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Transforming growth factor-β signaling through the Smad proteins: Role in systemic sclerosis
Autoimmunity Reviews 5 (2006) 563 – 569 www.elsevier.com/locate/autrev
Transforming growth factor-β signaling through the Smad proteins: Role in systemic sclerosis Franck Verrecchia ⁎, Alain Mauviel, Dominique Farge INSERM U697, Hôpital Saint-Louis, Pavillon Bazin, 1 avenue Claude Vellefaux, 75010 Paris, France Received 17 May 2006; accepted 8 June 2006 Available online 5 July 2006
Abstract Transforming growth factor-β (TGF-β) plays a critical role in the development of tissue fibrosis. Its expression is consistently elevated in affected organs and correlates with increased extracellular matrix deposition. During the last few years, tremendous progress has been made in understanding the molecular aspects of intracellular signaling downstream of the TGF-β receptors. In particular, Smad proteins, TGF-β receptor kinase substrates that translocate into the cell nucleus to act as transcription factors, have been studied extensively. Their role in the transcriptional regulation of type I collagen and other extracellular matrix (ECM) genes expression, and in the development of fibrosis is of critical importance because it may lead to novel therapeutic strategies for the treatment of these multi-organ tissue reactions to injury. Systemic sclerosis (SSc) is a complex autoimmune disease characterized by pathological remodelling of connective tissues correlated to the activation of TGF-β/Smad signaling pathway. This review focuses on the mechanisms underlying Smad modulation of gene expression and how they relate to fibrotic process. Potential implications for the development of therapeutic approaches against tissue fibrosis during SSc are discussed. © 2006 Elsevier B.V. All rights reserved. Keywords: Collagen genes; Fibrosis; Transforming growth factor-β; Smad; Systemic sclerosis
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The transforming growth factor (TGF-β)/Smad signaling pathways . . 1.1. The TGF-β superfamily: structure and activation . . . . . . . . 1.2. TGF-β signaling through Smad proteins . . . . . . . . . . . . TGF-β/Smad in the fibrotic process . . . . . . . . . . . . . . . . . . 2.1. Transcriptional regulation of COL1A1 and COL1A2 by TGF-β 2.2. Regulation of ECM genes by Smad3 . . . . . . . . . . . . . . 2.3. TGF-β/Smad3 signaling in fibrotic responses (in vivo data) . . TGF-β/Smad and systemic sclerosis . . . . . . . . . . . . . . . . . . 3.1. Systemic sclerosis (SSc) . . . . . . . . . . . . . . . . . . . . 3.2. TGF-β/Smad in SSc . . . . . . . . . . . . . . . . . . . . . .
⁎ Corresponding author. Tel.: +33 153722076; fax: +33 153722051. E-mail address: [email protected] (F. Verrecchia). 1568-9972/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.autrev.2006.06.001
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4. Antifibrotic therapeutic Acknowledgments . . . . . Take-home messages . . . . References . . . . . . . . .
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1. The transforming growth factor (TGF-β)/Smad signaling pathways 1.1. The TGF-β superfamily: structure and activation Members of the TGF-β family are secreted polypeptides that regulate many different physiological processes, including embryonic development, homeostasis, wound healing, chemotaxis, and cell cycle control. Among the 60 TGF-β family members identified in multicellular organisms, three TGF-βs, five activins, and at least eight BMPs (Bone Morphogenetics Proteins) encoded by different genes have been described. TGFβ1, 2, and 3 are the three highly homologous TGF-βs isoforms expressed in mammal cells which often have similar biological activities in vitro, while eliciting distinct biological responses in vivo. TGF-βs are secreted as latent precursors (LTGF-β), requiring activation into a mature form to allow receptor binding and subsequent activation of the signal transduction pathways. The LTGF-β molecules consist of 390–414 amino acids. They contain the latency-associated peptide (LAP) region, an amino-terminal hydrophobic signal peptide region of 249 residues, and the potentially bioactive Cterminal region, which contains 112 amino acids. LTGFβ is usually secreted as a large latent complex covalently bound to LTGF-β-binding protein (LTBP) via the LAP region, or as a small latent complex without LTBP. The LAP confers latency to the complex, whereas LTBP serves to bind TGF-β to the extracellular matrix (ECM) and enables proteolytic activation. Activation of the TGF-β is a complex process, involving conformational changes of LTGF-β, induced either by cleavage of the LAP by various proteases or by physical interactions of the LAP with other proteins such as thrombospondin. Thus, the TGF-β activators are proteins intimately associated with the wound healing response. For example, the plasmin proteases MMP-2 and MMP-9, which promote matrix degradation, activate TGF-β [1,2]. 1.2. TGF-β signaling through Smad proteins Upon activation, TGF-β family members initiate their cellular action by binding to “type I” and “type II” receptors cell surface complexes, each made of one small cysteine-rich extracellular region plus an intracel-
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lular region consisting mainly of their kinase domains. Type I receptors have a region rich in glycine and serine residues (GS domain) preceding the receptor kinase domain. In the absence of a ligand, type I and type II receptors exist as homodimers at the cell surface. Ligand binding allows the formation of a stable receptor complex, consisting of two receptors of each type and permitting phosphorylation of the GS domain by the type II receptor kinases. This phosphorylation stimulates the type I receptor kinases, resulting in the phosphorylation of Smad proteins and subsequent downstream signaling. In humans, the number of TGF-β ligands exceeds the number of type II and type I receptors. Thus, combinatorial interactions of type I and type II receptors in functional receptor complexes allow for the diversity and selectivity in ligand binding as well as in intracellular signaling. Several accessory cell surface proteins further define the binding efficiency and specificity of the ligand to the receptor complex [1,3,4]. After ligand activation, signaling from receptors to nucleus occurs predominantly by phosphorylation of cytoplasmic mediators belonging to the Smad family. Type I receptors specifically recognize and phosphorylate the ligand-specific receptor-activated Smad (R-Smad). These R-Smads are recruited to activate type I receptors by a membrane bound cytoplasmic protein called SARA (Smad Anchor for Receptor Activation). R-Smads include Smad1, Smad5, and Smad8 downstream of the BMP, and Smad2 and Smad3 downstream of the TGF-β and activin. Phosphorylation of R-Smad by type I receptors occurs on two serine residues within a conserved –SS(M/V)S– motif at their C-terminus. Upon phosphorylation by type I receptors, R-Smad forms heteromeric complex with the Common-Smad, e.g. Co-Smad (Smad4 in vertebrates) and translocates into the nucleus. R-Smad and Smad4 contain a conversed MH1 and C-terminal MH2 domains, flanking a divergent middle linker domain. MH1 and MH2 domains can interact with transcription factors, whereas the C-terminus of the R-Smad interacts with the coactivators CREBbinding protein (CBP) or p300. A third group of Smad proteins, the inhibitory Smads (I-Smads), Smad6 and Smad7, prevent R-Smad phosphorylation and/or nuclear translocation of R-Smads, and recruit E3-type ubiquitin ligases to the receptors complexes, ultimately leading to their degradation [1,3,4] (Fig. 1).
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Fig. 1. The Smad signaling pathway. A. Schematic representation of the Smad signaling cascade downstream of the TGF-β receptors. After ligand binding to TGF-β type II receptor (TβRII) (1), recruitment (2), and phosphorylation (3) of TβRI occurs, followed by activation of the Smad cascade. TβRI acts as the transducer receptor, which phosphorylate the R-Smad (4). Phosphorylated R-Smad associate with Co-Smad (5). The resulting heterocomplex then translocate into the nucleus (6), where it binds DNA and acts as a transcription factor to regulate the expression of specific gene targets (7). I-Smads prevent R-Smad phosphorylation and/or nuclear translocation of R-Smads, and recruit E3-type ubiquitin ligases to the receptors complexes, ultimately leading to their degradation (8). B. Structural domain of Smad3. Smad3 consists of two conserve globular domains known as MH1 (Mad homology 1) and MH2 domains, linked by a linker region. In the basal state, Smad3 remain in an inactive conformation through an autoinhibitory MH1/MH2 interaction. Phosphorylation of the C-terminal SSXS motif by activated TβRI results in Smad3 activation, heteromerization with Smad4, and subsequent translocation into the cell nucleus. The Smad3 domain in MH1 recognizes the DNA sequence CAGAC, the MH2 domain is involved in protein/protein interactions with Smad4, transcriptional coactivators and corepressors.
At the regulatory DNA binding sequence of genes, Smad proteins activate transcription through physical interaction and functional cooperation of DNA-binding Smads with sequence-specific transcription factors and the coactivators CBP and p300. The R-Smads MH1 domain can bind directly to DNA except in the case of Smad2 where a 30 amino acid insertion in this domain prevents DNA binding. The minimal Smad-binding element (SBE) contains only four base pairs, 5′-AGAC-3′, but there are reports of binding to other G/C-rich sequences [1,3,4].
removal of collagen. TGF-β, as the major contributor to the ECM metabolic modulation, drives pro-fibrotic responses in vitro and in vivo by enhancing ECM gene expression and repressing that of catabolic enzymes, enhancing fibroblasts proliferation and inducing the myofibroblast phenotype. The combined action of TGF-β on the genes implicated in the ECM formation and degradation is mostly exerted at the transcriptional level [2,7,8]. 2.1. Transcriptional regulation of COL1A1 and COL1A2 by TGF-β [8,9]
2. TGF-β/Smad in the fibrotic process Fibrosis is a complex tissue disease whose predominant characteristic is the excessive deposition of extracellular matrix (ECM) components, especially collagens, the major fibrous proteins in ECM [5,6]. The net accumulation of collagens in tissue fibrosis is a result of an imbalance between the factors leading to enhanced production and deposition, or impaired degradation and
Several studies have shown that excessive tissue deposition during the fibrotic process is largely due to an increase in the rate of transcription of type I collagen genes, COL1A1 and COL1A2 [6]. Original works demonstrated that TGF-β-responsive sequences regarding the human promoter of COL1A1 are located between 174 and 84 bp from the transcription start site, whose region contains a binding site for Sp1. In addition, the
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human COL1A2 promoter with a 135-bp region, within 330 bp of the transcription start site, was first shown to confer responsiveness to TGF-β [8,9]. The minimal TGFβ-response element was further refined to the region between nucleotides -271 and -235. The latter contains potential overlapping cis-element for Smad, AP-1 and NFκB [10–12]. Cooperation between Smad3 and Sp1 to transactivate the COL1A2 promoter has been described. It has also been shown that Smad–p300/CBP interactions are a key event in TGF-β driven COL1A2 gene transactivation [13,14].
Smad3 appears to be a key element in the signal transduction pathways involved in the in vivo process of fibrosis [17]. Indeed, Smad3 null mice are protected against radiation-induced fibrosis of the skin [23], and a second Smad3 null line demonstrated attenuated lung fibrosis induced by bleomycin [24]. Overexpression of Smad7, which prevents the phosphorylation and the activation of Smad3, has also been shown to protect against fibrosis in a bleomycin model [25].
2.2. Regulation of ECM genes by Smad3
3.1. Systemic sclerosis (SSc)
By the end of the year 2000, only approximately 12 genes were known to contain Smad-responsive regions, binding Smad complexes directly or indirectly. All Smad targets identified downstream TGF-β were Smad3-dependent including PAI-1 and COL1A2 genes. Using a combined cDNA microarray promoter transactivation approach, we have identified new Smad3/4 gene targets in cultured dermal fibroblasts: COL1A1, COL3A1, COL5A2, COL6A1, COL6A3, and TIMP-1. In addition, we identified 49 immediate-early TGF-β target genes. Their activation by TGF-β is rapid and does not require protein neo-synthesis or JNK activity. These genes represent potential novel Smad targets. Therefore, the Smad signaling pathway is crucial for the simultaneous activation of skin fibrillar collagen genes (COL1A1, COL1A2, COL3A1 and COL5A2) by TGF-β [15]. Besides playing a large part in the regulation of the expression of ECM components, Smads have been identified as capable of mediating the inhibitory activity of TGF-β on interstitial collagenase (matrix metalloproteinase-1, MMP-1) gene activation by pro-inflammatory cytokines, such as IL-1β [16].
Systemic sclerosis (SSc) is a heterogenous and generalized connective tissue disorder characterized by microvascular and larger vessel lesions, with consequent induration and thickening of the skin, fibrotic degenerative changes in muscles, joints and viscera, mainly the intestinal tract, the heart, the lungs and the kidneys. Excessive production and changes in the architecture of connective tissue components in the dermis and in the subcutaneous tissue are considered as the hallmark of the disease. These changes are caused by variable degrees of extracellular matrix accumulation. Although its exact pathogenesis remains unknown, many experimental and in vivo data support the concept that an unknown original antigen stimulation or genetic susceptibility may contribute to altered endothelial function and blood vessel reactivity – which are clinically evidenced by the presence of a Raynaud phenomenon – with consequent inflammation and autoimmunity reactions [26–28]. This chronic autoimmune, vascular, and fibrotic disease is heterogenous and characterized by initial predominant T-cell activation with altered repertoires, production of antigen-specific autoantibodies and cytokines, predominantly TH2, chemokines and growth factors release with pro-fibrotic properties. All these factors in turn contribute to diffuse microvascular injury, fibroblast activation, and increased production of collagen, leading to diffuse sclerosis within the skin and internal organs, namely the heart, the lungs, the kidneys and the intestinal tract. Two main clinical subsets, namely limited and diffuse cutaneous forms, can be distinguished by the extent of skin involvement, their autoantibody profile and the pattern of organ involvement. In severe forms of the disease, especially rapidly progressive diffuse cutaneous SSc, the 5-year mortality is estimated to be 40–50% [26–30]. The most abundant extracellular matrix protein (ECM) in tissues affected by systemic sclerosis seems to be the type 1 collagen [27]. In this context, it has been shown that fibroblasts culture from the affected skin
2.3. TGF-β/Smad3 signaling in fibrotic responses (in vivo data) TGF-β has been implicated as being an important mediator in a number of fibrotic diseases [17,18]. For example, expression of TGF-β and TGF-β receptors is elevated in the human fibroblasts of hypertrophic scars and of keloids [19,20]. The most direct evidence supporting the involvement of TGF-β in fibrosis are data showing that agents blocking the TGF-β function reduce the fibrotic response. In mice, progressive TGFβ1-induced lung fibrosis is blocked by an orally active TβRI kinase inhibitor [21] and the administration of TGF-β antibodies prevents skin and lung fibrosis in murine sclerodermatous graft-vs-host disease [22].
3. TGF-β/Smad and systemic sclerosis
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produce excessive amounts of various collagens associated with increased transcription rates of the corresponding genes or increased stability of collagen mRNA [27]. Although the mechanisms involved in the pathological increase of collagen expression in SSc have not been entirely elucidated, extensive recent efforts have been devoted to study the role of TGF-β signaling pathway by Smad proteins. 3.2. TGF-β/Smad in SSc The multifunctional cytokine, TGF-β and its intracellular pathways has been examined in patients with SSc. Some data have reported an increase of circulating TGF-β levels. Most of them observed a TGF-β immunoreactivity in affected scleroderma skin, which was present in the early inflammatory lesions before dense fibrosis had occurred [31]. Regarding the TGF-β signaling pathway, TGF-β receptors may be overexpressed or upregulated in SSc [31]. Immunohistochemistral analysis of skin biopsies performed in non-lesional areas from SSc patients and analysis of fibroblast cultures showed that Smad2 and Smad3 expression and their nuclear translocation were increased in these SSc patients [32]. However in these studies, no direct correlation between the status of Smad expression and the severity of the disease had been established. More recently, we showed that the extent of skin fibrosis, as clinically assessed by the modified Rodnan skin score, was directly correlated with the activation of TGF-β signaling pathway, as estimated by the levels of the Smad3 phosphorylation forms in protein extracts from SSc and normal fibroblasts obtained by explanting skin punch biopsy from SSc patients or healthy donors (Verrecchia et al., ACR Meeting 2005). One of the characterized mechanisms for inhibiting TGF-β signaling through the Smad pathway involves endogenous Smad7. Dong et al. reported a reduction of Smad7 expression in SSc derived fibroblast cultures as compared to fibroblast cultures from unaffected areas of the same patients, suggesting that defective Smad7 feedback inhibition could play a role in TGF-β hyperresponsiveness in SSc [33]. In contrast with these results, and as previously observed by Mori et al. [32], we found no difference between SSc and normal tissues regarding Smad4 and Smad7 expression when using immunohistochemistry analysis (Verrecchia et al., ACR Meeting, 2005). Variations in the two studies could partly be explained by differences in the skin biopsy sites, which in our work were obtained from lesional areas. In diffuse SSc, skin involvement concerns the whole body surface, although at variable severity degrees according to the various sites. Thus, sustained activation of TGF-β sig-
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naling pathway in fibroblast cultures may represent an intrinsic defect in SSc fibroblasts, that may contribute to the development of autonomous SSc lesions in the absence of altered circulatory or systemic factors. 4. Antifibrotic therapeutic strategies in SSc Several animal models, which may not fully be extrapolated to human disease, provided tools to study the antifibrotic effects of several drugs. In this rare and heterogenous disease prospective clinical trials are hard to perform. In a majority of SSc patients, clinical necessity often requires the simultaneous or successive use of vascular, immunosuppressive and antifibrotic therapies, and their respective benefits are hard to distinguish. To date no single treatment has yet been shown to prevent disease progression and to reverse fibrosis in SSc patients. Nonetheless, experimental data provide arguments to use therapeutic strategies aiming at immunomodulation and inhibition of TGF-β signaling. An increasing body of evidence demonstrates that inhibiting TGF-β signaling especially through blocking Smad3 can decrease fibrotic responses both in vitro and in vivo. Halofuginone, a semisynthetic plant alkaloid, and ALK5/TβRI small molecule inhibitors, have been shown to reduce the expression of collagen by fibroblasts and to prevent fibrotic responses in several animal models. Imatinib (Gleevec), a tyrosine kinase receptor inhibitor is also being tested [34]. Pirfenidone, an arginase enzyme inhibitor, downregulating some of TGF-β-dependent patterns of gene expression, was recently tested successfully in a double blind placebo control trial in patients with idiopathic pulmonary fibrosis [35]. The apparent effectiveness of anti-TGF-β antagonists in blocking fibrosis in animal models led to test the use of anti-TGF-β in diffuse SSc with successful safety and tolerability. However, modifications of TGF-β signaling may vary according to the clinical stage and subset of SSc [34]. Immunosuppressive therapy, given at an early stage of SSc disease, could be beneficial [36] and we recently reported (Verrechia et al., ACR 2005) that the use of broad spectrum immunosuppression could also interact with TGF-β modulation. Indeed, since 1998, we – as part of an European collaborative effort under the auspices of the EBMT (European Bone Marrow Transplant Association) — EULAR (European League Against Rheumatism) – and several other North American groups under the CIBMTR (Center for International Blood and Marrow Transplant Research), have used intensive myelo- and immunosuppression followed by autologous hematopoietic stem cell transplantation (HSCT) to treat severe SSc [37,38]. The early results from the phase I/II clinical trials
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showed a significant clinical regression of skin fibrosis and durable clinical improvement up to 7 years after autologous HSCT. When analyzing the regression of skin fibrosis after HSCT, we found a decrease in the extent of fibrosis first within in the papillary dermis, then in the deep dermis according to the extent of the disease regression. These results suggest that blockade of the autoimmune response after HSCT inhibits the signals leading to increased ECM synthesis. These promising clinical and histological results showing regression of skin fibrosis after HSCT will have to be confirmed in a larger number of patients after HSCT, as currently enrolled in the ongoing phase III European ASTIS trial (http://www.astistrial.com) [39], as well as in a similar study being started in the USA (SCOT trial), which uses irradiation with lung and kidney shielding in addition to anti-thymocytes globulins and cyclophosphamide as used in the ASTIS trial. Acknowledgments This work was supported by the Groupe Français de Recherche sur la Sclérodermie (GFRS, www.sclerodermie. org), the Association Française contre la Sclérodermie (ASF), INSERM (Institut Nationale de la Santé Et de la Recherche Médicale), Délégation Régionale à la Recherche Clinique (DRRC), Assistance Publique-Hôpitaux de Paris (AP-HP), the French Ministry of Health (Programme Hospitalier de Recherche Clinique: PHRC 1997 AOM 97030) and the Etablissement Français des Greffes (2003). Take-home messages • Exaggerated tissue deposition during the fibrosis process is largely due to an increase in the rate of transcription of type I collagen genes. • The Smad signaling pathway is crucial for simultaneous activation of fibrillar collagen genes by TGF-β • Smad3 has been implicated as being an important mediator in a number of fibrotic conditions. • A crucial element in the pathogenesis of SSc is the abnormal regulation of expression of the genes involved in the production and deposition of type I collagen. • Smad3 play a crucial role in TGF-β responsiveness to type 1 collagen gene. • Inhibition of TGF-β signaling can decrease the fibrotic responses. References [1] Feng XH, Derynck R. Specificity and versatility in TGFsignaling through Smads. Annu Rev Cell Dev Biol 2005.
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[31] Denton CP, Abraham DJ. Transforming growth factor-beta and connective tissue growth factor: key cytokines in scleroderma pathogenesis. Curr Opin Rheumatol 2001;13:505–11. [32] Mori Y, Chen SJ, Varga J. Expression and regulation of intracellular SMAD signaling in scleroderma skin fibroblasts. Arthritis Rheum 2003;48:1964–78. [33] Dong C, Zhu S, Wang T, Yoon W, Li Z, Alvarez RJ, et al. Deficient Smad7 expression: a putative molecular defect in scleroderma. Proc Natl Acad Sci U S A 2002;99:3908–13. [34] Denton CP, Black CM. Targeted therapy comes of age in scleroderma. Trends Immunol 2005;26:596–602. [35] Azuma A, Nukiwa T, Tsuboi E, Suga M, Abe S, Nakata K, et al. Double-blind, placebo-controlled trial of pirfenidone in patients with idiopathic pulmonary fibrosis. Am J Respir Crit Care Med 2005;171:1040–7. [36] McSweeney PA, Nash RA, Sullivan KM, Storek J, Crofford LJ, Dansey R, et al. High-dose immunosuppressive therapy for severe systemic sclerosis: initial outcomes. Blood 2002;100:1602–10. [37] Farge D, Marolleau JP, Zohar S, Marjanovic Z, Cabane J, Mounier N, et al. Autologous bone marrow transplantation in the treatment of refractory systemic sclerosis: early results from a French multicentre phase I–II study. Br J Haematol 2002;119:726–39. [38] Farge D, Passweg J, van Laar JM, Marjanovic Z, Besenthal C, Finke J, et al. Autologous stem cell transplantation in the treatment of systemic sclerosis: report from the EBMT/EULAR registry. Ann Rheum Dis 2004;63:974–81. [39] van Laar JM, Farge D, Tyndall A. Autologous Stem cell Transplantation International Scleroderma (ASTIS) trial: hope on the horizon for patients with severe systemic sclerosis. Ann Rheum Dis 2005;64:1515.
Oxidized low-density lipoprotein autoantibodies, chronic infections, and carotid atherosclerosis in a populationbased study Atherosclerosis is a chronic immuno-inflammatory disease wherein both oxidized lipids and infectious agents are incriminated as possible contributors. This is why associations between immune reactions to oxidized low-density lipoproteins (oxLDLs), chronic infections, and carotid atherosclerosis as quantified by ultrasound was investigated. Here, Mayer M. et. al. (J Am Cardiol 2006; 47:2436-43) measured immunoglobulin IgG and IgM autoantibody titers to copper-oxidized-LDL and malondialdehyde-LDL (oxLDL-AB), IgG and IgM apolipoprotein B-100immune complexes (ApoB-IC), and titers of antibodies to E. coli and chlamydial LPS, mycobacterial heat shock protein 65 (mHSP65), and evaluated their relationship to cardiovascular risk factors, chronic infections, and incident/progressive carotid atherosclerosis. The oxLDL-AB and ApoB-IC levels remained stable over time as indicated by strong correlations between 1995 and 2000 measurements (p < 0.001 each). Significant associations existed between all oxLDL markers and antibody titers to pathogens, especially to E. coli-LPS and mHSP65. The IgG oxLDL markers were positively and IgM markers were inversely associated with incident and progressive carotid atherosclerosis in univariate analyses but were not independent predictors in multivariate analyses. This study provides evidence for an association between human oxLDL markers and chronic infections. In this study, neither IgG nor IgM oxLDL autoantibodies were independently predictive of atherosclerosis progression in the carotid arteries.
Transforming growth factor-β signaling through the Smad proteins: Role in systemic sclerosis Franck Verrecchia ⁎, Alain Mauviel, Dominique Farge INSERM U697, Hôpital Saint-Louis, Pavillon Bazin, 1 avenue Claude Vellefaux, 75010 Paris, France Received 17 May 2006; accepted 8 June 2006 Available online 5 July 2006
Abstract Transforming growth factor-β (TGF-β) plays a critical role in the development of tissue fibrosis. Its expression is consistently elevated in affected organs and correlates with increased extracellular matrix deposition. During the last few years, tremendous progress has been made in understanding the molecular aspects of intracellular signaling downstream of the TGF-β receptors. In particular, Smad proteins, TGF-β receptor kinase substrates that translocate into the cell nucleus to act as transcription factors, have been studied extensively. Their role in the transcriptional regulation of type I collagen and other extracellular matrix (ECM) genes expression, and in the development of fibrosis is of critical importance because it may lead to novel therapeutic strategies for the treatment of these multi-organ tissue reactions to injury. Systemic sclerosis (SSc) is a complex autoimmune disease characterized by pathological remodelling of connective tissues correlated to the activation of TGF-β/Smad signaling pathway. This review focuses on the mechanisms underlying Smad modulation of gene expression and how they relate to fibrotic process. Potential implications for the development of therapeutic approaches against tissue fibrosis during SSc are discussed. © 2006 Elsevier B.V. All rights reserved. Keywords: Collagen genes; Fibrosis; Transforming growth factor-β; Smad; Systemic sclerosis
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The transforming growth factor (TGF-β)/Smad signaling pathways . . 1.1. The TGF-β superfamily: structure and activation . . . . . . . . 1.2. TGF-β signaling through Smad proteins . . . . . . . . . . . . TGF-β/Smad in the fibrotic process . . . . . . . . . . . . . . . . . . 2.1. Transcriptional regulation of COL1A1 and COL1A2 by TGF-β 2.2. Regulation of ECM genes by Smad3 . . . . . . . . . . . . . . 2.3. TGF-β/Smad3 signaling in fibrotic responses (in vivo data) . . TGF-β/Smad and systemic sclerosis . . . . . . . . . . . . . . . . . . 3.1. Systemic sclerosis (SSc) . . . . . . . . . . . . . . . . . . . . 3.2. TGF-β/Smad in SSc . . . . . . . . . . . . . . . . . . . . . .
⁎ Corresponding author. Tel.: +33 153722076; fax: +33 153722051. E-mail address: [email protected] (F. Verrecchia). 1568-9972/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.autrev.2006.06.001
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4. Antifibrotic therapeutic Acknowledgments . . . . . Take-home messages . . . . References . . . . . . . . .
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1. The transforming growth factor (TGF-β)/Smad signaling pathways 1.1. The TGF-β superfamily: structure and activation Members of the TGF-β family are secreted polypeptides that regulate many different physiological processes, including embryonic development, homeostasis, wound healing, chemotaxis, and cell cycle control. Among the 60 TGF-β family members identified in multicellular organisms, three TGF-βs, five activins, and at least eight BMPs (Bone Morphogenetics Proteins) encoded by different genes have been described. TGFβ1, 2, and 3 are the three highly homologous TGF-βs isoforms expressed in mammal cells which often have similar biological activities in vitro, while eliciting distinct biological responses in vivo. TGF-βs are secreted as latent precursors (LTGF-β), requiring activation into a mature form to allow receptor binding and subsequent activation of the signal transduction pathways. The LTGF-β molecules consist of 390–414 amino acids. They contain the latency-associated peptide (LAP) region, an amino-terminal hydrophobic signal peptide region of 249 residues, and the potentially bioactive Cterminal region, which contains 112 amino acids. LTGFβ is usually secreted as a large latent complex covalently bound to LTGF-β-binding protein (LTBP) via the LAP region, or as a small latent complex without LTBP. The LAP confers latency to the complex, whereas LTBP serves to bind TGF-β to the extracellular matrix (ECM) and enables proteolytic activation. Activation of the TGF-β is a complex process, involving conformational changes of LTGF-β, induced either by cleavage of the LAP by various proteases or by physical interactions of the LAP with other proteins such as thrombospondin. Thus, the TGF-β activators are proteins intimately associated with the wound healing response. For example, the plasmin proteases MMP-2 and MMP-9, which promote matrix degradation, activate TGF-β [1,2]. 1.2. TGF-β signaling through Smad proteins Upon activation, TGF-β family members initiate their cellular action by binding to “type I” and “type II” receptors cell surface complexes, each made of one small cysteine-rich extracellular region plus an intracel-
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lular region consisting mainly of their kinase domains. Type I receptors have a region rich in glycine and serine residues (GS domain) preceding the receptor kinase domain. In the absence of a ligand, type I and type II receptors exist as homodimers at the cell surface. Ligand binding allows the formation of a stable receptor complex, consisting of two receptors of each type and permitting phosphorylation of the GS domain by the type II receptor kinases. This phosphorylation stimulates the type I receptor kinases, resulting in the phosphorylation of Smad proteins and subsequent downstream signaling. In humans, the number of TGF-β ligands exceeds the number of type II and type I receptors. Thus, combinatorial interactions of type I and type II receptors in functional receptor complexes allow for the diversity and selectivity in ligand binding as well as in intracellular signaling. Several accessory cell surface proteins further define the binding efficiency and specificity of the ligand to the receptor complex [1,3,4]. After ligand activation, signaling from receptors to nucleus occurs predominantly by phosphorylation of cytoplasmic mediators belonging to the Smad family. Type I receptors specifically recognize and phosphorylate the ligand-specific receptor-activated Smad (R-Smad). These R-Smads are recruited to activate type I receptors by a membrane bound cytoplasmic protein called SARA (Smad Anchor for Receptor Activation). R-Smads include Smad1, Smad5, and Smad8 downstream of the BMP, and Smad2 and Smad3 downstream of the TGF-β and activin. Phosphorylation of R-Smad by type I receptors occurs on two serine residues within a conserved –SS(M/V)S– motif at their C-terminus. Upon phosphorylation by type I receptors, R-Smad forms heteromeric complex with the Common-Smad, e.g. Co-Smad (Smad4 in vertebrates) and translocates into the nucleus. R-Smad and Smad4 contain a conversed MH1 and C-terminal MH2 domains, flanking a divergent middle linker domain. MH1 and MH2 domains can interact with transcription factors, whereas the C-terminus of the R-Smad interacts with the coactivators CREBbinding protein (CBP) or p300. A third group of Smad proteins, the inhibitory Smads (I-Smads), Smad6 and Smad7, prevent R-Smad phosphorylation and/or nuclear translocation of R-Smads, and recruit E3-type ubiquitin ligases to the receptors complexes, ultimately leading to their degradation [1,3,4] (Fig. 1).
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Fig. 1. The Smad signaling pathway. A. Schematic representation of the Smad signaling cascade downstream of the TGF-β receptors. After ligand binding to TGF-β type II receptor (TβRII) (1), recruitment (2), and phosphorylation (3) of TβRI occurs, followed by activation of the Smad cascade. TβRI acts as the transducer receptor, which phosphorylate the R-Smad (4). Phosphorylated R-Smad associate with Co-Smad (5). The resulting heterocomplex then translocate into the nucleus (6), where it binds DNA and acts as a transcription factor to regulate the expression of specific gene targets (7). I-Smads prevent R-Smad phosphorylation and/or nuclear translocation of R-Smads, and recruit E3-type ubiquitin ligases to the receptors complexes, ultimately leading to their degradation (8). B. Structural domain of Smad3. Smad3 consists of two conserve globular domains known as MH1 (Mad homology 1) and MH2 domains, linked by a linker region. In the basal state, Smad3 remain in an inactive conformation through an autoinhibitory MH1/MH2 interaction. Phosphorylation of the C-terminal SSXS motif by activated TβRI results in Smad3 activation, heteromerization with Smad4, and subsequent translocation into the cell nucleus. The Smad3 domain in MH1 recognizes the DNA sequence CAGAC, the MH2 domain is involved in protein/protein interactions with Smad4, transcriptional coactivators and corepressors.
At the regulatory DNA binding sequence of genes, Smad proteins activate transcription through physical interaction and functional cooperation of DNA-binding Smads with sequence-specific transcription factors and the coactivators CBP and p300. The R-Smads MH1 domain can bind directly to DNA except in the case of Smad2 where a 30 amino acid insertion in this domain prevents DNA binding. The minimal Smad-binding element (SBE) contains only four base pairs, 5′-AGAC-3′, but there are reports of binding to other G/C-rich sequences [1,3,4].
removal of collagen. TGF-β, as the major contributor to the ECM metabolic modulation, drives pro-fibrotic responses in vitro and in vivo by enhancing ECM gene expression and repressing that of catabolic enzymes, enhancing fibroblasts proliferation and inducing the myofibroblast phenotype. The combined action of TGF-β on the genes implicated in the ECM formation and degradation is mostly exerted at the transcriptional level [2,7,8]. 2.1. Transcriptional regulation of COL1A1 and COL1A2 by TGF-β [8,9]
2. TGF-β/Smad in the fibrotic process Fibrosis is a complex tissue disease whose predominant characteristic is the excessive deposition of extracellular matrix (ECM) components, especially collagens, the major fibrous proteins in ECM [5,6]. The net accumulation of collagens in tissue fibrosis is a result of an imbalance between the factors leading to enhanced production and deposition, or impaired degradation and
Several studies have shown that excessive tissue deposition during the fibrotic process is largely due to an increase in the rate of transcription of type I collagen genes, COL1A1 and COL1A2 [6]. Original works demonstrated that TGF-β-responsive sequences regarding the human promoter of COL1A1 are located between 174 and 84 bp from the transcription start site, whose region contains a binding site for Sp1. In addition, the
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human COL1A2 promoter with a 135-bp region, within 330 bp of the transcription start site, was first shown to confer responsiveness to TGF-β [8,9]. The minimal TGFβ-response element was further refined to the region between nucleotides -271 and -235. The latter contains potential overlapping cis-element for Smad, AP-1 and NFκB [10–12]. Cooperation between Smad3 and Sp1 to transactivate the COL1A2 promoter has been described. It has also been shown that Smad–p300/CBP interactions are a key event in TGF-β driven COL1A2 gene transactivation [13,14].
Smad3 appears to be a key element in the signal transduction pathways involved in the in vivo process of fibrosis [17]. Indeed, Smad3 null mice are protected against radiation-induced fibrosis of the skin [23], and a second Smad3 null line demonstrated attenuated lung fibrosis induced by bleomycin [24]. Overexpression of Smad7, which prevents the phosphorylation and the activation of Smad3, has also been shown to protect against fibrosis in a bleomycin model [25].
2.2. Regulation of ECM genes by Smad3
3.1. Systemic sclerosis (SSc)
By the end of the year 2000, only approximately 12 genes were known to contain Smad-responsive regions, binding Smad complexes directly or indirectly. All Smad targets identified downstream TGF-β were Smad3-dependent including PAI-1 and COL1A2 genes. Using a combined cDNA microarray promoter transactivation approach, we have identified new Smad3/4 gene targets in cultured dermal fibroblasts: COL1A1, COL3A1, COL5A2, COL6A1, COL6A3, and TIMP-1. In addition, we identified 49 immediate-early TGF-β target genes. Their activation by TGF-β is rapid and does not require protein neo-synthesis or JNK activity. These genes represent potential novel Smad targets. Therefore, the Smad signaling pathway is crucial for the simultaneous activation of skin fibrillar collagen genes (COL1A1, COL1A2, COL3A1 and COL5A2) by TGF-β [15]. Besides playing a large part in the regulation of the expression of ECM components, Smads have been identified as capable of mediating the inhibitory activity of TGF-β on interstitial collagenase (matrix metalloproteinase-1, MMP-1) gene activation by pro-inflammatory cytokines, such as IL-1β [16].
Systemic sclerosis (SSc) is a heterogenous and generalized connective tissue disorder characterized by microvascular and larger vessel lesions, with consequent induration and thickening of the skin, fibrotic degenerative changes in muscles, joints and viscera, mainly the intestinal tract, the heart, the lungs and the kidneys. Excessive production and changes in the architecture of connective tissue components in the dermis and in the subcutaneous tissue are considered as the hallmark of the disease. These changes are caused by variable degrees of extracellular matrix accumulation. Although its exact pathogenesis remains unknown, many experimental and in vivo data support the concept that an unknown original antigen stimulation or genetic susceptibility may contribute to altered endothelial function and blood vessel reactivity – which are clinically evidenced by the presence of a Raynaud phenomenon – with consequent inflammation and autoimmunity reactions [26–28]. This chronic autoimmune, vascular, and fibrotic disease is heterogenous and characterized by initial predominant T-cell activation with altered repertoires, production of antigen-specific autoantibodies and cytokines, predominantly TH2, chemokines and growth factors release with pro-fibrotic properties. All these factors in turn contribute to diffuse microvascular injury, fibroblast activation, and increased production of collagen, leading to diffuse sclerosis within the skin and internal organs, namely the heart, the lungs, the kidneys and the intestinal tract. Two main clinical subsets, namely limited and diffuse cutaneous forms, can be distinguished by the extent of skin involvement, their autoantibody profile and the pattern of organ involvement. In severe forms of the disease, especially rapidly progressive diffuse cutaneous SSc, the 5-year mortality is estimated to be 40–50% [26–30]. The most abundant extracellular matrix protein (ECM) in tissues affected by systemic sclerosis seems to be the type 1 collagen [27]. In this context, it has been shown that fibroblasts culture from the affected skin
2.3. TGF-β/Smad3 signaling in fibrotic responses (in vivo data) TGF-β has been implicated as being an important mediator in a number of fibrotic diseases [17,18]. For example, expression of TGF-β and TGF-β receptors is elevated in the human fibroblasts of hypertrophic scars and of keloids [19,20]. The most direct evidence supporting the involvement of TGF-β in fibrosis are data showing that agents blocking the TGF-β function reduce the fibrotic response. In mice, progressive TGFβ1-induced lung fibrosis is blocked by an orally active TβRI kinase inhibitor [21] and the administration of TGF-β antibodies prevents skin and lung fibrosis in murine sclerodermatous graft-vs-host disease [22].
3. TGF-β/Smad and systemic sclerosis
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produce excessive amounts of various collagens associated with increased transcription rates of the corresponding genes or increased stability of collagen mRNA [27]. Although the mechanisms involved in the pathological increase of collagen expression in SSc have not been entirely elucidated, extensive recent efforts have been devoted to study the role of TGF-β signaling pathway by Smad proteins. 3.2. TGF-β/Smad in SSc The multifunctional cytokine, TGF-β and its intracellular pathways has been examined in patients with SSc. Some data have reported an increase of circulating TGF-β levels. Most of them observed a TGF-β immunoreactivity in affected scleroderma skin, which was present in the early inflammatory lesions before dense fibrosis had occurred [31]. Regarding the TGF-β signaling pathway, TGF-β receptors may be overexpressed or upregulated in SSc [31]. Immunohistochemistral analysis of skin biopsies performed in non-lesional areas from SSc patients and analysis of fibroblast cultures showed that Smad2 and Smad3 expression and their nuclear translocation were increased in these SSc patients [32]. However in these studies, no direct correlation between the status of Smad expression and the severity of the disease had been established. More recently, we showed that the extent of skin fibrosis, as clinically assessed by the modified Rodnan skin score, was directly correlated with the activation of TGF-β signaling pathway, as estimated by the levels of the Smad3 phosphorylation forms in protein extracts from SSc and normal fibroblasts obtained by explanting skin punch biopsy from SSc patients or healthy donors (Verrecchia et al., ACR Meeting 2005). One of the characterized mechanisms for inhibiting TGF-β signaling through the Smad pathway involves endogenous Smad7. Dong et al. reported a reduction of Smad7 expression in SSc derived fibroblast cultures as compared to fibroblast cultures from unaffected areas of the same patients, suggesting that defective Smad7 feedback inhibition could play a role in TGF-β hyperresponsiveness in SSc [33]. In contrast with these results, and as previously observed by Mori et al. [32], we found no difference between SSc and normal tissues regarding Smad4 and Smad7 expression when using immunohistochemistry analysis (Verrecchia et al., ACR Meeting, 2005). Variations in the two studies could partly be explained by differences in the skin biopsy sites, which in our work were obtained from lesional areas. In diffuse SSc, skin involvement concerns the whole body surface, although at variable severity degrees according to the various sites. Thus, sustained activation of TGF-β sig-
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naling pathway in fibroblast cultures may represent an intrinsic defect in SSc fibroblasts, that may contribute to the development of autonomous SSc lesions in the absence of altered circulatory or systemic factors. 4. Antifibrotic therapeutic strategies in SSc Several animal models, which may not fully be extrapolated to human disease, provided tools to study the antifibrotic effects of several drugs. In this rare and heterogenous disease prospective clinical trials are hard to perform. In a majority of SSc patients, clinical necessity often requires the simultaneous or successive use of vascular, immunosuppressive and antifibrotic therapies, and their respective benefits are hard to distinguish. To date no single treatment has yet been shown to prevent disease progression and to reverse fibrosis in SSc patients. Nonetheless, experimental data provide arguments to use therapeutic strategies aiming at immunomodulation and inhibition of TGF-β signaling. An increasing body of evidence demonstrates that inhibiting TGF-β signaling especially through blocking Smad3 can decrease fibrotic responses both in vitro and in vivo. Halofuginone, a semisynthetic plant alkaloid, and ALK5/TβRI small molecule inhibitors, have been shown to reduce the expression of collagen by fibroblasts and to prevent fibrotic responses in several animal models. Imatinib (Gleevec), a tyrosine kinase receptor inhibitor is also being tested [34]. Pirfenidone, an arginase enzyme inhibitor, downregulating some of TGF-β-dependent patterns of gene expression, was recently tested successfully in a double blind placebo control trial in patients with idiopathic pulmonary fibrosis [35]. The apparent effectiveness of anti-TGF-β antagonists in blocking fibrosis in animal models led to test the use of anti-TGF-β in diffuse SSc with successful safety and tolerability. However, modifications of TGF-β signaling may vary according to the clinical stage and subset of SSc [34]. Immunosuppressive therapy, given at an early stage of SSc disease, could be beneficial [36] and we recently reported (Verrechia et al., ACR 2005) that the use of broad spectrum immunosuppression could also interact with TGF-β modulation. Indeed, since 1998, we – as part of an European collaborative effort under the auspices of the EBMT (European Bone Marrow Transplant Association) — EULAR (European League Against Rheumatism) – and several other North American groups under the CIBMTR (Center for International Blood and Marrow Transplant Research), have used intensive myelo- and immunosuppression followed by autologous hematopoietic stem cell transplantation (HSCT) to treat severe SSc [37,38]. The early results from the phase I/II clinical trials
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showed a significant clinical regression of skin fibrosis and durable clinical improvement up to 7 years after autologous HSCT. When analyzing the regression of skin fibrosis after HSCT, we found a decrease in the extent of fibrosis first within in the papillary dermis, then in the deep dermis according to the extent of the disease regression. These results suggest that blockade of the autoimmune response after HSCT inhibits the signals leading to increased ECM synthesis. These promising clinical and histological results showing regression of skin fibrosis after HSCT will have to be confirmed in a larger number of patients after HSCT, as currently enrolled in the ongoing phase III European ASTIS trial (http://www.astistrial.com) [39], as well as in a similar study being started in the USA (SCOT trial), which uses irradiation with lung and kidney shielding in addition to anti-thymocytes globulins and cyclophosphamide as used in the ASTIS trial. Acknowledgments This work was supported by the Groupe Français de Recherche sur la Sclérodermie (GFRS, www.sclerodermie. org), the Association Française contre la Sclérodermie (ASF), INSERM (Institut Nationale de la Santé Et de la Recherche Médicale), Délégation Régionale à la Recherche Clinique (DRRC), Assistance Publique-Hôpitaux de Paris (AP-HP), the French Ministry of Health (Programme Hospitalier de Recherche Clinique: PHRC 1997 AOM 97030) and the Etablissement Français des Greffes (2003). Take-home messages • Exaggerated tissue deposition during the fibrosis process is largely due to an increase in the rate of transcription of type I collagen genes. • The Smad signaling pathway is crucial for simultaneous activation of fibrillar collagen genes by TGF-β • Smad3 has been implicated as being an important mediator in a number of fibrotic conditions. • A crucial element in the pathogenesis of SSc is the abnormal regulation of expression of the genes involved in the production and deposition of type I collagen. • Smad3 play a crucial role in TGF-β responsiveness to type 1 collagen gene. • Inhibition of TGF-β signaling can decrease the fibrotic responses. References [1] Feng XH, Derynck R. Specificity and versatility in TGFsignaling through Smads. Annu Rev Cell Dev Biol 2005.
[2] Verrecchia F, Mauviel A. Control of connective tissue gene expression by TGF beta: role of Smad proteins in fibrosis. Curr Rheumatol Rep 2002;4:143–9. [3] Shi Y, Massague J. Mechanisms of TGF-beta signaling from cell membrane to the nucleus. Cell 2003;113:685–700. [4] ten Dijke P, Hill CS. New insights into TGF-beta-Smad signalling. Trends Biochem Sci 2004;29:265–73. [5] Trojanowska M, LeRoy EC, Eckes B, Krieg T. Pathogenesis of fibrosis: type 1 collagen and the skin. J Mol Med 1998;76:266–74. [6] Uitto J, Kouba D. Cytokine modulation of extracellular matrix gene expression: relevance to fibrotic skin diseases. J Dermatol Sci 2000;24(Suppl 1):S60–9. [7] Verrecchia F, Mauviel A. Transforming growth factor-beta signaling through the Smad pathway: role in extracellular matrix gene expression and regulation. J Invest Dermatol 2002;118:211–5. [8] Verrecchia F, Mauviel A. TGF-beta and TNF-alpha: antagonistic cytokines controlling type I collagen gene expression. Cell Signal 2004;16:873–80. [9] Ghosh AK. Factors involved in the regulation of type I collagen gene expression: implication in fibrosis. Exp Biol Med (Maywood) 2002;227:301–14. [10] Chung KY, Agarwal A, Uitto J, Mauviel A. An AP-1 binding sequence is essential for regulation of the human alpha2(I) collagen (COL1A2) promoter activity by transforming growth factor-beta. J Biol Chem 1996;271:3272–8. [11] Verrecchia F, Tacheau C, Wagner EF, Mauviel A. A central role for the JNK pathway in mediating the antagonistic activity of pro-inflammatory cytokines against transforming growth factorbeta-driven SMAD3/4-specific gene expression. J Biol Chem 2003;278:1585–93. [12] Verrecchia F, Wagner EF, Mauviel A. Distinct involvement of the Jun-N-terminal kinase and NF-kappaB pathways in the repression of the human COL1A2 gene by TNF-alpha. EMBO Rep 2002;3:1069–74. [13] Chen SJ, Yuan W, Mori Y, Levenson A, Trojanowska M, Varga J. Stimulation of type I collagen transcription in human skin fibroblasts by TGF-beta: involvement of Smad 3. J Invest Dermatol 1999;112:49–57. [14] Poncelet AC, Schnaper HW. Sp1 and Smad proteins cooperate to mediate transforming growth factor-beta 1-induced alpha 2(I) collagen expression in human glomerular mesangial cells. J Biol Chem 2001;276:6983–92. [15] Verrecchia F, Chu ML, Mauviel A. Identification of novel TGFbeta/Smad gene targets in dermal fibroblasts using a combined cDNA microarray/promoter transactivation approach. J Biol Chem 2001;276:17058–62. [16] Yuan W, Varga J. Transforming growth factor-beta repression of matrix metalloproteinase-1 in dermal fibroblasts involves Smad3. J Biol Chem 2001;276:38502–10. [17] Flanders KC. Smad3 as a mediator of the fibrotic response. Int J Exp Pathol 2004;85:47–64. [18] Leask A, Abraham DJ. TGF-beta signaling and the fibrotic response. Faseb J 2004;18:816–27. [19] Chin GS, Liu W, Peled Z, Lee TY, Steinbrech DS, Hsu M, et al. Differential expression of transforming growth factor-beta receptors I and II and activation of Smad 3 in keloid fibroblasts. Plast Reconstr Surg 2001;108:423–9. [20] Schmid P, Itin P, Cherry G, Bi C, Cox DA. Enhanced expression of transforming growth factor-beta type I and type II receptors in wound granulation tissue and hypertrophic scar. Am J Pathol 1998;152:485–93.
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Oxidized low-density lipoprotein autoantibodies, chronic infections, and carotid atherosclerosis in a populationbased study Atherosclerosis is a chronic immuno-inflammatory disease wherein both oxidized lipids and infectious agents are incriminated as possible contributors. This is why associations between immune reactions to oxidized low-density lipoproteins (oxLDLs), chronic infections, and carotid atherosclerosis as quantified by ultrasound was investigated. Here, Mayer M. et. al. (J Am Cardiol 2006; 47:2436-43) measured immunoglobulin IgG and IgM autoantibody titers to copper-oxidized-LDL and malondialdehyde-LDL (oxLDL-AB), IgG and IgM apolipoprotein B-100immune complexes (ApoB-IC), and titers of antibodies to E. coli and chlamydial LPS, mycobacterial heat shock protein 65 (mHSP65), and evaluated their relationship to cardiovascular risk factors, chronic infections, and incident/progressive carotid atherosclerosis. The oxLDL-AB and ApoB-IC levels remained stable over time as indicated by strong correlations between 1995 and 2000 measurements (p < 0.001 each). Significant associations existed between all oxLDL markers and antibody titers to pathogens, especially to E. coli-LPS and mHSP65. The IgG oxLDL markers were positively and IgM markers were inversely associated with incident and progressive carotid atherosclerosis in univariate analyses but were not independent predictors in multivariate analyses. This study provides evidence for an association between human oxLDL markers and chronic infections. In this study, neither IgG nor IgM oxLDL autoantibodies were independently predictive of atherosclerosis progression in the carotid arteries.