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1.  A synthetic PPAR-γ agonist triterpenoid ameliorates experimental fibrosis: PPAR-γ-independent suppression of fibrotic responses 
Annals of the rheumatic diseases  2013;73(2):446-454.
Background
Persistent fibroblast activation initiated by transforming growth factor β (TGF-β) is a fundamental event in the pathogenesis of systemic sclerosis (SSc), and its pharmacological inhibition represents a potential therapeutic strategy. The nuclear receptor peroxisome proliferator-activated receptor-gamma (PPAR-γ) exerts potent fibrotic activities. The synthetic triterpenoid oleanane 2-cyano-3,12-dioxoolean-1,9-dien-28-oic (CDDO) is a PPAR-γ agonist with potential effects on TGF-β signaling and dermal fibrosis.
Objective
To examine the modulation of fibrogenesis by CDDO in explanted fibroblasts, skin organ cultures and murine models of scleroderma.
Material and methods
The effects of CDDO on experimental fibrosis induced by bleomycin injection or by overexpression of type I constitutively active TGF-β receptor was evaluated. Modulation of fibrotic gene expression was examined in human skin organ cultures. To delineate the mechanisms underlying the anti-fibrotic effects of CDDO, explanted skin fibroblasts cultured in 2-dimensional monolayers or in 3-dimensional full-thickness human skin equivelants were studied.
Results
CDDO significantly ameliorated dermal fibrosis in two complementary mouse models of scleroderma, as well as in human skin organ cultures and in 3-dimensional human skin equivalents. In 2-dimensional monolayer cultures, CDDO abrogated fibrogenic responses in explanted normal human skin fibroblasts. These CDDO effects occurred via disruption of Smad-dependent transcription and were associated with inhibition of Akt activation. In scleroderma fibroblasts, CDDO attenuated collagen synthesis. Remarkably, the anti-fibrotic effects of CDDO were independent of PPAR-γ.
Conclusion
The PPAR-γ agonist triterpenoid CDDO attenuates fibrogenesis by antagonistically targeting canonical TGF-β/Smad and Akt signaling in a PPAR-γ-independent manner. These findings identify this synthetic triterpenoid as a potential new therapy for the control of fibrosis.
doi:10.1136/annrheumdis-2012-202716
PMCID: PMC4028127  PMID: 23515440
CDDO; triterpenoid; fibrosis; PPAR-γ; TGF-β; fibroblast; murine scleroderma
2.  Smad-independent transforming growth factor-ß signaling in fibroblasts via c-Abl and Egr-1: selective modulation by imatinib mesylate 
Oncogene  2009;28(10):1285-1297.
The non-receptor protein tyrosine kinase c-Abl regulates cell proliferation and survival. Recent studies provide evidence that implicate c-Abl as a mediator for fibrotic responses induced by Transforming growth factor-ß (TGF-ß), but the precise mechanisms underlying this novel oncogene function are unknown. Here we report that when expressed in normal fibroblasts, a constitutively active mutant of Abl mutant that causes chronic myelogenous leukemia stimulated the expression and transcriptional activity of the early growth response factor Egr-1. Mouse embryonic fibroblasts lacking c-Abl were resistant to TGF-ß. Sensitivity of these cells to TGF-ß could be rescued by wildtype c-Abl, but not by a kinase-deficient mutant form of c-Abl. Furthermore, Abl kinase activity was necessary for the induction of Egr-1 by TGF-ß in normal fibroblasts, and Egr-1 was required for stimulation of collagen by Bcr-Abl. Lesional skin fibroblasts in mice with bleomycin-induced scleroderma displayed evidence of c-Abl activation in situ, and elevated phospho-c-Abl correlated with increased local expression of Egr-1. Collectively, these results position Egr-1 downstream of c-Abl in the fibrotic response, delineate a novel Egr-1-dependent intracellular signaling mechanism that underlies the involvement of c-Abl in TGF-ß responses, and identify Egr-1 as a target of inhibition by imatinib. Furthermore, the findings demonstrate in situ activation of c-Abl paralleling the up-regulation tissue expression of Egr-1 in fibrosis. Pharmacological targeting of c-Abl and its downstream effector pathways may therefore represent a novel therapeutic approach to blocking TGF-ß-dependent fibrotic processes.
doi:10.1038/onc.2008.479
PMCID: PMC4006376  PMID: 19151753
c-Abl; imatinib mesylate; TGF-ß; Egr-1; fibrosis; fibroblast; Type I collagen
3.  Fibrosis in systemic sclerosis: Emerging concepts and implications for targeted therapy 
Autoimmunity reviews  2010;10(5):267-275.
Systemic sclerosis (SSc) is a complex and incompletely understood disease associated with fibrosis in multiple organs. Recent findings identify transforming growth factor-ß (TGF-ß), Wnt ligands, toll-like receptor-mediated signaling, hypoxia, type I interferon, type 2 immune responses and mechanical stress as extracellular cues that modulate fibroblast function and differentiation, and as potential targets for therapy. Moreover, fibrillin-1 has a major role in storing and regulating the bioavailability of TGF-ß and other cytokines, and fibrillin-1 mutations are implicated in a congenital form of scleroderma called stiff skin syndrome. Fibrosis is due not only to the activation of tissue-resident fibroblasts and their transdifferentiation into myofibroblasts, but also the differentiation of bone marrow-derived fibrocytes, and transition of endothelial and epithelial cells, pericytes and adipocytes into activated mesenchymal cells. These responses are modulated by signaling mediators and microRNAs that amplify or inhibit TGF-ß and Wnt signaling. Gain-of-function and loss-of-function abnormalities of these mediators may account for the characteristic activated phenotype of SSc fibroblasts. The nuclear orphan receptor PPAR-γ plays a particularly important role in limiting the duration and intensity of fibroblast activation and differentiation, and impaired PPAR-γ expression or function in SSc may underlie the uncontrolled progression of fibrosis.
Identifying the perturbations in signaling pathways, mediators and differentiation programs that are responsible for SSc tissue damage allows their selective targeting. This in turn opens the door for therapies utilizing novel compounds, or drug repurposing by innovative uses of already-approved drugs. In view of the heterogeneous clinical presentation and unpredictable course of SSc, as well as its complex pathogenesis, only robust clinical trials incorporating the judicious application of biomarkers will be able to clarify the clinical utility of these innovative approaches.
doi:10.1016/j.autrev.2010.09.015
PMCID: PMC3998379  PMID: 20863909
Fibrosis; Systemic sclerosis; Fibroblast; TGF-ß; Wnt; Collagen
4.  Egr-1: new conductor for the tissue repair orchestra directs harmony (regeneration) or cacophony (fibrosis) 
The Journal of pathology  2012;229(2):286-297.
Fibroblasts and myofibroblasts are the key effector cells executing physiologic tissue repair leading to regeneration on one hand, and pathological fibrogenesis leading to chronic fibrosing conditions on the other. Recent studies identify the multifunctional transcription factor Early Growth Response-1(Egr-1) as an important mediator of fibroblast activation triggered by diverse stimuli. Egr-1 has potent stimulatory effects on fibrotic gene expression, and aberrant Egr-1 expression or function is associated with animal models of fibrosis and human fibrotic disorders including emphysema, pulmonary fibrosis, pulmonary hypertension and systemic sclerosis. Pharmacological suppression or genetic targeting of Egr-1 blocks fibrotic responses in vitro and ameliorates experimental fibrosis in the skin and lung. In contrast, Egr-1 appear to acts as a negative regulator of hepatic fibrosis in mouse models, suggesting a context-dependent role in fibrosis. The Egr-1-binding protein Nab2 is an endogenous inhibitor of Egr-1-mediated signaling, and abrogates the stimulation of fibrotic responses induced by transforming growth factor-ß (TGF-ß). Moreover, mice deficient in Nab2 show excessive collagen accumulation in the skin. These observations highlight a previously unsuspected fundamental physiologic function for the Egr-1/Nab2 signaling axis in regulating fibrogenesis, and suggest that Egr-1 may be a potential novel therapeutic target in human diseases complicated by fibrosis. This review summarizes recent advances in understanding the regulation and complex functional role of Egr-1 and its related proteins and inhibitors in pathological fibrosis.
doi:10.1002/path.4131
PMCID: PMC3965177  PMID: 23132749
Egr-1; Nab2; TGF-ß; fibrosis; scleroderma; fibroblast; myofibroblast; p300; c-Abl
5.  Connective tissue growth factor/CCN2-null mouse embryonic fibroblasts retain intact transforming growth factor-beta responsiveness 
Experimental cell research  2007;314(5):1094-1104.
Background
The matricellular protein connective tissue growth factor (CCN2) has been implicated in pathological fibrosis, but its physiologic role remains elusive. In vitro, transforming growth factor-β (TGF-β) induces CCN2 expression in mesenchymal cells. Because CCN2 can enhance pro-fibrotic responses elicited by TGF-β, it has been proposed that CCN2 functions as an essential downstream signaling mediator for TGF-β. To explore this notion, we characterized TGF-β-induced activation of fibroblasts from CCN2-null (CCN2−/−) mouse embryos.
Methods
The regulation of CCN2 expression was examined in vivo in a model of fibrosis induced by bleomycin. Cellular TGF-β signal transduction and regulation of collagen gene expression were examined in CCN2−/− MEFs by immunohistochemistry, Northern, Western and RT-PCR analysis, immunocytochemistry and transient transfection assays.
Results
Bleomycin-induced skin fibrosis in the mouse was associated with substantial CCN2 up-regulation in lesional fibroblasts. Whereas in vitro proliferation rate of CCN2−/− MEFs was markedly reduced compared to wild type MEFs, TGF-β-induced activation of the Smad pathways, including Smad2 phosphorylation, Smad2/3 and Smad4 nuclear accumulation and Smad-dependent transcriptional responses, were unaffected by loss of CCN2. The stimulation of COL1A2 and fibronectin mRNA expression and promoter activity, and of corresponding protein levels, showed comparable time and dose-response in wild type and CCN2−/− MEFs, whereas stimulation of alpha-smooth muscle actin and myofibroblast transdifferentiation showed subtle impairment in MEFs lacking CCN2.
Conclusion
Whereas endogenous CCN2 plays a role in regulation of proliferation and TGF-β-induced myofibroblast transdifferentation, it appears to be dispensable for Smad-dependent stimulation of collagen and extracellular matrix synthesis in murine embryonic fibroblasts.
doi:10.1016/j.yexcr.2007.12.010
PMCID: PMC3963386  PMID: 18201696
TGF-β; CTGF/CCN2; fibrosis; fibroblast; Type I collagen
6.  Transforming growth factor-ß as a therapeutic target in systemic sclerosis 
Nature reviews. Rheumatology  2009;5(4):200-206.
SUMMARY
Transforming growth factor-ß (TGF-ß) is a pleiotropic cytokine with vital homeostatic functions. Aberrant TGF-ß expression or activity is implicated in the pathogenesis of fibrosis in patients with systemic sclerosis (SSc), thus TGF-ß represents a molecular therapeutic target. Multiple strategies are available for blocking the TGF-ß pathway. A monoclonal antibody targeting TGF-ß has been evaluated in a small clinical trial for early SSc with disappointing results. Antibodies to the αvß6 integrin that prevent latent TGF-ß activation, however, show promise in preclinical studies. Small molecules inhibiting TGF-ß receptor activity are effective in animal models of fibrosis. Imatinib mesylate and related tyrosine kinase inhibitors that are currently used in cancer therapy also block TGF-ß pathways and abrogate fibrotic responses. Furthermore, some commonly used drugs have shown unanticipated anti-TGF-ß activity and, therefore, could have anti-fibrotic effects. Since TGF-ß has important physiologic functions for tissue homeostasis, blocking TGF-ß activity might lead to spontaneous immune activation, epithelial hyperplasia and impaired wound healing. Loss of immune tolerance is a potential concern in an autoimmune disease such as SSc. Novel insights from microarray-based gene expression analysis and studies of genetic polymorphisms in TGF-ß signaling could aid in identifying those patients who are most likely to respond to anti-TGF-ß treatment. Anti-TGF-ß interventions promise to have a major impact on the treatment of SSc. Significant concerns regarding efficacy, safety, questions regarding appropriate dosing and timing of therapy, identification of responders, and of biomarkers of safety and efficacy are critical challenges ahead.
doi:10.1038/nrrheum.2009.26
PMCID: PMC3959159  PMID: 19337284
7.  Understanding fibrosis in systemic sclerosis: shifting paradigms, emerging opportunities 
Nature reviews. Rheumatology  2011;8(1):42-54.
Fibrosis in multiple organs is a prominent pathological finding and distinguishing hallmark of systemic sclerosis (SSc). Findings during the past 5 years have contributed to a more complete understanding of the complex cellular and molecular underpinning of fibrosis in SSc. Fibroblasts, the principal effector cells, are activated in the profibrotic cellular milieu by cytokines and growth factors, developmental pathways, endothelin 1 and thrombin. Innate immune signaling via Toll-like receptors, matrix-generated biomechanical stress signaling via integrins, hypoxia and oxidative stress seem to be implicated in perpetuating the process. Beyond chronic fibroblast activation, fibrosis represents a failure to terminate tissue repair, coupled with an expanded population of mesenchymal cells originating from bone marrow and transdifferentiation of epithelial cells, endothelial cells and pericytes. In addition, studies have identified intrinsic alterations in SSc fibroblasts resulting from epigenetic changes, as well as altered microRNA expression that might underlie the cell-autonomous, persistent activation phenotype of these cells. Precise characterization of the deregulated extracellular and intracellular signaling pathways, mediators and cellular differentiation programs that contribute to fibrosis in SSc will facilitate the development of selective, targeted therapeutic strategies. Effective antifibrotic therapy will ultimately involve novel compounds and repurposing of drugs that are already approved for other indications.
doi:10.1038/nrrheum.2011.149
PMCID: PMC3954787  PMID: 22025123
8.  Molecular Signatures in Skin Associated with Clinical Improvement During Mycophenolate Treatment in Systemic Sclerosis 
Heterogeneity in systemic sclerosis/SSc confounds clinical trials. We previously identified ‘intrinsic’ gene expression subsets by analysis of SSc skin. Here we test the hypotheses that skin gene expression signatures including intrinsic subset are associated with skin score/MRSS improvement during mycophenolate mofetil (MMF) treatment. Gene expression and intrinsic subset assignment were measured in 12 SSc patients’ biopsies and ten controls at baseline, and from serial biopsies of one cyclophosphamide-treated patient, and nine MMF-treated patients. Gene expression changes during treatment were determined using paired t-tests corrected for multiple hypothesis testing. MRSS improved in four of seven MMF-treated patients classified as the inflammatory intrinsic subset. Three patients without MRSS improvement were classified as normal-like or fibroproliferative intrinsic subsets. 321 genes (FDR <5%) were differentially expressed at baseline between patients with and without MRSS improvement during treatment. Expression of 571 genes (FDR <10%) changed between pre- and post-MMF treatment biopsies for patients demonstrating MRSS improvement. Gene expression changes in skin are only seen in patients with MRSS improvement. Baseline gene expression in skin, including intrinsic subset assignment, may identify SSc patients whose MRSS will improve during MMF treatment, suggesting that gene expression in skin may allow targeted treatment in SSc.
doi:10.1038/jid.2013.130
PMCID: PMC3714324  PMID: 23677167
9.  A TGFβ-Responsive Gene Signature Is Associated with a Subset of Diffuse Scleroderma with Increased Disease Severity 
The Journal of investigative dermatology  2009;130(3):10.1038/jid.2009.318.
Systemic sclerosis is a complex disease with widespread skin fibrosis and variable visceral organ involvement. Since transforming growth factor-β (TGFβ) has been implicated in driving fibrosis in systemic sclerosis, a mechanism-derived gene expression signature was used to assay TGFβ-responsive gene expression in the skin of patients with systemic sclerosis (SSc). Primary dermal fibroblasts from patients with diffuse SSc (dSSc) and healthy controls were treated with TGFβ, and the genome-wide gene expression was measured on DNA microarrays over a time course of 24 hours. Eight hundred and ninety-four probes representing 674 uniquely annotated genes were identified as TGFβ responsive. Expression of the TGFβ-responsive signature was examined in skin biopsies from 17 dSSc, seven limited SSc (lSSc), three morphea patients, and six healthy controls. The TGFβ-responsive signature was expressed in 10 out of 17 dSSc skin biopsies, but was not found in lSSc, morphea, or healthy control biopsies. Expression of dSSC the TGFβ-responsive signature stratifies patients into two major groups, one of which corresponds to the “diffuse-proliferation” intrinsic subset that showed higher modified Rodnan skin score and a higher likelihood of scleroderma lung disease. The TGFβ-responsive signature is found in only a subset of dSSc patients who could be targeted by specific therapies.
doi:10.1038/jid.2009.318
PMCID: PMC3867816  PMID: 19812599
10.  Imatinib Mesylate Causes Genome-wide Transcriptional Changes in Systemic Sclerosis Fibroblasts in vitro 
Clinical and experimental rheumatology  2012;30(2 0 71):S86-S96.
Objective
Systemic sclerosis (SSc) is a heterogeneous multifactorial disease dominated by progressive skin and internal organ fibrosis that is driven in part by Transforming Growth Factor-beta (TGF-β). An important downstream target of TGF-β is the Abelson (c-Abl) tyrosine kinase, and its inhibition by imatinib mesylate (Gleevec)attenuates fibrosis in mice. Here we examined the effect of c-Abl activation and blockade in explanted healthy control and SSc fibroblasts.
Methods
Skin biopsies and explanted fibroblasts from healthy subjects and patients with SSc were studied. Changes in genome-wide expression patterns in imatinib-treated control and SSc fibroblasts were analyzed by DNA microarray.
Results
Treatment of control fibroblasts with TGF-β resulted in activation of c-Abl and stimulation of fibrotic gene expression that was prevented by imatinib. Moreover, imatinib reduced basal collagen gene expression in SSc but not control fibroblasts. No significant differences in tissue levels of c-Abl and phospho-c-Abl were detected between SSc and control skin biopsies. In vitroimatinib induced dramatic changes in the expression of genes involved in fibrosis, cardiovascular disease, inflammation, and lipid and cholesterol metabolism. Remarkably, of the 587-imatinib-responsive genes, 91% showed significant change in SSc fibroblasts, but only 12% in control fibroblasts.
Conclusion
c-Abl plays a key role in fibrotic responses. Imatinib treatment results in dramatic changes in gene expression in SSc fibroblasts but has only modest effects in control fibroblasts. These data provide novel insights into the mechanisms underlying the antifibrotic effect of imatinib in SSc.
PMCID: PMC3860597  PMID: 22691216
11.  Post-epidemic eosinophilia myalgia syndrome associated with L-Tryptophan 
Arthritis and rheumatism  2011;63(11):10.1002/art.30514.
Eosinophilia–myalgia syndrome (EMS) is characterized by subacute onset of myalgias and peripheral eosinophilia, followed by chronic neuropathy and skin induration. An epidemic of EMS in 1989 was linked to L-tryptophan consumption originating from a single source. Following the Food and Drug Administration (FDA) ban on the sale of L-tryptophan, the incidence of EMS declined rapidly. Moreover, no new cases have been published since the FDA ban was lifted in 2005. We report the clinical, histopathological and immunogenetic features of a new case of L-tryptophan-associated EMS along with evidence of activated transforming growth factor-ß and interleukin-4 signaling in the lesional skin.
doi:10.1002/art.30514
PMCID: PMC3848710  PMID: 21702023
12.  p300 is elevated in systemic sclerosis and its expression is positively regulated by TGF-β: epigenetic feed-forward amplification of fibrosis 
Fibrosis, the hallmark of systemic sclerosis (SSc), is characterized by persistent fibroblast activation triggered by transforming growth factor-β (TGF-β). Since the acetyltransferase p300 plays a key role in fibrosis and its availability governs the intensity of fibrotic responses, we investigated p300 expression in SSc and the molecular basis of its regulation. We found that expression of p300 was markedly elevated in SSc skin biopsies, and was induced by TGF-β in explanted normal skin fibroblasts. Stimulation of p300 by TGF-β was independent of Smads, and involved the early-immediate transcription factor Egr-1, a key regulator of profibrotic TGF-β signaling. Indeed, Egr-1 was both sufficient and necessary for p300 regulation in vitro and in vivo. Increased p300 accumulation in TGF-β-treated fibroblasts was associated with histone hyperacetylation, whereas p300 depletion, or selective pharmacological blockade of its acetyltransferase activity, attenuated TGF-β-induced responses. Moreover, TGF-β enhanced both p300 recruitment and in vivo histone H4 acetylation at the COL1A2 locus. These findings implicate p300-mediated histone acetylation as a fundamental epigenetic mechanism in fibrogenesis, and place Egr-1 upstream in TGF-β-driven stimulation of p300 gene expression. The results establish a firm link between fibrosis with aberrant p300 expression and epigenetic activity to our knowledge previously unreported. Targeted disruption of p300-mediated histone acetylation might therefore represent a viable anti-fibrotic strategy.
doi:10.1038/jid.2012.479
PMCID: PMC3626729  PMID: 23303459
Acetyltransferase p300; TGF-β; fibroblast; systemic sclerosis; fibrosis; EGR-1; epigenetics
13.  Draft Genome Sequences of Burkholderia cenocepacia ET12 Lineage Strains K56-2 and BC7 
Genome Announcements  2013;1(5):e00841-13.
The Burkholderia cepacia complex (BCC) is a group of closely related bacteria that are responsible for respiratory infections in immunocompromised humans, most notably those with cystic fibrosis (CF). We report the genome sequences for Burkholderia cenocepacia ET12 lineage CF isolates K56-2 and BC7.
doi:10.1128/genomeA.00841-13
PMCID: PMC3798455  PMID: 24136849
14.  Distinct human antibody response to the biological warfare agent Burkholderia mallei 
Virulence  2012;3(6):510-514.
The genetic similarity between Burkholderia mallei (glanders) and Burkholderia pseudomallei (melioidosis) had led to the general assumption that pathogenesis of each bacterium would be similar. In 2000, the first human case of glanders in North America since 1945 was reported in a microbiology laboratory worker. Leveraging the availability of pre-exposure sera for this individual and employing the same well-characterized protein array platform that has been previously used to study a large cohort of melioidosis patients in southeast Asia, we describe the antibody response in a human with glanders. Analysis of 156 peptides present on the array revealed antibodies against 17 peptides with a > 2-fold increase in this infection. Unexpectedly, when the glanders data were compared with a previous data set from B. pseudomallei infections, there were only two highly increased antibodies shared between these two infections. These findings have implications in the diagnosis and treatment of B. mallei and B. pseudomallei infections.
doi:10.4161/viru.22056
PMCID: PMC3524150  PMID: 23076276
Burkholderia mallei; Burkholderia pseudomallei; glanders; melioidosis; protein microarray
15.  The MUC5B Variant Is Associated with Idiopathic Pulmonary Fibrosis but Not with Systemic Sclerosis Interstitial Lung Disease in the European Caucasian Population 
PLoS ONE  2013;8(8):e70621.
A polymorphism on the MUC5B promoter (rs35705950) has been associated with idiopathic pulmonary fibrosis (IPF) but not with systemic sclerosis (SSc) with interstitial lung disease (ILD). We genotyped the MUC5B promoter in the first 142 patients of the French national prospective cohort of IPF, in 981 French patients with SSc (346 ILD), 598 Italian patients with SSc (207 ILD), 1383 French controls and 494 Italian controls. A meta-analysis was performed including all American data available. The T risk allele was present in 41.9% of the IPF patients, 10.8% of the controls (P = 2×10–44), OR 6.3 [4.6–8.7] for heterozygous patients and OR 21.7 [10.4–45.3] for homozygous patients. Prevalence of the T allele was not modified according to age, gender, smoking in IPF patients. However, none of the black patients with IPF presented the T allele. The prevalence of the T risk allele was similar between French (10%) and Italian (12%) cohorts of SSc whatever the presence of an ILD (11.1% and 13.5%, respectively). Meta-analysis confirmed the similarity between French, Italian and American cohorts of IPF or SSc-ILD. This study confirms 1) an association between the T allele risk and IPF, 2) an absence of association with SSc-ILD, suggesting different pathophysiology.
doi:10.1371/journal.pone.0070621
PMCID: PMC3734256  PMID: 23940607
16.  Wnt/β-catenin signaling is hyperactivated in systemic sclerosis and induces Smad-dependent fibrotic responses in mesenchymal cells 
Arthritis and rheumatism  2012;64(8):2734-2745.
Introduction
Fibrosis in human diseases and animal models is associated with aberrant Wnt/β-catenin pathway activation. The regulation, activity, mechanism of action and significance of Wnt/β-catenin signaling in the context of systemic sclerosis (SSc) has not been characterized.
Methods
Expression of Wnt signaling pathway components in SSc skin biopsies was analyzed. The regulation of profibrotic responses by canonical Wnt/ß-catenin was examined in explanted human mesenchymal cells. Fibrotic responses were studied by proliferation, migration and gel contraction assays. The fate specification of subcutaneous preadipocytes by canonical Wnt signaling was evaluated.
Results
Analysis of published genome-wide expression datasets revealed elevated expression of the Wnt receptor Fzd2 and the Wnt target Lef1, and decreased expression of Wnt antagonists Dkk2 and Wif1 in skin biopsies from subsets of dcSSc patients. Immunohistochemistry showed increased nuclear β-catenin expression in these biopsies. In vitro, Wnt3a induced ß-catenin activation, stimulated fibroblast proliferation, migration, gel contraction and myofibroblast differentiation, and profibrotic gene expression. Genetic and pharmacological approaches were used to demonstrate that these profibrotic responses involved autocrine TGF-β signaling via Smads. In contrast, in explanted subcutaneous preadipocytes Wnt3a repressed adipogenesis and promoted myofibroblast differentiation.
Conclusions
Canonical Wnt signaling was hyperactivated in SSc skin biopsies, and in explanted mesenchymal cells Wnt3a stimulated fibrogenic responses while suppressing adipogenesis. Together, these results indicate that Wnts have potent profibrotic effects and canonical Wnt signaling plays an important role in the pathogenesis of fibrosis and lipoatrophy in SSc.
doi:10.1002/art.34424
PMCID: PMC3553791  PMID: 22328118
17.  Draft Genome Sequence Determination for Cystic Fibrosis and Chronic Granulomatous Disease Burkholderia multivorans Isolates 
Journal of Bacteriology  2012;194(22):6356-6357.
Burkholderia multivorans is a Gram-negative bacterium and a member of the Burkholderia cepacia complex, which is frequently associated with respiratory infections in people with cystic fibrosis (CF) and chronic granulomatous disease (CGD). We are reporting the genome sequences of 4 B. multivorans strains, 2 from CF patients and 2 from CGD patients.
doi:10.1128/JB.01306-12
PMCID: PMC3486389  PMID: 23105085
18.  Proteasomal inhibition after injury prevents fibrosis by modulating TGF-β1 signalling 
Thorax  2011;67(2):139-146.
Background
The development of organ fibrosis after injury requires activation of transforming growth factor β1 which regulates the transcription of profibrotic genes. The systemic administration of a proteasomal inhibitor has been reported to prevent the development of fibrosis in the liver, kidney and bone marrow. It is hypothesised that proteasomal inhibition would prevent lung and skin fibrosis after injury by inhibiting TGF-β1-mediated transcription.
Methods
Bortezomib, a small molecule proteasome inhibitor in widespread clinical use, was administered to mice beginning 7 days after the intratracheal or intradermal administration of bleomycin and lung and skin fibrosis was measured after 21 or 40 days, respectively. To examine the mechanism of this protection, bortezomib was administered to primary normal lung fibroblasts and primary lung and skin fibroblasts obtained from patients with idiopathic pulmonary fibrosis and scleroderma, respectively.
Results
Bortezomib promoted normal repair and prevented lung and skin fibrosis when administered beginning 7 days after the initiation of bleomycin. In primary human lung fibroblasts from normal individuals and patients with idiopathic pulmonary fibrosis and in skin fibroblasts from a patient with scleroderma, bortezomib inhibited TGF-β1-mediated target gene expression by inhibiting transcription induced by activated Smads. An increase in the abundance and activity of the nuclear hormone receptor PPARγ, a repressor of Smad-mediated transcription, contributed to this response.
Conclusions
Proteasomal inhibition prevents lung and skin fibrosis after injury in part by increasing the abundance and activity of PPARγ. Proteasomal inhibition may offer a novel therapeutic alternative in patients with dysregulated tissue repair and fibrosis.
doi:10.1136/thoraxjnl-2011-200717
PMCID: PMC3595535  PMID: 21921091
20.  Prevalence, Prognosis, and Factors Associated with Left Ventricular Diastolic Dysfunction in Systemic Sclerosis 
Clinical and experimental rheumatology  2012;30(2 Suppl 71):S30-S37.
Objectives
To determine the frequency, associated clinical factors, and prognostic significance of left ventricular (LV) diastolic dysfunction in systemic sclerosis (SSc).
Methods
We studied 153 consecutive patients with SSc and divided the study sample into those with and without LV diastolic dysfunction using established age-based normal cut-offs for lateral tissue Doppler early mitral annular (E’) velocity, a marker of impaired relaxation and diastolic dysfunction. We compared clinical characteristics, laboratory tests, pulmonary function tests, and echocardiographic data between those with and without LV diastolic dysfunction. We used multivariable linear regression analyses to determine the factors most associated with lateral tissue Doppler E’ velocity. We also performed multivariable Cox regression analyses to determine whether or not tissue Doppler E’ velocity was independently associated with mortality.
Results
LV diastolic dysfunction was present in 23% of the subjects whereas LV systolic dysfunction was present in 5.2% of subjects. Factors independently associated with LV diastolic dysfunction on multivariable analysis included SSc disease duration, age, coronary artery disease, and systemic hypertension. During a mean follow-up of 1.9±1.3 years LV diastolic dysfunction was independently associated with increased risk of death (hazard ratio [HR] 3.2, 95% confidence interval [CI] 1.1–9.5, p=0.034 per each standard deviation decrease in tissue Doppler E’ velocity).
Conclusions
LV diastolic dysfunction in SSc is independently associated with disease duration and is a marker of increased risk of death.
PMCID: PMC3507505  PMID: 22338601
systemic sclerosis; diastolic dysfunction; echocardiography; mortality
21.  Validity of Two New Patient Reported Outcome Measures in Systemic Sclerosis: the PROMIS-29 Profile and the FACIT-Dyspnea 
Arthritis care & research  2011;63(11):1620-1628.
Objective
Many patient-reported outcome (PRO) instruments used in systemic sclerosis (SSc) trials are limited by lack of validation, licensing fees, and complicated scoring systems. We assessed the construct validity for discriminative purposes of two new PRO instruments-the Patient Reported Outcomes Measurement Information System 29-item Health Profile (PROMIS-29) and the Functional Assessment of Chronic Illness Therapy-Dyspnea short form (FACIT-Dyspnea), measuring health status and dyspnea in SSc patients.
Methods
Seventy-three patients participated in a cross-sectional study at a tertiary SSc program. PROMIS-29, FACIT-Dyspnea, and legacy PRO instruments used in clinical trials (Medical Research Council [MRC] Dyspnea Score, St. George’s Respiratory Questionnaire [SGRQ], Health Assessment Questionnaire-Disability Index [HAQ-DI] and Short-Form 36 [SF-36]) were administered. Composite severity scores using an adaptation of the Medsger Disease Severity Index were generated using clinical, diagnostic and laboratory information. PROMIS-29 and FACIT-Dyspnea scores were compared with legacy PRO measures and composite severity scores.
Results
The mean (range) patient age (84% women) was 51 years (22–72). The mean (range) SSc disease duration from the onset of the first non-Raynaud symptom was 7 years (0–45). Spearman correlation coefficients across FACIT-Dyspnea and PROMIS Physical Functioning scores with legacy PRO instruments were generally high (range=0.64–0.86); those between PROMIS and FACIT-Dyspnea with composite disease severity scores were more modest, but statistically significant (range=0.33–0.48, p<0.01).
Conclusion
PROMIS-29 and FACIT-Dyspnea are valid instruments to measure the health status of SSc patients. PROMIS-29 and FACIT-Dyspnea may be preferable to legacy instruments because they are freely available in multiple languages, and simple to administer, score and interpret.
doi:10.1002/acr.20591
PMCID: PMC3205420  PMID: 22034123
22.  Nuclear β-Catenin Is Increased in Systemic Sclerosis Pulmonary Fibrosis and Promotes Lung Fibroblast Migration and Proliferation 
Pulmonary fibrosis is a disease that results in loss of normal lung architecture, but the signaling events that drive tissue destruction are incompletely understood. Wnt/β-catenin signaling is important in normal lung development, but whether abnormal signaling occurs in lung fibrosis due to systemic sclerosis and the consequences of β-catenin signaling toward the fibrogenic phenotype remain poorly defined. In this study, we show nuclear β-catenin accumulation in fibroblastic foci from lungs of patients with systemic sclerosis–associated advanced pulmonary fibrosis. Forced activation of β-catenin signaling in three independently derived sources of normal human lung fibroblasts promotes proliferation and migratory activities but is not sufficient to activate classic markers of fibroblast activation, such as TGF-β, type 1 collagen, α-smooth muscle actin, and connective tissue growth factor. These findings indicate that activation of β-catenin signaling in pulmonary fibroblasts may be a common feature of lung fibrosis, contributing to fibroproliferative and migratory activities associated with the disease.
doi:10.1165/rcmb.2010-0113OC
PMCID: PMC3262680  PMID: 21454805
Wnt/β-catenin signaling; scleroderma; fibrosis
23.  The adipokine adiponectin has potent anti-fibrotic effects mediated via adenosine monophosphate-activated protein kinase: novel target for fibrosis therapy 
Arthritis Research & Therapy  2012;14(5):R229.
Introduction
Fibrosis in scleroderma is associated with collagen deposition and myofibroblast accumulation. Peroxisome proliferator activated receptor gamma (PPAR-γ), a master regulator of adipogenesis, inhibits profibrotic responses induced by transforming growth factor-ß (TGF-β), and its expression is impaired in scleroderma. The roles of adiponectin, a PPAR-γ regulated pleiotropic adipokine, in regulating the response of fibroblasts and in mediating the effects of PPAR-γ are unknown.
Methods
Regulation of fibrotic gene expression and TGF-ß signaling by adiponectin and adenosine monophosphate protein-activated (AMP) kinase agonists were examined in normal fibroblasts in monolayer cultures and in three-dimensional skin equivalents. AdipoR1/2 expression on skin fibroblasts was determined by real-time quantitative PCR.
Results
Adiponectin, an adipokine directly regulated by PPAR-γ, acts as a potent anti-fibrotic signal in normal and scleroderma fibroblasts that abrogates the stimulatory effects of diverse fibrotic stimuli and reduces elevated collagen gene expression in scleroderma fibroblasts. Adiponectin responses are mediated via AMP kinase, a fuel-sensing cellular enzyme that is necessary and sufficient for down-regulation of fibrotic genes by blocking canonical Smad signaling. Moreover, we demonstrate that endogenous adiponectin accounts, at least in part, for the anti-fibrotic effects exerted by ligands of PPAR-γ.
Conclusions
These findings reveal a novel link between cellular energy metabolism and extracellular matrix homeostasis converging on AMP kinase. Since the levels of adiponectin as well as its receptor are impaired in scleroderma patients with progressive fibrosis, the present results suggest a potential role for defective adiponectin expression or function in progressive fibrogenesis in scleroderma and other chronic fibrosing conditions. Restoring the adiponectin signaling axis in fibroblasts might, therefore, represent a novel pharmacological approach to controlling fibrosis.
doi:10.1186/ar4070
PMCID: PMC3580540  PMID: 23092446
24.  Pulmonary Hypertension Assessment and Recognition of Outcomes in Scleroderma (PHAROS): Baseline Characteristics and Description of Study Population 
The Journal of rheumatology  2011;38(10):2172-2179.
Objective
Pulmonary arterial hypertension (PAH) increases mortality in systemic sclerosis (SSc). The multicenter PHAROS (Pulmonary Hypertension Assessment and Recognition of Outcomes in Scleroderma) registry prospectively follows SSc subjects at high-risk for, or with incident pulmonary hypertension (PH). Herein, we describe registry design and baseline characteristics of subjects enrolled during the first 18 months since study inception.
Methods
High-risk subjects are enrolled and classified as Pre-PAH if they have 1) carbon monoxide diffusing capacity (DLCO) < 55% predicted, 2) % predicted forced vital capacity/DLCO ratio ≥1.6, or 3) an estimated right ventricular systolic pressure > 35 mm Hg on echocardiography. Subjects with right heart catheterization confirmed incident PH (mean pulmonary artery pressure ≥ 25 mm Hg within previous 6 months) are subclassified into PAH, pulmonary venous hypertension secondary to left-sided heart disease (PVH), and PH due to interstitial lung disease (PH-ILD). Baseline and biannual demographic, clinical, and laboratory data as well as patient-reported health questionnaires are collected.
Results
There are 237 subjects enrolled in PHAROS. The majority are Caucasian (73%) and women (87%). There are 166 Pre-PAH and 71 Definite PH subjects (49 PAH, 7 PVH, and 15 PH-ILD).
Conclusion
PHAROS is the largest U.S. and Canadian cohort of SSc subjects at high-risk for or with incident PAH. PAH-specific therapies are approved for 49/71 subjects with RHC-confirmed-PAH. Analyses of PHAROS registry data will permit identification of risk factors for the development of PAH amongst SSc patients at high-risk for PAH and enhance our understanding of the course of SSc-PAH.
doi:10.3899/jrheum.101243
PMCID: PMC3230328  PMID: 21844142
Systemic scleroderma; Pulmonary hypertension; Registry; Pulmonary arterial hypertension
25.  Text data extraction for a prospective, research-focused data mart: implementation and validation 
Background
Translational research typically requires data abstracted from medical records as well as data collected specifically for research. Unfortunately, many data within electronic health records are represented as text that is not amenable to aggregation for analyses. We present a scalable open source SQL Server Integration Services package, called Regextractor, for including regular expression parsers into a classic extract, transform, and load workflow. We have used Regextractor to abstract discrete data from textual reports from a number of ‘machine generated’ sources. To validate this package, we created a pulmonary function test data mart and analyzed the quality of the data mart versus manual chart review.
Methods
Eleven variables from pulmonary function tests performed closest to the initial clinical evaluation date were studied for 100 randomly selected subjects with scleroderma. One research assistant manually reviewed, abstracted, and entered relevant data into a database. Correlation with data obtained from the automated pulmonary function test data mart within the Northwestern Medical Enterprise Data Warehouse was determined.
Results
There was a near perfect (99.5%) agreement between results generated from the Regextractor package and those obtained via manual chart abstraction. The pulmonary function test data mart has been used subsequently to monitor disease progression of patients in the Northwestern Scleroderma Registry. In addition to the pulmonary function test example presented in this manuscript, the Regextractor package has been used to create cardiac catheterization and echocardiography data marts. The Regextractor package was released as open source software in October 2009 and has been downloaded 552 times as of 6/1/2012.
Conclusions
Collaboration between clinical researchers and biomedical informatics experts enabled the development and validation of a tool (Regextractor) to parse, abstract and assemble structured data from text data contained in the electronic health record. Regextractor has been successfully used to create additional data marts in other medical domains and is available to the public.
doi:10.1186/1472-6947-12-106
PMCID: PMC3537747  PMID: 22970696
Medical informatics; Information storage and retrieval; Information systems; Electronic health records; Automatic data processing

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