dysregulated CCN2 has been associated with cancer, cardiovascular disease, and tissue fibrosis. In this study, genetic manipulation of the CCN2 gene was employed to investigate the role of CCN2 expression in vitro and in experimentally-induced models of pulmonary fibrosis and pulmonary arterial hypertension (PAH). Knocking down CCN2 using siRNA reduced expression of pro-fibrotic markers (fibronectin p < 0.01, collagen type I p < 0.05, α-SMA p < 0.0001, TIMP-1 p < 0.05 and IL-6 p < 0.05) in TGF-β-treated lung fibroblasts derived from systemic sclerosis patients. In vivo studies were performed in mice using a conditional gene deletion strategy targeting CCN2 in a fibroblast-specific and time-dependent manner in two models of lung disease. CCN2 deletion significantly reduced pulmonary interstitial scarring and fibrosis following bleomycin-instillation, as assessed by fibrotic scores (wildtype bleomycin 3.733 ± 0.2667 vs CCN2 knockout (KO) bleomycin 4.917 ± 0.3436, p < 0.05) and micro-CT. In the well-established chronic hypoxia/Sugen model of pulmonary hypertension, CCN2 gene deletion resulted in a significant decrease in pulmonary vessel remodelling, less right ventricular hypertrophy and a reduction in the haemodynamic measurements characteristic of PAH (RVSP and RV/LV + S were significantly reduced (p < 0.05) in CCN2 KO compared to WT mice in hypoxic/SU5416 conditions). These results support a prominent role for CCN2 in pulmonary fibrosis and in vessel remodelling associated with PAH. Therefore, therapeutics aimed at blocking CCN2 function are likely to benefit several forms of severe lung disease.
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