Publications by authors named "Markella Ponticos"

27 Publications

  • Page 1 of 1

"Epigenome-wide methylation profile of chronic kidney disease-derived arterial DNA uncovers novel pathways in disease-associated cardiovascular pathology."

Epigenetics 2020 Sep 15:1-11. Epub 2020 Sep 15.

Department of Renal Medicine, Division of Medicine, UCL, London, UK.

Chronic kidney disease (CKD) related cardiovascular disease (CVD) is characterized by vascular remodelling with well-established structural and functional changes in the vascular wall such as arterial stiffness, matrix deposition, and calcification. These phenotypic changes resemble pathology seen in ageing, and are likely to be mediated by sustained alterations in gene expression, which may be caused by epigenetic changes such as tissue-specific DNA methylation. We aimed to investigate tissue specific changes in DNA methylation that occur in CKD-related CVD. Genome-wide DNA methylation changes were examined in bisulphite converted genomic DNA isolated from the vascular media of CKD and healthy arteries. Methylation-specific PCR was used to validate the array data, and the association between DNA methylation and gene and protein expression was examined. The DNA methylation age was compared to the chronological age in both cases and controls. Three hundred and nineteen differentially methylated regions (DMR) were identified spread across the genome. Pathway analysis revealed that DMRs associated with genes were involved in embryonic and vascular development, and signalling pathways such as TGFβ and FGF. Expression of top differentially methylated gene showed a significant negative correlation with DNA methylation. Interestingly, DNA methylation age and chronological age were highly correlated, but there was no evidence of accelerated age-related DNA methylation in the arteries of CKD patients. In conclusion, we demonstrated that differential DNA methylation in the arterial tissue of CKD patients represents a potential mediator of arterial pathology and may be used to uncover novel pathways in the genesis of CKD-associated complications.
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http://dx.doi.org/10.1080/15592294.2020.1819666DOI Listing
September 2020

Analysis of Anti-RNA Polymerase III Antibody-positive Systemic Sclerosis and Altered GPATCH2L and CTNND2 Expression in Scleroderma Renal Crisis.

J Rheumatol 2020 11 15;47(11):1668-1677. Epub 2020 Mar 15.

S.G. Guerra, PhD, H. Chinque, BSc, V. Acquaah, BSc, M. Ponticos, V.H. Ong, PhD, FRCP, K. Khan, BSc, S.I. Nihtyanova, MD, C. Fonseca, MD, C.P. Denton, PhD, FRCP, Professor of Experimental Rheumatology, UCL Centre for Rheumatology and Connective Tissue Diseases, London, UK;

Objective: Scleroderma renal crisis (SRC) is a life-threatening complication of systemic sclerosis (SSc) strongly associated with anti-RNA polymerase III antibody (ARA) autoantibodies. We investigated genetic susceptibility and altered protein expression in renal biopsy specimens in ARA-positive patients with SRC.

Methods: ARA-positive patients (n = 99) with at least 5 years' follow-up (49% with a history of SRC) were selected from a well characterized SSc cohort (n = 2254). Cases were genotyped using the Illumina Human Omni-express chip. Based on initial regression analysis, 9 single-nucleotide polymorphisms (SNP) were chosen for validation in a separate cohort of 256 ARA-positive patients (40 with SRC). Immunostaining of tissue sections from SRC or control kidney was used to quantify expression of candidate proteins based upon genetic analysis of the discovery cohort.

Results: Analysis of 641,489 SNP suggested association of POU2F1 (rs2093658; = 1.98 × 10), CTNND2 (rs1859082; = 5.58 × 10), HECW2 (rs16849716; = 1.2 × 10), and GPATCH2L (rs935332; = 4.92 × 10) with SRC. Further, the validation cohort showed an association between rs935332 within the region, with SRC ( = 0.025). Immunostaining of renal biopsy sections showed increased tubular expression of GPATCH2L ( = 0.026) and glomerular expression of CTNND2 ( = 0.026) in SRC samples (n = 8) compared with normal human kidney controls (n = 8), despite absence of any genetic replication for the associated SNP.

Conclusion: Increased expression of 2 candidate proteins, GPATCH2L and CTNND2, in SRC compared with control kidney suggests a potential role in pathogenesis of SRC. For GPATCH2L, this may reflect genetic susceptibility in ARA-positive patients with SSc based upon 2 independent cohorts.
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http://dx.doi.org/10.3899/jrheum.190945DOI Listing
November 2020

Insights into myofibroblasts and their activation in scleroderma: opportunities for therapy?

Curr Opin Rheumatol 2018 11;30(6):581-587

Centre for Rheumatology and Connective Tissue Diseases, Research Department of Inflammation, Division of Medicine, University College London, London, UK.

Purpose Of Review: The persistence of myofibroblasts is a key feature of fibrosis and in fibrotic diseases including scleroderma. This review evaluates the emerging concepts of the origins and cell populations that contribute to myofibroblasts and the molecular mechanisms that govern phenotypic conversion and that highlight opportunities for new interventional treatments in scleroderma.

Recent Findings: Studies have defined heterogeneity in fibroblast-like cells that can develop into myofibroblast in normal wound healing, scarring and fibrosis. Characterizing these distinct cell populations and their behaviour has been a key focus. In addition, the overarching impact of epigenetic regulation of genes associated with inflammatory responses, cell signalling and cell communication and the extracellular matrix (ECM) has provided important insights into the formation of myofibroblast and their function. Important new studies include investigations into the relationship between inflammation and myofibroblast production and further evidence has been gathered that reveal the importance of ECM microenvironment, biomechanical sensing and mechanotransduction.

Summary: This review highlights our current understanding and outlines the increasing complexity of the biological processes that leads to the appearance of the myofibroblast in normal functions and in diseased tissues. We also focus on areas of special interest in particular, studies that have therapeutic potential in fibrosis and scleroderma.
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http://dx.doi.org/10.1097/BOR.0000000000000543DOI Listing
November 2018

Molecular Basis for Dysregulated Activation of NKX2-5 in the Vascular Remodeling of Systemic Sclerosis.

Arthritis Rheumatol 2018 06 24;70(6):920-931. Epub 2018 Apr 24.

University College London, London, UK.

Objective: NKX2-5 is a homeobox transcription factor that is required for the formation of the heart and vessels during development, with significant postnatal down-regulation and reactivation in disease states, characterized by vascular remodeling. The purpose of this study was to investigate mechanisms that activate NKX2-5 expression in diseased vessels, such as systemic sclerosis (scleroderma; SSc)-associated pulmonary hypertension (PH), and to identify genetic variability that potentially underlies susceptibility to specific vascular complications.

Methods: We explored NKX2-5 expression in biopsy samples from patients with SSc-associated PH and in pulmonary artery smooth muscle cells (PASMCs) from patients with scleroderma. Disease-associated putative functional single-nucleotide polymorphisms (SNPs) at the NKX2-5 locus were cloned and studied in reporter gene assays. SNP function was further examined through protein-DNA binding assays, chromatin immunoprecipitation assays, and RNA silencing analyses.

Results: Increased NKX2-5 expression in biopsy samples from patients with SSc-associated PH was localized to remodeled vessels and PASMCs. Meta-analysis of 2 independent scleroderma cohorts revealed an association of rs3131917 with scleroderma (P = 0.029). We demonstrated that disease-associated SNPs are located in a novel functional enhancer, which increases NKX2-5 transcriptional activity through the binding of GATA-6, c-Jun, and myocyte-specific enhancer factor 2C. We also characterized an activator/coactivator transcription-enhancer factor domain 1 (TEAD1)/Yes-associated protein 1 (YAP1) complex, which was bound at rs3095870, another functional SNP, with TEAD1 binding the risk allele and activating the transcription of NKX2-5.

Conclusion: NKX2-5 is genetically associated with scleroderma, pulmonary hypertension, and fibrosis. Functional evidence revealed a regulatory mechanism that results in NKX2-5 transcriptional activation in PASMCs through the interaction of an upstream promoter and a novel downstream enhancer. This mechanism can act as a model for NKX2-5 activation in cardiovascular disease characterized by vascular remodeling.
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http://dx.doi.org/10.1002/art.40419DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6001790PMC
June 2018

STAT3 controls COL1A2 enhancer activation cooperatively with JunB, regulates type I collagen synthesis posttranscriptionally, and is essential for lung myofibroblast differentiation.

Mol Biol Cell 2018 01 15;29(2):84-95. Epub 2017 Nov 15.

Centre for Rheumatology and Connective Tissue Diseases, Division of Medicine, University College London, London NW3 2PF, United Kingdom

Fibroblast differentiation is a key cellular process that underlies the process of fibrosis, a deadly complication of fibrotic diseases like scleroderma (SSc). This transition coincides with the overproduction of collagen type I (COL1) and other extracellular matrix proteins. High-level expression of the collagen type 1α2 subunit (COL1A2), requires the engagement of a far-upstream enhancer, whose activation is strongly dependent on the AP1 factor JunB. We now report that STAT3 also binds the COL1A2 enhancer and is essential for RNA polymerase recruitment, without affecting JunB binding. STAT3 is required for the increased COL1A2 expression observed in myofibroblasts. We also report that TGFβ partially activates STAT3 and show that inhibiting STAT3 potently blocks TGFβ signaling, matrix remodeling, and TGFβ-induced myofibroblast differentiation. Activation of STAT3 with IL6 transsignaling alone, however, only increased COL1A2 protein expression, leaving COL1A2 mRNA levels unchanged. Our results suggest that activated STAT3 is not the limiting factor for collagen enhancer activation in human lung fibroblasts. Yet, a certain threshold level of STAT3 activity is essential to support activation of the COL1A2 enhancer and TGFβ signaling in fibroblasts. We propose that STAT3 operates at the posttranscriptional as well as the transcriptional level.
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http://dx.doi.org/10.1091/mbc.E17-06-0342DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5909935PMC
January 2018

Aldehyde dehydrogenase inhibition blocks mucosal fibrosis in human and mouse ocular scarring.

JCI Insight 2016 08 4;1(12):e87001. Epub 2016 Aug 4.

NIH Research (NIHR) Biomedical Research Centre at Moorfields Eye Hospital NHS Foundation Trust and the UCL Institute of Ophthalmology, London, United Kingdom.

Mucous membrane pemphigoid (MMP) is a systemic mucosal scarring disease, commonly causing blindness, for which there is no antifibrotic therapy. Aldehyde dehydrogenase family 1 (ALDH1) is upregulated in both ocular MMP (OMMP) conjunctiva and cultured fibroblasts. Application of the ALDH metabolite, retinoic acid (RA), to normal human conjunctival fibroblasts in vitro induced a diseased phenotype. Conversely, application of ALDH inhibitors, including disulfiram, to OMMP fibroblasts in vitro restored their functionality to that of normal controls. ALDH1 is also upregulated in the mucosa of the mouse model of scarring allergic eye disease (AED), used here as a surrogate for OMMP, in which topical application of disulfiram decreased fibrosis in vivo. These data suggest that progressive scarring in OMMP results from ALDH/RA fibroblast autoregulation, that the ALDH1 subfamily has a central role in immune-mediated ocular mucosal scarring, and that ALDH inhibition with disulfiram is a potential and readily translatable antifibrotic therapy.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5033866PMC
http://dx.doi.org/10.1172/jci.insight.87001DOI Listing
August 2016

Data on CUX1 isoforms in idiopathic pulmonary fibrosis lung and systemic sclerosis skin tissue sections.

Data Brief 2016 Sep 10;8:1377-80. Epub 2016 Aug 10.

Royal Free and University College Medical School, London, UK.

This data article contains complementary figures related to the research article entitled, "Transforming growth factor-β-induced CUX1 isoforms are associated with fibrosis in systemic sclerosis lung fibroblasts" (Ikeda et al. (2016) [2], http://dx.doi.org/10.1016/j.bbrep.2016.06.022), which presents that TGF-β increased CUX1 binding in the proximal promoter and enhancer of the COL1A2 and regulated COL1. Further, in the scleroderma (SSc) lung and diffuse alveolar damage lung sections, CUX1 localized within the α- smooth muscle actin (α-SMA) positive cells (Fragiadaki et al., 2011) [1], "High doses of TGF-beta potently suppress type I collagen via the transcription factor CUX1" (Ikeda et al., 2016) [2]. Here we show that CUX1 isoforms are localized within α-smooth muscle actin-positive cells in SSc skin and idiopathic pulmonary fibrosis (IPF) lung tissue sections. In particular, at the granular and prickle cell layers in the SSc skin sections, CUX1 and α-SMA are co-localized. In addition, at the fibrotic loci in the IPF lung tissue sections, CUX1 localized within the α-smooth muscle actin (α-SMA) positive cells.
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http://dx.doi.org/10.1016/j.dib.2016.08.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4993856PMC
September 2016

Transforming growth factor--induced CUX1 isoforms are associated with fibrosis in systemic sclerosis lung fibroblasts.

Biochem Biophys Rep 2016 Sep 2;7:246-252. Epub 2016 Jul 2.

Royal Free and University College Medical School, London, UK.

In the enhancer region of the human type I collagen alpha 2 () gene, we identified cis-elements for the transcription factor CUX1. However, the role of CUX1 in fibrosis remains unclear. Here we investigated the role of CUX1 in the regulation of COL1 expression and delineated the mechanisms underlying the regulation of expression by CUX1 in systemic sclerosis (SSc) lung fibroblasts. The binding of CUX1 to the enhancer region was assessed using electrophoretic mobility shift assays after treatment with transforming growth factor (TGF)-. Subsequently, the protein expression levels of CUX1 isoforms were determined using Western blotting. Finally, the expression levels of COL1 and fibrosis-related cytokines, including CTGF, ET-1, Wnt1 and -catenin were determined. The binding of CUX1 isoforms to the enhancer region increased after TGF- treatment. TGF- also increased the protein levels of the CUX1 isoforms p200, p150, p110, p75, p30 and p28. Moreover, SSc lung fibroblasts showed higher levels of CUX1 isoforms than normal lung fibroblasts, and treatment of SSc lung fibroblasts with a cathepsin L inhibitor (IW-CHO) decreased COL1 protein expression and reduced cell size, as measured using immunocytochemistry. In SSc and diffuse alveolar damage lung tissue sections, CUX1 localised within α-smooth muscle actin-positive cells. Our results suggested that CUX1 isoforms play vital roles in connective tissue deposition during wound repair and fibrosis.
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http://dx.doi.org/10.1016/j.bbrep.2016.06.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5613511PMC
September 2016

Endoplasmic reticulum stress enhances fibrosis through IRE1α-mediated degradation of miR-150 and XBP-1 splicing.

EMBO Mol Med 2016 07 1;8(7):729-44. Epub 2016 Jul 1.

Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden Department of Radiology, Uppsala University Hospital, Uppsala, Sweden

ER stress results in activation of the unfolded protein response and has been implicated in the development of fibrotic diseases. In this study, we show that inhibition of the ER stress-induced IRE1α signaling pathway, using the inhibitor 4μ8C, blocks TGFβ-induced activation of myofibroblasts in vitro, reduces liver and skin fibrosis in vivo, and reverts the fibrotic phenotype of activated myofibroblasts isolated from patients with systemic sclerosis. By using IRE1α(-/-) fibroblasts and expression of IRE1α-mutant proteins lacking endoribonuclease activity, we confirmed that IRE1α plays an important role during myofibroblast activation. IRE1α was shown to cleave miR-150 and thereby to release the suppressive effect that miR-150 exerted on αSMA expression through c-Myb. Inhibition of IRE1α was also demonstrated to block ER expansion through an XBP-1-dependent pathway. Taken together, our results suggest that ER stress could be an important and conserved mechanism in the pathogenesis of fibrosis and that components of the ER stress pathway may be therapeutically relevant for treating patients with fibrotic diseases.
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http://dx.doi.org/10.15252/emmm.201505925DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4931288PMC
July 2016

A Role of Myocardin Related Transcription Factor-A (MRTF-A) in Scleroderma Related Fibrosis.

PLoS One 2015 8;10(5):e0126015. Epub 2015 May 8.

Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts, United States of America.

In scleroderma (systemic sclerosis, SSc), persistent activation of myofibroblast leads to severe skin and organ fibrosis resistant to therapy. Increased mechanical stiffness in the involved fibrotic tissues is a hallmark clinical feature and a cause of disabling symptoms. Myocardin Related Transcription Factor-A (MRTF-A) is a transcriptional co-activator that is sequestered in the cytoplasm and translocates to the nucleus under mechanical stress or growth factor stimulation. Our objective was to determine if MRTF-A is activated in the disease microenvironment to produce more extracellular matrix in progressive SSc. Immunohistochemistry studies demonstrate that nuclear translocation of MRTF-A in scleroderma tissues occurs in keratinocytes, endothelial cells, infiltrating inflammatory cells, and dermal fibroblasts, consistent with enhanced signaling in multiple cell lineages exposed to the stiff extracellular matrix. Inhibition of MRTF-A nuclear translocation or knockdown of MRTF-A synthesis abolishes the SSc myofibroblast enhanced basal contractility and synthesis of type I collagen and inhibits the matricellular profibrotic protein, connective tissue growth factor (CCN2/CTGF). In MRTF-A null mice, basal skin and lung stiffness was abnormally reduced and associated with altered fibrillar collagen. MRTF-A has a role in SSc fibrosis acting as a central regulator linking mechanical cues to adverse remodeling of the extracellular matrix.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0126015PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4425676PMC
February 2016

Failed degradation of JunB contributes to overproduction of type I collagen and development of dermal fibrosis in patients with systemic sclerosis.

Arthritis Rheumatol 2015 Jan;67(1):243-53

University College London Medical School, London, UK.

Objective: The excessive deposition of extracellular matrix, including type I collagen, is a key aspect in the pathogenesis of connective tissue diseases such as systemic sclerosis (SSc; scleroderma). To further our understanding of the mechanisms governing the dysregulation of type I collagen production in SSc, we investigated the role of the activator protein 1 (AP-1) family of transcription factors in regulating COL1A2 transcription.

Methods: The expression and nuclear localization of AP-1 family members (c-Jun, JunB, JunD, Fra-1, Fra-2, and c-Fos) were examined by immunohistochemistry and Western blotting in dermal biopsy specimens and explanted skin fibroblasts from patients with diffuse cutaneous SSc and healthy controls. Gene activation was determined by assessing the interaction of transcription factors with the COL1A2 enhancer using transient transfection of reporter gene constructs, electrophoretic mobility shift assays, chromatin immunoprecipitation analysis, and RNA interference involving knockdown of individual AP-1 family members. Inhibition of fibroblast mammalian target of rapamycin (mTOR), Akt, and glycogen synthase kinase 3β (GSK-3β) signaling pathways was achieved using small-molecule pharmacologic inhibitors.

Results: Binding of JunB to the COL1A2 enhancer was observed, with its coalescence directed by activation of gene transcription through the proximal promoter. Knockdown of JunB reduced enhancer activation and COL1A2 expression in response to transforming growth factor β. In SSc dermal fibroblasts, increased mTOR/Akt signaling was associated with inactivation of GSK-3β, leading to blockade of JunB degradation and, thus, constitutively high expression of JunB.

Conclusion: In patients with SSc, the accumulation of JunB resulting from altered mTOR/Akt signaling and a failure of proteolytic degradation underpins the aberrant overexpression of type I collagen. These findings identify JunB as a potential target for antifibrotic therapy in SSc.
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http://dx.doi.org/10.1002/art.38897DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4312903PMC
January 2015

Extracellular matrix synthesis in vascular disease: hypertension, and atherosclerosis.

J Biomed Res 2014 Jan 20;28(1):25-39. Epub 2013 Sep 20.

Department of Biochemistry, Boston University School of Medicine, Boston, MA 02118, USA.

Extracellular matrix (ECM) within the vascular network provides both a structural and regulatory role. The ECM is a dynamic composite of multiple proteins that form structures connecting cells within the network. Blood vessels are distended by blood pressure and, therefore, require ECM components with elasticity yet with enough tensile strength to resist rupture. The ECM is involved in conducting mechanical signals to cells. Most importantly, ECM regulates cellular function through chemical signaling by controlling activation and bioavailability of the growth factors. Cells respond to ECM by remodeling their microenvironment which becomes dysregulated in vascular diseases such hypertension, restenosis and atherosclerosis. This review examines the cellular and ECM components of vessels, with specific emphasis on the regulation of collagen type I and implications in vascular disease.
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http://dx.doi.org/10.7555/JBR.27.20130064DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3904172PMC
January 2014

Connective tissue growth factor causes EMT-like cell fate changes in vivo and in vitro.

J Cell Sci 2013 May 22;126(Pt 10):2164-75. Epub 2013 Mar 22.

University of Texas M. D. Anderson Cancer Center, Department of Genetics, Houston, TX, USA.

Connective tissue growth factor (CTGF) plays an important role in the pathogenesis of chronic fibrotic diseases. However, the mechanism by which paracrine effects of CTGF control the cell fate of neighboring epithelial cells is not known. In this study, we investigated the paracrine effects of CTGF overexpressed in fibroblasts of Col1a2-CTGF transgenic mice on epithelial cells of skin and lung. The skin and lungs of Col1a2-CTGF transgenic mice were examined for phenotypic markers of epithelial activation and differentiation and stimulation of signal transduction pathways. In addition to an expansion of the dermal compartment in Col1a2-CTGF transgenic mice, the epidermis was characterized by focal hyperplasia, and basal cells stained positive for αSMA, Snail, S100A4 and Sox9, indicating that these cells had undergone a change in their genetic program. Activation of phosphorylated p38 and phosphorylated Erk1/2 was observed in the granular and cornified layers of the skin. Lung fibrosis was associated with a marked increase in cells co-expressing epithelial and mesenchymal markers in the lesional and unaffected lung tissue of Col1a2-CTGF mice. In epithelial cells treated with TGFβ, CTGF-specific siRNA-mediated knockdown suppressed Snail, Sox9, S100A4 protein levels and restored E-cadherin levels. Both adenoviral expression of CTGF in epithelial cells and treatment with recombinant CTGF induced EMT-like morphological changes and expression of α-SMA. Our in vivo and in vitro data supports the notion that CTGF expression in mesenchymal cells in the skin and lungs can cause changes in the differentiation program of adjacent epithelial cells. We speculate that these changes might contribute to fibrogenesis.
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http://dx.doi.org/10.1242/jcs.111302DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3672936PMC
May 2013

Connective tissue growth factor (CCN2) in blood vessels.

Vascul Pharmacol 2013 Mar 4;58(3):189-93. Epub 2013 Feb 4.

Division of Medicine-Inflammation, Royal Free & University College Medical School, Centre for Rheumatology & Connective Tissue Diseases, Rowland Hill StreetLondon, NW3 2PF, UK.

The CCN family comprise the products of six immediate-early response genes (Cyr61, Ctgf, Nov and Wisp1-3) and are multi-functional proteins, characterised by four discrete protein modules in which reside functional domains: an insulin-like growth factor binding protein-like module (IGFBP) but has low affinity for IGFBPs, a von Willebrand factor type C repeat module (VWC) which mediates integrin and growth factor binding, a thrombospondin type-1 repeat module (TSP-1), and a cysteine-knot-containing module (CT). These modules mediate a host of interactions such as growth factor binding, integrin recognition, and interaction(s) with heparin and proteoglycans (reviewed in Holbourn et al., 2008; Chen and Lau, 2009). The CCN family are involved in many normal and pathological cellular processes and have a plethora of functions including cell proliferation, angiogenesis, wound healing, and fibrogenesis, tumourigenesis. In addition, many roles have been described for CCN family members in the cardiovascular system (Table 1). The focus of this review is the role of connective tissue growth factor (CCN2, CTGF) in blood vessels and in vascular pathology.
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http://dx.doi.org/10.1016/j.vph.2013.01.004DOI Listing
March 2013

Elevated CCN2 expression in scleroderma: a putative role for the TGFβ accessory receptors TGFβRIII and endoglin.

J Cell Commun Signal 2011 Aug 19;5(3):173-7. Epub 2011 Jul 19.

Centre for Rheumatology and Connective Tissue Diseases, UCL Medical School, Royal Free Campus, London, UK, NW3 2PF,

The ability of TGFβ1 to act as a potent pro-fibrotic mediator is well established, potently inducing the expression of fibrogenic genes including type I collagen (COL1A2) and CCN2. Previously we have shown elevated expression of the TGFβ accessory receptor, endoglin on Systemic Sclerosis (SSc) dermal fibroblasts. Here we sought to assess the cell surface expression of the TGFβ receptor complex on SSc dermal fibroblasts (SDF), and investigate their role in maintaining the elevated expression of CCN2. SDF exhibited elevated expression of the TGFβ accessory receptors betaglycan/TGFβRIII and endoglin, but not type I or type II receptors. To determine the effect of altered receptor repertoire on TGFβ responses, we investigated the effect of exogenous TGFβ on expression of two pro-fibrotic genes. SDF exhibited higher basal expression of COL1A2 and CCN2 compared to healthy controls. TGFβ induced a marked increase in the expression of these genes in normal dermal fibroblasts, whereas SDF exhibited only a modest increase. We next sought to determine if higher basal expression in SDF was a result of autocrine expression of TGFβ. Surprisingly basal expression was not affected by a pan-neutralizing TGFβ antibody. To explore if altered accessory receptor expression alone could account for these changes, we determined their effects on CCN2 promoter activity. Endoglin inhibited CCN2 promoter activity in response to TGFβ. TGFβRIII alone or in combination with endoglin was sufficient to enhance basal CCN2 promoter activity. Thus TGFβ accessory receptors may play a significant role in the altered expression of fibrogenic genes in SDF.
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http://dx.doi.org/10.1007/s12079-011-0140-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3145876PMC
August 2011

Pivotal role of connective tissue growth factor in lung fibrosis: MAPK-dependent transcriptional activation of type I collagen.

Arthritis Rheum 2009 Jul;60(7):2142-55

Royal Free and University College Medical School, London, UK.

Objective: Connective tissue growth factor (CTGF; CCN2) is overexpressed in systemic sclerosis (SSc) and has been hypothesized to be a key mediator of the pulmonary fibrosis frequently observed in this disease. CTGF is induced by transforming growth factor beta (TGFbeta) and is a mediator of some profibrotic effects of TGFbeta in vitro. This study was undertaken to investigate the role of CTGF in enhanced expression of type I collagen in bleomycin-induced lung fibrosis, and to delineate the mechanisms of action underlying the effects of CTGF on Col1a2 (collagen gene type I alpha2) in this mouse model and in human pulmonary fibroblasts.

Methods: Transgenic mice that were carrying luciferase and beta-galactosidase reporter genes driven by the Col1a2 enhancer/promoter and the CTGF promoter, respectively, were injected with bleomycin to induce lung fibrosis (or saline as control), and the extracted pulmonary fibroblasts were incubated with CTGF blocking agents. In vitro, transient transfection, promoter/reporter constructs, and electrophoretic mobility shift assays were used to determine the mechanisms of action of CTGF in pulmonary fibroblasts.

Results: In the mouse lung tissue, CTGF expression and promoter activity peaked 1 week after bleomycin challenge, whereas type I collagen expression and Col1a2 promoter activity peaked 2 weeks postchallenge. Fibroblasts isolated from the mouse lungs 14 days after bleomycin treatment retained a profibrotic expression pattern, characterized by greatly elevated levels of type I collagen and CTGF protein and increased promoter activity. In vitro, inhibition of CTGF by specific small interfering RNA and neutralizing antibodies reduced the collagen protein expression and Col1a2 promoter activity. Moreover, in vivo, anti-CTGF antibodies applied after bleomycin challenge significantly reduced the Col1a2 promoter activity by approximately 25%. The enhanced Col1a2 promoter activity in fibroblasts from bleomycin-treated lungs was partly dependent on Smad signaling, whereas CTGF acted on the Col1a2 promoter by a mechanism that was independent of the Smad binding site, but was, instead, dependent on the ERK-1/2 and JNK MAPK pathways. The CTGF effect was mapped to the proximal promoter region surrounding the inverted CCAAT box, possibly involving CREB and c-Jun. In human lung fibroblasts, the human COL1A2 promoter responded in a similar manner, and the mechanisms of action also involved ERK-1/2 and JNK signaling.

Conclusion: Our results clearly define a direct profibrotic effect of CTGF and demonstrate its contribution to lung fibrosis through transcriptional activation of Col1a2. Blocking strategies revealed the signaling mechanisms involved. These findings show CTGF to be a rational target for therapy in fibrotic diseases such as SSc.
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http://dx.doi.org/10.1002/art.24620DOI Listing
July 2009

JunB mediates enhancer/promoter activity of COL1A2 following TGF-beta induction.

Nucleic Acids Res 2009 Sep 26;37(16):5378-89. Epub 2009 Jun 26.

Department of Medicine, Centre for Rheumatology, University College London Royal Free Campus, London NW3 2PF, UK.

Transcriptional control of the genes coding for collagen type I is regulated by a complex interaction between a distal enhancer and a proximal promoter. In this study, we have dissected the molecular mechanism of this interaction by defining a specific sequence within the enhancer that respond in fibroblasts to transforming growth factor-beta (TGF-beta). We show that TGF-beta activates COL1A2 gene via a non-canonical (Smad-independent) signalling pathway, which requires enhancer/promoter co-operation. This interaction involves exchange of cJun/Jun B transcription factor occupancy of a critical enhancer site resulting in the stabilization of enhancer/promoter coalescence. Moreover, using transgenesis, we show that interference in this mechanism results in the abolition of COL1A2 fibroblast expression in vivo. These data are therefore relevant to the control of collagen type I in vivo both in embryonic development, in adult connective tissue homeostasis, and in tissue repair and scarring pathologies.
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http://dx.doi.org/10.1093/nar/gkp544DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2760791PMC
September 2009

Neuronal regulators and vascular dysfunction in Raynaud's phenomenon and systemic sclerosis.

Curr Vasc Pharmacol 2009 Jan;7(1):34-9

Centre for Rheumatology and Connective Tissue Diseases, Royal Free and University College Medical School, Hampstead, London, NW3 2PF, UK.

Raynaud's phenomenon (RP) results from an exaggerated cutaneous vasospastic response to cold or emotional stress. The mechanisms that lead to impaired cutaneous vascular tone are complex. The regulation of cutaneous vasoconstriction and vasodilation, involves altered sympathetic nerve activity and a host of neuronal regulators, including adrenergic and non-adrenergic, as well as REDOX signalling and other signalling such as the RhoA/ROCK pathway. This review summarises the literature concerning the regulation of vascular tone by neurohumoral factors that may be involved in RP and systemic sclerosis (SSc).
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http://dx.doi.org/10.2174/157016109787354105DOI Listing
January 2009

A polymorphism in the CTGF promoter region associated with systemic sclerosis.

N Engl J Med 2007 Sep;357(12):1210-20

Centre for Rheumatology, Royal Free and University College Medical School, London, United Kingdom.

Background: Systemic sclerosis (scleroderma) is a life-threatening autoimmune disease that is characterized by the presence of specific autoantibodies and fibrosis of the skin and major internal organs.

Methods: We genotyped a polymorphism (G-945C) in the promoter of the connective-tissue growth factor (CTGF) gene in 1000 subjects in two groups: group 1, consisting of 200 patients with systemic sclerosis and 188 control subjects; and group 2, consisting of 300 patients with systemic sclerosis and 312 control subjects. The combined groups represented an estimated 10% of patients with systemic sclerosis in the United Kingdom. We tested the effect of the polymorphism on the transcription of CTGF.

Results: The GG genotype was significantly more common in patients with systemic sclerosis than in control subjects in both groups, with an odds ratio for the combined group of 2.2 (95% confidence interval [CI], 1.5 to 3.2; P<0.001 for trend). Analysis of the combined group of patients with systemic sclerosis showed a significant association between homozygosity for the G allele and the presence of anti-topoisomerase I antibodies (odds ratio, 3.3; 95% CI, 2.0 to 5.6; P<0.001) and fibrosing alveolitis (odds ratio, 3.1; 95% CI, 1.9 to 5.0; P<0.001). We observed that the substitution of cytosine for guanine created a binding site of the transcriptional regulators Sp1 and Sp3. The C allele has high affinity for Sp3 and is associated with severely reduced transcriptional activity. A chromatin immunoprecipitation assay showed a marked shift in the ratio of Sp1 to Sp3 binding at this region, demonstrating functional relevance in vivo.

Conclusions: The G-945C substitution represses CTGF transcription, and the -945G allele is significantly associated with susceptibility to systemic sclerosis.
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http://dx.doi.org/10.1056/NEJMoa067655DOI Listing
September 2007

Increased endogenous angiogenic response and hypoxia-inducible factor-1alpha in human critical limb ischemia.

J Vasc Surg 2006 Jan;43(1):125-33

Vascular Unit, Department of Surgery, The Royal Free Hospital, The Royal Free & University College Medical School, University College London, UK.

Objective: The potent physiologic endogenous angiogenic response to ischemic stimuli is often suboptimal, and therefore, a better understanding of the basic mechanisms is essential for the use in therapeutic angiogenesis. Hypoxia-inducible factor-1 (HIF-1) is a major transcription factor that promotes ischemia-driven angiogenesis and is induced when the HIF-1alpha subunit is upregulated. However, little is known about the endogenous angiogenic response and the role of HIF-1alpha in human critical limb ischemia (CLI). We aimed to investigate the extent of the angiogenic response and the expression of HIF-1alpha in the lower limbs of CLI patients.

Method: Skeletal muscle biopsy specimens were obtained from the lower limbs of 12 patients with CLI and 12 patients without limb ischemia (controls), with ethical committee approval. Microvessel density (MVD) was determined by using endothelial marker anti-CD31, and HIF-1alpha expressions were determined by immunohistochemistry. MVD was measured as the median number of microvessels in x200 magnification fields. Five random fields per section and three sections per biopsy specimen were analyzed. Enzyme-linked immunoabsorbent assay and Western blotting were used to quantify the HIF-1alpha levels. Colocalization between cell-specific antigens was investigated by double immunofluorescence labelling by using confocal microscopy. Statistical analyses were performed with the Mann-Whitney U test.

Results: The CLI group have significantly higher MVD, with an increase of 2.7-fold compared with the controls (P < 0.001). HIF-1alpha expression was significantly increased in CLI muscles (P < 0.001) and was localized to vascular endothelial cells.

Conclusions: Our findings suggest that the endogenous angiogenic response occurs in CLI. The increased HIF-1alpha level and colocalization to vascular endothelial cells suggest that HIF-1alpha plays a role in the physiologic endogenous angiogenic response in CLI. Therefore, augmentation of the HIF-1alpha pathway may be an important aspect in therapeutic angiogenesis.
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http://dx.doi.org/10.1016/j.jvs.2005.08.042DOI Listing
January 2006

Chemokine receptor CCR2 expression by systemic sclerosis fibroblasts: evidence for autocrine regulation of myofibroblast differentiation.

Arthritis Rheum 2005 Dec;52(12):3772-82

Royal Free and University College Medical School, London, UK.

Objective: To investigate expression of the chemokine receptor CCR2 on key cell types involved in the pathogenesis of systemic sclerosis (SSc) and to assess the potential for autocrine activation of SSc dermal fibroblasts via CCL2/CCR2.

Methods: Chemokine receptor expression in skin biopsy tissues and explanted dermal fibroblasts from a well-characterized cohort of SSc patients was examined using immunohistochemistry and flow cytometry techniques. Autocrine regulation of the expression of fibrotic markers in CCR2+ SSc fibroblast cell lines was assessed using specific ligand or receptor antagonists.

Results: We identified strong CCR2 expression in skin biopsy samples of early-stage diffuse cutaneous SSc (dcSSc), but not late-stage dcSSc or limited cutaneous SSc. Double labeling confirmed up-regulation of CCL2/CCR2 on myofibroblasts, pericytes, lymphocytes, macrophages, and endothelial cells. Explanted dermal fibroblasts from early dcSSc tissues expressed CCR2 and CXCR2 in 55% and 66% of cell strains, respectively. There was no expression in control fibroblasts. CCR2+ fibroblasts demonstrated a profibrotic phenotype, with overexpression of alpha-smooth muscle actin (alpha-SMA), connective tissue growth factor (CTGF), and CCL2. Flow cytometric analysis identified a subset of CCR2+ SSc fibroblasts expressing the myofibroblast marker alpha-SMA. In these cultures, specific inhibition of CCL2 or CCR2 attenuated the overexpression of alpha-SMA, but not CTGF or plasminogen activator inhibitor 1.

Conclusion: Our results show that CCR2 is up-regulated in early dcSSc on cell types known to be activated in the disease, which is consistent with a key role in SSc pathogenesis. CCR2 expression on SSc fibroblasts appears to regulate the expression of CCL2 and alpha-SMA. Our findings suggest potential autocrine regulation of key profibrotic properties via a CCL2/CCR2 loop in early-stage dcSSc.
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http://dx.doi.org/10.1002/art.21396DOI Listing
December 2005

Connective tissue remodeling: cross-talk between endothelins and matrix metalloproteinases.

Curr Vasc Pharmacol 2005 Oct;3(4):369-79

Centre for Rheumatology and Connective Tissue Diseases, Department of Medicine, Royal Free and University College Medical School, London, UK.

Connective tissue remodeling is achieved by a complex process involving several cell types, a plethora of growth factors, cytokines, chemokines and turnover of extracellular matrix (ECM). The main enzymes that degrade ECM molecules are matrix metalloproteinases (MMPs) and their activities are regulated by endogenous inhibitors, the tissue inhibitors of metalloproteinases (TIMPs). Recent studies have indicated that endothelins and their receptor expression affects tissue remodeling and repair. Endothelins are rapidly produced by endothelial cells in response to tissue injury and they have potent vasoconstrictive properties. They also promote tissue remodeling through activation of resident connective tissue cells and controlling the production of MMPs and TIMPs by the activated cells. In this review we present the cross-talk between the endothelins and the MMP-TIMP system and their implications in controlling the normal and abnormal tissue remodeling.
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http://dx.doi.org/10.2174/157016105774329480DOI Listing
October 2005

Regulation of collagen type I in vascular smooth muscle cells by competition between Nkx2.5 and deltaEF1/ZEB1.

Mol Cell Biol 2004 Jul;24(14):6151-61

Centre for Rheumatology, Department of Medicine, University College of London, United Kingdom.

A major component of the vessel wall of large arteries and veins is the extracellular matrix (ECM), which consists of collagens, elastin, and proteoglycans. Collagen type I is one of the most abundant of the ECM proteins. We have previously shown that the pro-collagen type I alpha 2 gene contains an enhancer which confers tissue-specific expression in the majority of collagen-producing cells, including blood vessels. In this paper, we delineate a specific vascular smooth muscle cell (vSMC) element: a 100-bp sequence around -16.6 kb upstream of the transcription start site that regulates collagen expression exclusively in vSMCs. Furthermore, we show that the expression is activated through the binding of the homeodomain protein Nkx2.5, which is further potentiated in the presence of GATA6. In contrast, this element was repressed by the binding of the zinc-finger protein deltaEF1/ZEB1. We propose a model of regulation where the activating transcription factor Nkx2.5 and the repressor deltaEF1/ZEB1 compete for an overlapping DNA binding site. This element is important in understanding the molecular mechanisms of vessel remodeling and is a potential target for intervention in vascular diseases where there is excessive deposition of collagen in the vessel wall.
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http://dx.doi.org/10.1128/MCB.24.14.6151-6161.2004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC434261PMC
July 2004

Extra-cellular matrix in vascular networks.

Cell Prolif 2004 Jun;37(3):207-20

Renal Medicine, Imperial College London, Hammersmith Campus, London, W12 ONN, UK.

The vascular network is a series of linked conduits of blood vessels composed of the endothelium, a monolayer of cells that adorn the vessel lumen and surrounding layer(s) of mesenchymal cells (vascular smooth muscle, pericytes and fibroblasts). In addition to providing structural support, the mesenchymal cells are essential for vessel contractility. The extracellular matrix is a major constituent of blood vessels and provides a framework in which these various cell types are attached and embedded. The composition and organization of vascular extracellular matrix is primarily controlled by the mesenchymal cells, and is also responsible for the mechanical properties of the vessel wall, forming complex networks of structural proteins which are highly regulated. The extracellular matrix also plays a central role in cellular adhesion, differentiation and proliferation. This review examines the cellular and extracellular matrix components of vessels, with specific emphasis on the regulation of collagen type I and implications in vascular disease.
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http://dx.doi.org/10.1111/j.1365-2184.2004.00306.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6496925PMC
June 2004

Col1a2 enhancer regulates collagen activity during development and in adult tissue repair.

Matrix Biol 2004 Feb;22(8):619-28

Muscle Cell Biology Group, MRC Clinical Science Centre, Imperial College, Hammersmith Hospital Campus, Du Cane Road, London W12 0NN, UK.

An enhancer region in the type I collagen alpha 2 chain (pro-Col1a2) promoter has been previously identified approximately -17 kb away from the transcription start site. This upstream region termed the far-upstream-enhancer contains three DNAse I hypersensitive sites and has been shown to be conserved between mouse and human genes. In this study, we used transgenic mice harbouring the complete promotor sequence of the pro-Col1a2 gene up to -17 kb to examine the role of this enhancer in the expression and regulation of the collagen gene during development and in adult tissues pre and post injury. By careful histological mapping of the collagen type I endogenous gene distribution with that of the transgene driven by the mouse far upstream enhancer, we are able to show that in early days of collagen expression, E8.5-9.5, the endogenous gene preceded transgene expression. However, by E11.5 the overall pattern becomes synchronous with a few exceptions. In adult tissue, both endogenous and transgene expression are attenuated and both are reactivated in parallel in various organs by physical injury or fibrogenic cytokine injection. These findings suggest that the enhancer is central to the activation of the collagen type I and that mice harbouring this enhancer/reporter provide a useful model to follow collagen gene transcription activity and for investigating cellular activity in tissue fibrosis.
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http://dx.doi.org/10.1016/j.matbio.2003.12.002DOI Listing
February 2004

Prostacyclin derivatives prevent the fibrotic response to TGF-beta by inhibiting the Ras/MEK/ERK pathway.

FASEB J 2002 Dec 4;16(14):1949-51. Epub 2002 Oct 4.

Centre for Rheumatology, Royal Free Hospital and University College School of Medicine, London NW3 2PF, UK.

The SMAD-mediated induction of connective tissue growth factor (CTGF), a fibroproliferative cytokine, by transforming growth factor (TGF)beta is required for the development of sustained fibrosis in humans. Here, we show that in fibroblasts, activation of the Ras/MEK/ERK pathway is required for the SMAD-mediated induction of CTGF by TGFbeta2. We then show that activation of protein kinase A (PKA) in fibroblasts is able to block Ras/MEK/ERK signaling and abolish the fibrotic response. Previously, we found that prostacyclin agonists were able to prevent the induction of CTGF in fibroblasts, and in patients with the fibrotic disease scleroderma. Here, we confirm the in vitro and in vivo antifibrotic effects of prostacyclin derivatives and show that these effects are due to PKA-dependent inhibition of the Ras/MEK/ERK pathway. Ras/MEK/ERK does not directly affect SMAD signaling. The coordinate and varied biological responses to TGFbeta are in part due to the interactions of signaling pathways within target cells. Specific inhibition of fibroblast Ras/MEK/ERK signaling might prevent fibrosis while leaving other physiological effects of TGFbeta unaltered.
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http://dx.doi.org/10.1096/fj.02-0204fjeDOI Listing
December 2002

Identification of the key regions within the mouse pro-alpha 2(I) collagen gene far-upstream enhancer.

J Biol Chem 2002 Mar 27;277(11):9286-92. Epub 2001 Dec 27.

Medical Research Council Clinical Sciences Centre, Imperial College, Hammersmith Campus, London W12 ONN, United Kingdom.

Studies using transgenic mice have shown that the mouse pro-alpha2(I) collagen gene contains a far-upstream enhancer, which directs expression in the majority of collagen I-producing cells during development and in response to tissue injury. In this study, we have investigated the minimal functional region required for the enhancer effect and studied the role of the three hypersensitive sites (HS3-HS5) that overlap this region. The results of deletion experiments indicate that the minimal functional unit of this enhancer is a 1.5-kb region between -17.0 and -15.45 kb from the transcription start site. This region includes the core sequences of HS3 and HS4 but not HS5. The HS4 sequences are essential for the functional integrity of the enhancer, whereas HS3 represents tissue-specific elements that direct expression in mesenchymal cells of internal tissues and body wall muscles. The HS3 region appears to bind a complex of transcription factors illustrated by large regions of protected sequences. A 400-bp sequence located between -17.0 and -16.6 is also essential for the enhancer because its deletion results in increased susceptibility to the chromatin environment.
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http://dx.doi.org/10.1074/jbc.M111040200DOI Listing
March 2002