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Venous endothelial cells are molecularly and functionally distinct from their arterial counterparts. Although veins are often considered the default endothelial state, genetic manipulations can modulate both acquisition and loss of venous fate, suggesting that venous identity is the result of active transcriptional regulation. However, little is known about this process. Here we show that BMP signalling controls venous identity via the ALK3/BMPR1A receptor and SMAD1/SMAD5. Perturbations to TGF-β and BMP signalling in mice and zebrafish result in aberrant vein formation and loss of expression of the venous-specific gene Ephb4, with no effect on arterial identity. Analysis of a venous endothelium-specific enhancer for Ephb4 shows enriched binding of SMAD1/5 and a requirement for SMAD binding motifs. Further, our results demonstrate that BMP/SMAD-mediated Ephb4 expression requires the venous-enriched BMP type I receptor ALK3/BMPR1A. Together, our analysis demonstrates a requirement for BMP signalling in the establishment of Ephb4 expression and the venous vasculature.

Objective: Identify gene changes in articular cartilage of the medial tibial plateau (MTP) at 2, 4 and 8 weeks after destabilisation of the medial meniscus (DMM) in mice. Compare our data with previously published datasets to ascertain dysregulated pathways and genes in osteoarthritis (OA)., Design: RNA was extracted from the ipsilateral and contralateral MTP cartilage, amplified, labelled and hybridized on Illumina WGv2 microarrays. Results were confirmed by real-time polymerase chain reaction (PCR) for selected genes., Results: Transcriptional analysis and network reconstruction revealed changes in extracellular matrix and cytoskeletal genes induced by DMM. TGFβ signalling pathway and complement and coagulation cascade genes were regulated at 2 weeks. Fibronectin (Fn1) is a hub in a reconstructed network at 2 weeks. Regulated genes decrease over time. By 8 weeks fibromodulin (Fmod) and tenascin N (Tnn) are the only dysregulated genes present in the DMM operated knees. Comparison with human and rodent published gene sets identified genes overlapping between our array and eight other studies., Conclusions: Cartilage contributes a minute percentage to the RNA extracted from the whole joint (<0.2%), yet is sensitive to changes in gene expression post-DMM. The post-DMM transcriptional reprogramming wanes over time dissipating by 8 weeks. Common pathways between published gene sets include focal adhesion, regulation of actin cytoskeleton and TGFβ. Common genes include Jagged 1 (Jag1), Tetraspanin 2 (Tspan2), neuroblastoma, suppression of tumourigenicity 1 (Nbl1) and N-myc downstream regulated gene 2 (Ndrg2). The concomitant genes and pathways we identify may warrant further investigation as biomarkers or modulators of OA., (Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.)

We previously identified transforming growth factor (TGF)-β signaling as a fibronectin-independent mechanism of type I collagen fibrillogenesis following adult liver injury. To address the contribution of TGF-β signaling during the development of liver fibrosis, we generated adult mice lacking TGF-β type II receptor (TGF-βIIR) from the liver. TGF-βIIR knockout livers indeed showed a dominant effect in reducing fibrosis, but fibrosis still remained approximately 45% compared with control and fibronectin knockout livers. Unexpectedly, this was accompanied by significant up-regulation of connective tissue growth factor mRNA levels. Organized type I collagen networks in TGF-βIIR knockout livers colocalized well with fibronectin. We provide evidence that elimination of TGF-βIIR is not sufficient to completely prevent liver fibrosis. Our results indicate a TGF-β-independent mechanism of type I collagen production and suggest connective tissue growth factor as its potent mediator. We advocate combined elimination of TGF-β signaling and connective tissue growth factor as a potential therapeutic target by which to attenuate liver fibrosis., (Copyright © 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Fibrosis is excessive scarring caused by the accumulation of extracellular matrix proteins and is a common end pathway in many chronic diseases. Endothelin-1 is a possible contributor to the persistent fibrotic phenotype of fibroblasts isolated from fibrotic lesions. In this report we used a specific dual endothelin A/B receptor antagonist, bosentan, to determine the role of endogenous endothelin signaling in maintaining the profibrotic phenotype of lung fibroblasts from scleroderma patients. Bosentan treatment of lung fibroblasts cultured from normal individuals and individuals with scleroderma was assessed using Affymetrix genome-wide expression profiling, real-time polymerase chain reaction and Western blot analysis and revealed that approximately one-third of the transcripts elevated greater than two-fold in fibrotic fibroblasts were reduced by Bosentan treatment. Genes whose overexpression in fibrotic fibroblasts that were dependent on endogenous endothelin signaling included the matrix or matrix-associated genes type I collagen, fibronectin and CCN2. The elevated adhesive property of fibrotic fibroblasts was also reduced by endothelin receptor antagonism. Basal expression of collagen, fibronectin and CCN2 and adhesion to matrix was not affected. Thus endogenous endothelin signaling contributes to the fibrotic phenotype of fibrotic fibroblasts, suggesting that antagonizing endothelin receptors may be of benefit in combating fibrotic disease.

We have generated transgenic mice expressing a kinase-deficient type II transforming growth factor-beta (TGFbeta) receptor selectively on fibroblasts (TbetaRIIDeltak-fib). These mice develop dermal and pulmonary fibrosis. In the present study we explore activation of TGFbeta signaling pathways in this strain and examine the profibrotic properties of explanted transgenic fibroblasts including myofibroblast differentiation and abnormal metalloproteinase production. Gene expression profiles of littermate wild type or transgenic fibroblasts were compared using high-density gene arrays and validated by Taqman reverse transcriptase-PCR, Northern and Western blotting. Using a specific inhibitor (SD-208) we demonstrate that the abnormal phenotype of these cells is dependent upon TbetaRI kinase (ALK5) activity, and that transgenic fibroblasts show enhanced expression and activation of TGFbeta together with increased levels of wild type TbetaRII. Moreover, we confirm that transgene expression is itself regulated by TGFbeta and that expression at low levels facilitates signaling, whereas high level expression is inhibitory. For a subset of TGFbeta responsive genes basal up-regulation is normalized or suppressed by exogenous recombinant TGFbeta1 at time points coincident with increased transgene expression. These findings explain the profound refractoriness of TbetaRIIDeltak-fib fibroblasts to exogenous TGFbeta1, despite their activated phenotype. Thus, transgenic fibroblasts recapitulate many hallmark biochemical properties of fibrotic cells, including high level CTGF (CCN2) expression and type I collagen overproduction, altered MMP production, and myofibroblast differentiation. These cells also show an enhanced ability to contract collagen gel matrices. Our study demonstrates that altered high affinity TGFbeta receptor function may lead to ligand-dependent activation of downstream signaling, and provides further evidence of a pivotal role for sustained TGFbeta overactivity in fibrosis.

The endothelins are a family of endothelium-derived peptides that possess a variety of biological activities, including potent vasoconstriction. Endothelin-1 (ET-1) is up-regulated during tissue repair and pulmonary fibrosis. Here, we use genome-wide expression array analysis to show that the addition of ET-1 (100 nm, 4 h) to normal lung fibroblasts directly induces expression of matrix and matrix-associated genes, including the profibrotic protein CCN2 (connective tissue growth factor, or CTGF). ET-1 induces the MEK/ERK MAP kinase pathway in fibroblasts. Blockade of the MEK/ERK kinase pathway with U0126 abrogates the ability of ET-1 to induce expression of matrix and matrix-associated mRNAs and the CCN2 protein. The CCN2 promoter possesses an ET-1 response element, which maps to the previously identified basal control element-1 (BCE-1) site. Our results suggest that ET-1 induces a program of matrix synthesis in lung fibroblasts and that ET-1 may play a key role in connective tissue deposition during wound repair and in pulmonary fibrosis.