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1.  Angiogenin and SDF-1α serum concentration in patients with systemic sclerosis in relation to clinical status 
Systemic sclerosis (SSc) is a connective tissue disorder characterized by tissue hypoxia due to vascular changes and excessive fibrosis of the skin and internal organs. Damage to blood vessels and endothelium, as well as imbalance of vascular homeostasis, impairment of angiogenesis and vasculogenesis are observed in the course of the disease. The aim of the study was to investigate the pro-angiogenic factors angiogenin and SDF-1α in patients with SSc.
Material and methods
Serum samples were collected from 50 patients with dSSc (diffuse SSc) and lSSc (limited SSc) and from 38 patients used as a healthy control group. We explored: 1) how the serum concentrations of SDF-1α and angiogenin differ in the investigated groups; 2) the correlation among chemokines in SSc and the duration of the disease, Raynaud’s phenomenon, sclerosis of the skin and TSS (total skin score).
Patients with SSc showed statistically significantly higher serum angiogenin concentration and there was no correlation between duration of the disease and Raynaud’s phenomenon, skin sclerosis or TSS. There was also no difference or no correlation between serum level of SDF-1α and the investigated groups.
The increase in angiogenin concentration in the serum in patients with SSc may confirm endothelial damage caused by hypoxia and reduced vascular perfusion due to the course of SSc without contributing to compensatory revascularization.
PMCID: PMC3258685  PMID: 22291739
systemic sclerosis; angiogenesis; vasculogenesis; angiogenin; SDF-1α
2.  Impaired Cav-1 expression in SSc mesenchymal cells upregulates VEGF signaling: a link between vascular involvement and fibrosis 
Systemic sclerosis (SSc) is characterized by vascular alteration and fibrosis, the former probably leading to fibrosis via the ability of both endothelial cells and pericytes to differentiate toward myofibroblast. It is well known that vascular endothelial growth factor A (VEGF-A, hereafter referred to as VEGF) may induce a profibrotic phenotype on perivascular cells. Caveolin-1 (Cav-1) is involved in the regulation of VEGF signaling, playing a role in the transport of internalized VEGF receptor 2 (VEGFR2) toward degradation, thus decreasing VEGF signaling. In this work, we assessed the levels of Cav-1 in SSc bone marrow mesenchymal stem cells (SSc-MSCs), a pericyte surrogate, and correlate these results with VEGF signaling, focusing onpotential pathogenic pathways leading to fibrosis.
We explored the VEGF signaling assessing: (1) Cav-1 expression; (2) its co-localization with VEGFR2; (3) the activity of VEGFR2, by IF, immunoprecipitation, and western blot. In SSc-MSCs, Cav-1 levels were lower when compared to healthy controls (HC)-MSCs. Furthermore, the Cav-1/VEGFR2 co-localization and the ubiquitination of VEGFR2 were impaired in SSc-MSCs, suggesting a decreased degradation of the receptor and, as a consequence, the tyrosine phosphorylation of VEGFR2 and the PI3-kinase-Akt pathways were significantly increased when compared to HC. Furthermore, an increased connective tissue growth factor (CTGF) expression was observed in SSc-MSCs. Taken together, these data suggested the upregulation of VEGF signaling in SSc-MSCs. Furthermore, after silencing Cav-1 expression in HC-MSCs, an increased CTGF expression in HC-MSCs was observed, mirroring the results obtained in SSc-MSCs, and confirming the potential role that the lack of Cav-1 may play in the persistent VEGF signaling .
During SSc, the lower levels of Cav-1 may contribute to the pathogenesis of fibrosis via an upregulation of the VEGF signaling in perivascular cells which are shifted to a profibrotic phenotype.
PMCID: PMC4166421  PMID: 25237397
Systemic sclerosis; Mesenchymal stem cells; Pericytes; Caveolin-1
3.  CD109, a TGF-β co-receptor, attenuates extracellular matrix production in scleroderma skin fibroblasts 
Arthritis Research & Therapy  2012;14(3):R144.
Scleroderma or systemic sclerosis (SSc) is a complex connective tissue disease characterized by fibrosis of skin and internal organs. Transforming growth factor beta (TGF-β) plays a key role in the pathogenesis of SSc fibrosis. We have previously identified CD109 as a novel TGF-β co-receptor that inhibits TGF-β signaling. The aim of the present study was to determine the role of CD109 in regulating extracellular matrix (ECM) production in human SSc skin fibroblasts.
CD109 expression was determined in skin tissue and cultured skin fibroblasts of SSc patients and normal healthy subjects, using immunofluorescence, western blot and RT-PCR. The effect of CD109 on ECM synthesis was determined by blocking CD109 expression using CD109-specific siRNA or addition of recombinant CD109 protein, and analyzing the expression of ECM components by western blot.
The expression of CD109 proteinis markedly increased in SSc skin tissue in vivo and in SSc skin fibroblasts in vitro as compared to their normal counterparts. Importantly, both SSc and normal skin fibroblasts transfected with CD109-specific siRNA display increased fibronectin, collagen type I and CCN2 protein levels and enhanced Smad2/3 phosphorylation compared with control siRNA transfectants. Furthermore, addition of recombinant CD109 protein decreases TGF-β1-induced fibronectin, collagen type I and CCN2 levels in SSc and normal fibroblasts.
The upregulation of CD109 protein in SSc may represent an adaptation or consequence of aberrant TGF-β signaling in SSc. Our finding that CD109 is able to decrease excessive ECM production in SSc fibroblasts suggest that this molecule has potential therapeutic value for the treatment of SSc.
PMCID: PMC3446527  PMID: 22694813
4.  A Possible Contribution of Altered Cathepsin B Expression to the Development of Skin Sclerosis and Vasculopathy in Systemic Sclerosis 
PLoS ONE  2012;7(2):e32272.
Cathepsin B (CTSB) is a proteolytic enzyme potentially modulating angiogenic processes and extracellular matrix remodeling. While matrix metalloproteinases are shown to be implicated in tissue fibrosis and vasculopathy associated with systemic sclerosis (SSc), the role of cathepsins in this disease has not been well studied. The aim of this study is to evaluate the roles of CTSB in SSc. Serum pro-CTSB levels were determined by enzyme-linked immunosorbent assay in 55 SSc patients and 19 normal controls. Since the deficiency of transcription factor Fli1 in endothelial cells is potentially associated with the development of SSc vasculopathy, cutaneous CTSB expression was evaluated by immunostaining in Fli1+/− and wild type mice as well as in SSc and control subjects. The effects of Fli1 gene silencing and transforming growth factor-β (TGF-β) on CTSB expression were determined by real-time PCR in human dermal microvascular endothelial cells (HDMECs) and dermal fibroblasts, respectively. Serum pro-CTSB levels were significantly higher in limited cutaneous SSc (lcSSc) and late-stage diffuse cutaneous SSc (dcSSc) patients than in healthy controls. In dcSSc, patients with increased serum pro-CTSB levels showed a significantly higher frequency of digital ulcers than those with normal levels. CTSB expression in dermal blood vessels was increased in Fli1+/− mice compared with wild type mice and in SSc patients compared with healthy controls. Consistently, Fli1 gene silencing increased CTSB expression in HDMECs. In cultured dermal fibroblasts from early dcSSc, CTSB expression was decreased compared with normal fibroblasts and significantly reversed by TGF-β1 antisense oligonucleotide. In conclusion, up-regulation of endothelial CTSB due to Fli1 deficiency may contribute to the development of SSc vasculopathy, especially digital ulcers, while reduced expression of CTSB in lesional dermal fibroblasts is likely to be associated with skin sclerosis in early dcSSc.
PMCID: PMC3285678  PMID: 22384200
5.  Decreased expression of the endothelial cell-derived factor EGFL7 in systemic sclerosis: potential contribution to impaired angiogenesis and vasculogenesis 
Arthritis Research & Therapy  2013;15(5):R165.
Microvascular damage and defective angiogenesis and vasculogenesis have a major role in the pathogenesis of systemic sclerosis (SSc). Epidermal growth factor-like domain 7 (EGFL7) is a proangiogenic molecule which is predominantly expressed and secreted by endothelial cells and their progenitors and controls vascular development and integrity. In this study, we investigated the possible involvement of EGFL7 in SSc.
Serum EGFL7 levels from 60 patients with SSc and 35 age- and sex-matched healthy controls were examined by colorimetric sandwich enzyme-linked immunosorbent assay. The expression of EGFL7 in forearm skin biopsies (n = 16 SSc, n = 10 controls), cultured dermal microvascular endothelial cells (MVECs) (n = 3 SSc, n = 3 controls) and late-outgrowth peripheral blood endothelial progenitor cell (EPC)-derived endothelial cells (n = 15 SSc, n = 8 controls) was investigated by immunofluorescence and Western blotting.
Serum EGFL7 levels were detectable in 68.6% of healthy controls and 45% of SSc cases (P < 0.05). Circulating levels of EGFL7 were significantly decreased in SSc patients compared with healthy controls (P = 0.01). Serum levels of EGFL7 were significantly lower in both limited cutaneous SSc and diffuse cutaneous SSc patients than in controls (P = 0.02 and P = 0.04, respectively). In SSc, decreased serum EGFL7 levels were significantly correlated with the severity of nailfold capillary abnormalities. Patients with the most severe capillary changes and digital ulcers had serum EGFL7 levels significantly lower than healthy controls, while the EGFL7 levels did not differ significantly between controls and SSc patients with less capillary damage and lack of digital ulcers. Endothelial EGFL7 expression was strongly downregulated or even almost completely undetectable in SSc-affected dermis compared with controls (P < 0.001). In cultured SSc dermal MVECs and late-outgrowth peripheral blood EPC-derived endothelial cells, EGFL7 was significantly downregulated compared with cells obtained from healthy subjects (P < 0.01 and P < 0.001, respectively).
Our findings suggest that the loss of EGFL7 expression in endothelial cells and their progenitors might play a role in the development and progression of peripheral microvascular damage and the defective vascular repair process characteristic of SSc.
PMCID: PMC3978601  PMID: 24286167
6.  Correlation of Endostatin and Tissue Inhibitor of Metalloproteinases 2 (TIMP2) Serum Levels With Cardiovascular Involvement in Systemic Sclerosis Patients 
Mediators of Inflammation  2005;2005(3):144-149.
Fibrosis of oesophagus, lungs, heart, and kidney in the course of systemic sclerosis (SSc) may lead to dysfunction of the above organs or even patients death. Recent studies point out the role of angiogenesis and fibrosis disturbances in the pathogenesis of SSc. Heart fibrosis is one of the most important prognostic factors in SSc patients. So, the aim of our study was to examine cardiovascular dysfunction in SSc patients and its correlation with serum levels of vascular endothelial growth factor (VEGF), endostatin, and tissue inhibitor of metalloproteinase 2 (TIMP2). The study group comprised 34 patients (19 with limited scleroderma (lSSc) and 15 with diffuse scleroderma (dSSc)). The control group consisted of 20 healthy persons, age and sex matched. Internal organ involvement was assessed on the basis of specialist procedures. Serum VEGF, endostatin, and TIMP2 levels were evaluated by ELISA. We found cardiovascular changes in 15 patients with SSc (8 with lSSc and 7 with dSSc). The observed symptoms were of different characters and also coexisted with each other. Higher endostatin serum levels in all systemic sclerosis patients in comparison to the control group were demonstrated (P < .05). Also higher serum levels of endostatin and TIMP2 were observed in patients with cardiovascular changes in comparison to the patients without such changes (P < .05). The obtained results support the notion that angiogenesis and fibrosis disturbances may play an important role in SSc. Evaluation of endostatin and TIMP2 serum levels seems to be one of the noninvasive, helpful examinations of heart involvement in the course of systemic sclerosis.
PMCID: PMC1526474  PMID: 16106100
7.  Autoantibodies to angiotensin and endothelin receptors in systemic sclerosis induce cellular and systemic events associated with disease pathogenesis 
Vasculopathy, inflammatory fibrosis and functional autoantibodies (Abs) are major manifestations of systemic sclerosis (SSc). Abs directed against the angiotensin II type 1 receptor (AT1R) and endothelin-1 type A receptor (ETAR) are associated with characteristic disease features including vascular, inflammatory, and fibrotic complications indicating their role in SSc pathogenesis. Therefore, the impact of anti-AT1R and anti-ETAR Abs on initiation of inflammation and fibrosis was analyzed.
Anti-AT1R and anti-ETAR Ab-positive immunoglobulin G (IgG) from SSc patients (SSc-IgG) was used for experiments. Healthy donor IgG served as a normal control, and AT1R and ETAR activation was inhibited by antagonists. Protein expression was measured with ELISA, mRNA expression with real time-PCR, endothelial repair with a scratch assay, and collagen expression with immunocytochemistry. Transendothelial neutrophil migration was measured with a culture insert system, and neutrophil ROS activation with immunofluorescence. Neutrophils in bronchoalveolar lavage fluids (BALFs) were analyzed microscopically after passive transfer of SSc-IgG or NC-IgG into naïve C57BL/6J mice. KC plasma levels were quantified by a suspension array system. Histologic analyses were performed by using light microscopy.
Anti-AT1R and anti-ETAR Ab-positive SSc-IgG induced activation of human microvascular endothelial cells (HMEC-1). Elevated protein and mRNA levels of the proinflammatory chemokine interleukin-8 (IL-8, CXCL8) and elevated mRNA levels of the vascular cell adhesion molecule-1 (VCAM-1) were induced in HMEC-1. Furthermore, activation of HMEC-1 with SSc-IgG increased neutrophil migration through an endothelial cell layer and activation of reactive oxygen species (ROS). SSc-IgG decreased HMEC-1 wound repair and induced type I collagen production in healthy donor skin fibroblasts. Effects of migration, wound repair, and collagen expression were dependent on the Ab-levels. Passive transfer of anti-AT1R and anti-ETAR Ab-positive SSc-IgG into naïve C57BL/6J mice increased neutrophil BALF counts. In parallel, increased levels of the murine functional IL-8 homologue, chemokine KC, were found in the plasma of SSc-IgG-treated mice as well as structural alterations of the lungs.
We conclude that angiotensin and endothelin-receptor activation via anti-AT1R and anti-ETAR Abs mediate pathogenic effects, indicating their contribution to pathogenesis of SSc. Therefore, anti-AT1R and anti-ETAR Abs could provide novel targets for therapeutic intervention in the treatment of SSc.
PMCID: PMC3978438  PMID: 24472528
8.  Thrombospondin 1 in hypoxia-conditioned media blocks the growth of human microvascular endothelial cells and is increased in systemic sclerosis tissues 
Systemic sclerosis (SSc) is a chronic inflammatory autoimmune disease characterised by vascular dysfunction and damage, excess collagen deposition and subsequent organ manifestations. Vasculopathy is an early feature of the disease which leads to a chronic hypoxic environment in the tissues. Paradoxically, there is a lack of angiogenesis. We hypothesised that this may in part be due to a nonphysiological, overriding upregulation in antiangiogenic factors produced by the hypoxic tissues. We considered thrombospondin 1 (TSP-1) as a candidate antiangiogenic factor.
Conditioned media from human microvascular endothelial cells cultured in both normoxic and hypoxic environments were able to block endothelial cell proliferation, with the latter environment having a more profound effect. Filtration to remove > 100-kDa proteins or heparin-binding proteins from the conditioned media eliminated their antiproliferative effect. TSP-1 was expressed in high concentrations in the hypoxic media, as was vascular endothelial growth factor (VEGF). Depletion of TSP-1 from the media by immunoprecipitation reduced the antiproliferative effect. We then show that, in a dose-dependent fashion, recombinant TSP-1 blocks the proliferation of endothelial cells. Immunohistochemistry of skin biopsy material revealed that TSP-1 expression was significantly higher throughout the skin of patients with SSc compared with healthy controls.
Despite the environment of chronic tissue hypoxia in SSc, there is a paradoxical absence of angiogenesis. This is thought to be due in part to aberrant expression of antiangiogenic factors, including TSP-1. We have demonstrated that TSP-1 is released in high concentrations by hypoxic endothelial cells. The conditioned media from these cells is able to block proliferation and induce apoptosis in microvascular endothelial cells, an effect that is reduced when TSP-1 is immunoprecipitated out. Further, we have shown that recombinant TSP-1 is able to block proliferation and induce apoptosis at concentrations consistent with those found in the plasma of patients with SSc and that its effect occurs in the presence of elevated VEGF levels. Taken together, these data are consistent with a model wherein injured microvascular cells in SSc fail to repair because of dysregulated induction of TSP-1 in the hypoxic tissues.
PMCID: PMC3130666  PMID: 21635730
9.  Systemic sclerosis sera affect fibrillin-1 deposition by dermal blood microvascular endothelial cells: therapeutic implications of cyclophosphamide 
Systemic sclerosis (SSc) is a connective tissue disorder characterized by endothelial cell injury, autoimmunity and fibrosis. The following three fibrillin-1 alterations have been reported in SSc. (1) Fibrillin-1 microfibrils are disorganized in SSc dermis. (2) Fibrillin-1 microfibrils produced by SSc fibroblasts are unstable. (3) Mutations in the FBN1 gene and anti-fibrillin-1 autoantibodies have been reported in SSc. Fibrillin-1 microfibrils, which are abundantly produced by blood and lymphatic microvascular endothelial cells (B-MVECs and Ly-MVECs, respectively), sequester in the extracellular matrix the latent form of the potent profibrotic cytokine transforming growth factor β (TGF-β). In the present study, we evaluated the effects of SSc sera on the deposition of fibrillin-1 and microfibril-associated glycoprotein 1 (MAGP-1) and the expression of focal adhesion molecules by dermal B-MVECs and Ly-MVECs.
Dermal B-MVECs and Ly-MVECs were challenged with sera from SSc patients who were treatment-naïve or under cyclophosphamide (CYC) treatment and with sera from healthy controls. Fibrillin-1/MAGP-1 synthesis and deposition and the expression of αvβ3 integrin/phosphorylated focal adhesion kinase and vinculin/actin were evaluated by immunofluorescence and quantified by morphometric analysis.
Fibrillin-1 and MAGP-1 colocalized in all experimental conditions, forming a honeycomb pattern in B-MVECs and a dense mesh of short segments in Ly-MVECs. In B-MVECs, fibrillin-1/MAGP-1 production and αvβ3 integrin expression significantly decreased upon challenge with sera from naïve SSc patients compared with healthy controls. Upon challenge of B-MVECs with sera from CYC-treated SSc patients, fibrillin-1/MAGP-1 and αvβ3 integrin levels were comparable to those of cells treated with healthy sera. Ly-MVECs challenged with SSc sera did not differ from those treated with healthy control sera in the expression of any of the molecules assayed.
Because of the critical role of fibrillin-1 in sequestering the latent form of TGF-β in the extracellular matrix, its decreased deposition by B-MVECs challenged with SSc sera might contribute to dermal fibrosis. In SSc, CYC treatment might limit fibrosis through the maintenance of physiologic fibrillin-1 synthesis and deposition by B-MVECs.
PMCID: PMC3978697  PMID: 23962393
Systemic sclerosis; blood and lymphatic microvascular endothelial cells; fibrillin-1; focal adhesion molecules; cyclophosphamide
10.  Angiotensin receptor type 1 and endothelin receptor type A on immune cells mediate migration and the expression of IL-8 and CCL18 when stimulated by autoantibodies from systemic sclerosis patients 
Agonistic autoantibodies (Aabs) against the angiotensin II receptor type 1 (AT1R) and the endothelin receptor type A (ETAR) have been identified in patients with systemic sclerosis (SSc). In our present study, we examined the expression of the AT1R and the ETAR in human immune cells and the pathological effects mediated through these receptors by their corresponding Aabs.
Protein expression of AT1R and ETAR on peripheral blood mononuclear cells (PBMCs) from healthy individuals and SSc patients was analyzed using flow cytometry, and mRNA expression of both receptors in PBMCs from healthy donors was examined by real-time PCR. In addition, PBMCs from healthy donors were stimulated in vitro with affinity-purified immunoglobulin G (IgG) fractions from SSc patients positive for AT1R and ETAR Aabs, as well as with IgG from healthy donors serving as controls. Alterations in cell surface marker expression, cytokine secretion and chemotactic motility were analyzed using flow cytometry, enzyme-linked immunosorbent assays and chemotaxis assays, respectively. The results were correlated with the characteristics and clinical findings of the IgG donors.
Both AT1R and ETAR were expressed on PBMCs in humans. Protein expression of both receptors was decreased in SSc patients compared with that of healthy donors and declined during the course of disease. IgG fractions of SSc patients positive for AT1R and ETAR Aabs induced T-cell migration in an Aab level–dependent manner. Moreover, IgG of SSc patients stimulated PBMCs to produce more interleukin 8 (IL-8) and chemokine (C-C motif) ligand 18 (CCL18) than did the IgG of healthy donors. All effects were significantly reduced by selective AT1R and ETAR antagonists. Statistical analysis revealed an association of SSc-IgG induced high IL-8 concentrations with an early disease stage and of high CCL18 concentrations with lung fibrosis onset and vascular complications in the respective IgG donors.
In our present study, we could demonstrate the expression of both AT1R and ETAR on human peripheral T cells, B cells and monocytes. The decreased receptor expression in SSc patients, the inflammatory and profibrotic effects upon Aab stimulation of PBMCs in vitro and the associations with clinical findings suggest a role for Aab-induced activation of immune cells mediated by the AT1R and the ETAR in the pathogenesis or even the onset of the disease.
PMCID: PMC4060229  PMID: 24612997
11.  Altered monocyte and fibrocyte phenotype and function in scleroderma interstitial lung disease: reversal by caveolin-1 scaffolding domain peptide 
Interstitial lung disease (ILD) is a major cause of morbidity and mortality in scleroderma (systemic sclerosis, or SSc). Fibrocytes are a monocyte-derived cell population implicated in the pathogenesis of fibrosing disorders. Given the recently recognized importance of caveolin-1 in regulating function and signaling in SSc monocytes, in the present study we examined the role of caveolin-1 in the migration and/or trafficking and phenotype of monocytes and fibrocytes in fibrotic lung disease in human patients and an animal model. These studies fill a gap in our understanding of how monocytes and fibrocytes contribute to SSc-ILD pathology. We found that C-X-C chemokine receptor type 4-positive (CXCR4+)/collagen I-positive (ColI+), CD34+/ColI+ and CD45+/ColI+ cells are present in SSc-ILD lungs, but not in control lungs, with CXCR4+ cells being most prevalent. Expression of CXCR4 and its ligand, stromal cell-derived factor 1 (CXCL12), are also highly upregulated in SSc-ILD lung tissue. SSc monocytes, which lack caveolin-1 and therefore overexpress CXCR4, exhibit almost sevenfold increased migration toward CXCL12 compared to control monocytes. Restoration of caveolin-1 function by administering the caveolin scaffolding domain (CSD) peptide reverses this hypermigration. Similarly, transforming growth factor β-treated normal monocytes lose caveolin-1, overexpress CXCR4 and exhibit 15-fold increased monocyte migration that is CSD peptide-sensitive. SSc monocytes exhibit a different phenotype than normal monocytes, expressing high levels of ColI, CD14 and CD34. Because ColI+/CD14+ cells are prevalent in SSc blood, we looked for such cells in lung tissue and confirmed their presence in SSc-ILD lungs but not in normal lungs. Finally, in the bleomycin model of lung fibrosis, we show that CSD peptide diminishes fibrocyte accumulation in the lungs. Our results suggest that low caveolin-1 in SSc monocytes contributes to ILD via effects on cell migration and phenotype and that the hyperaccumulation of fibrocytes in SSc-ILD may result from the altered phenotype and migratory activity of their monocyte precursors.
PMCID: PMC3155832  PMID: 21722364
12.  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.
PMCID: PMC3998379  PMID: 20863909
Fibrosis; Systemic sclerosis; Fibroblast; TGF-ß; Wnt; Collagen
13.  Lack of association between three vascular endothelial growth factor gene polymorphisms and systemic sclerosis: results from a multicenter EUSTAR study of European Caucasian patients 
Annals of the Rheumatic Diseases  2006;66(2):257-259.
Systemic sclerosis (SSc) is characterised by disturbed vessel morphology and an overproduction of vascular endothelial growth factor (VEGF). The VEGF gene located on chromosome 6p21.3 has several polymorphisms.
To test the hypothesis that disturbed angiogenesis may be related to the genetic background of the VEGF gene.
Materials and methods
EUSTAR centres included European Caucasian patients with SSc and matched controls with osteoarthritis. The VEGF gene was genotyped by polymerase chain reaction, followed by restriction enzyme analysis. The 634 C/T and 936 C/G mutations and an 18‐base pair insertion/deletion at −2549 of the VEGF promoter region were tested.
416 patients with SSc and 249 controls were included in the study population. Of the patients with SSc, 42% had a diffuse cutaneous subtype, 16% had increased pulmonary arterial pressure and 61% had decreased carbon monoxide diffusion capacity. The genotype frequencies in the patients with SSc and in controls were in Hardy–Weinberg equilibrium. The allele and genotype frequencies of the polymorphisms did not differ between patients with SSc and controls. No association was found between these polymorphisms and disease phenotypes.
This study shows that there is no association between the three selected functional VEGF polymorphisms and SSc.
PMCID: PMC1798516  PMID: 16740682
14.  A loss of telocytes accompanies fibrosis of multiple organs in systemic sclerosis 
Systemic sclerosis (SSc) is a complex connective tissue disease characterized by fibrosis of the skin and various internal organs. In SSc, telocytes, a peculiar type of stromal (interstitial) cells, display severe ultrastructural damages and are progressively lost from the clinically affected skin. The aim of the present work was to investigate the presence and distribution of telocytes in the internal organs of SSc patients. Archival paraffin-embedded samples of gastric wall, myocardium and lung from SSc patients and controls were collected. Tissue sections were stained with Masson's trichrome to detect fibrosis. Telocytes were studied on tissue sections subjected to CD34 immunostaining. CD34/CD31 double immunofluorescence was performed to unequivocally differentiate telocytes (CD34-positive/CD31-negative) from vascular endothelial cells (CD34-positive/CD31-positive). Few telocytes entrapped in the fibrotic extracellular matrix were found in the muscularis mucosae and submucosa of SSc gastric wall. In the muscle layers and myenteric plexus, the network of telocytes was discontinuous or even completely absent around smooth muscle cells and ganglia. Telocytes were almost completely absent in fibrotic areas of SSc myocardium. In SSc fibrotic lung, few or no telocytes were observed in the thickened alveolar septa, around blood vessels and in the interstitial space surrounding terminal and respiratory bronchioles. In SSc, the loss of telocytes is not restricted to the skin, but it is a widespread process affecting multiple organs targeted by the fibrotic process. As telocytes are believed to be key players in the regulation of tissue/organ homoeostasis, our data suggest that telocyte loss might have important pathophysiological implications in SSc.
PMCID: PMC3930412  PMID: 24467430
telocytes; systemic sclerosis; scleroderma; fibrosis; gastric wall; myocardium; lung; CD34; immunohistochemistry
15.  Angiogenic and angiostatic factors in systemic sclerosis: increased levels of vascular endothelial growth factor are a feature of the earliest disease stages and are associated with the absence of fingertip ulcers 
Arthritis Research  2002;4(6):R11.
To examine whether the lack of sufficient neoangiogenesis in systemic sclerosis (SSc) is caused by a decrease in angiogenic factors and/or an increase in angiostatic factors, the potent proangiogenic molecules vascular endothelial growth factor (VEGF) and basic fibroblast growth factor, and the angiostatic factor endostatin were determined in patients with SSc and in healthy controls. Forty-three patients with established SSc and nine patients with pre-SSc were included in the study. Serum levels of VEGF, basic fibroblast growth factor and endostatin were measured by ELISA. Age-matched and sex-matched healthy volunteers were used as controls. Highly significant differences were found in serum levels of VEGF between SSc patients and healthy controls, whereas no differences could be detected for endostatin and basic fibroblast growth factor. Significantly higher levels of VEGF were detected in patients with Scl-70 autoantibodies and in patients with diffuse SSc. Patients with pre-SSc and short disease duration showed significant higher levels of VEGF than healthy controls, indicating that elevated serum levels of VEGF are a feature of the earliest disease stages. Patients without fingertip ulcers were found to have higher levels of VEGF than patients with fingertip ulcers. Levels of endostatin were associated with the presence of giant capillaries in nailfold capillaroscopy, but not with any other clinical parameter. The results show that the concentration of VEGF is already increased in the serum of SSc patients at the earliest stages of the disease. VEGF appears to be protective against ischemic manifestations when concentrations of VEGF exceed a certain threshold level.
PMCID: PMC153841  PMID: 12453314
basic fibroblast growth factor; endostatin; fingertip ulcers; systemic sclerosis; vascular endothelial growth factor
16.  A System Out of Breath: How Hypoxia Possibly Contributes to the Pathogenesis of Systemic Sclerosis 
Systemic sclerosis (SSc) is an autoimmune disease characterized by vascular alterations and immunological disturbances and fibrosis, the order of which remains to be fully determined. Clinically, patients show clear signs of hypoxia in skin and internal organs. The low oxygen tension is potentially caused by a yet to be indentified circuitry involving the three features that typify SSc. In addition, once present, the hypoxia creates a vicious circle of ongoing pathology. In this paper, we provide an overview of the evidence that points towards the mechanisms causing hypoxia in SSc. In addition, data that suggest how hypoxia itself may orchestrate worsening of symptoms is presented. Altogether, it is clear that hypoxia is an important hallmark in SSc patients. By providing an overview of the mechanisms at play and the possible therapeutic avenues that have emerged, we hope to stimulate researchers to provide novel clues into the conundrum in SSc patients.
PMCID: PMC3228323  PMID: 22162692
17.  Genome-Wide Scan Identifies TNIP1, PSORS1C1, and RHOB as Novel Risk Loci for Systemic Sclerosis 
PLoS Genetics  2011;7(7):e1002091.
Systemic sclerosis (SSc) is an orphan, complex, inflammatory disease affecting the immune system and connective tissue. SSc stands out as a severely incapacitating and life-threatening inflammatory rheumatic disease, with a largely unknown pathogenesis. We have designed a two-stage genome-wide association study of SSc using case-control samples from France, Italy, Germany, and Northern Europe. The initial genome-wide scan was conducted in a French post quality-control sample of 564 cases and 1,776 controls, using almost 500 K SNPs. Two SNPs from the MHC region, together with the 6 loci outside MHC having at least one SNP with a P<10−5 were selected for follow-up analysis. These markers were genotyped in a post-QC replication sample of 1,682 SSc cases and 3,926 controls. The three top SNPs are in strong linkage disequilibrium and located on 6p21, in the HLA-DQB1 gene: rs9275224, P = 9.18×10−8, OR = 0.69, 95% CI [0.60–0.79]; rs6457617, P = 1.14×10−7 and rs9275245, P = 1.39×10−7. Within the MHC region, the next most associated SNP (rs3130573, P = 1.86×10−5, OR = 1.36 [1.18–1.56]) is located in the PSORS1C1 gene. Outside the MHC region, our GWAS analysis revealed 7 top SNPs (P<10−5) that spanned 6 independent genomic regions. Follow-up of the 17 top SNPs in an independent sample of 1,682 SSc and 3,926 controls showed associations at PSORS1C1 (overall P = 5.70×10−10, OR:1.25), TNIP1 (P = 4.68×10−9, OR:1.31), and RHOB loci (P = 3.17×10−6, OR:1.21). Because of its biological relevance, and previous reports of genetic association at this locus with connective tissue disorders, we investigated TNIP1 expression. A markedly reduced expression of the TNIP1 gene and also its protein product were observed both in lesional skin tissue and in cultured dermal fibroblasts from SSc patients. Furthermore, TNIP1 showed in vitro inhibitory effects on inflammatory cytokine-induced collagen production. The genetic signal of association with TNIP1 variants, together with tissular and cellular investigations, suggests that this pathway has a critical role in regulating autoimmunity and SSc pathogenesis.
Author Summary
Systemic sclerosis (SSc) is a connective tissue disease characterized by generalized microangiopathy, severe immunologic alterations, and massive deposits of matrix components in the connective tissue. Epidemiological investigations indicate that SSc follows a pattern of multifactorial inheritance; however, only a few loci have been replicated in multiple studies. We undertook a two-stage genome-wide association study of SSc involving over 8,800 individuals of European ancestry. Combined analyses showed independent association at the known HLA-DQB1 region and revealed associations at PSORS1C1, TNIP1, and RHOB loci, in agreement with a strong immune genetic component. Because of its biological relevance, and previous reports of genetic association at this locus with other connective tissue disorders, we investigated TNIP1 expression. We observed a markedly reduced expression of the gene and its protein product in SSc, as well as its potential implication in control of extra-cellular matrix synthesis, providing a new clue for a link between inflammation/immunity and fibrosis.
PMCID: PMC3131285  PMID: 21750679
18.  Systems Level Analysis of Systemic Sclerosis Shows a Network of Immune and Profibrotic Pathways Connected with Genetic Polymorphisms 
PLoS Computational Biology  2015;11(1):e1004005.
Systemic sclerosis (SSc) is a rare systemic autoimmune disease characterized by skin and organ fibrosis. The pathogenesis of SSc and its progression are poorly understood. The SSc intrinsic gene expression subsets (inflammatory, fibroproliferative, normal-like, and limited) are observed in multiple clinical cohorts of patients with SSc. Analysis of longitudinal skin biopsies suggests that a patient's subset assignment is stable over 6–12 months. Genetically, SSc is multi-factorial with many genetic risk loci for SSc generally and for specific clinical manifestations. Here we identify the genes consistently associated with the intrinsic subsets across three independent cohorts, show the relationship between these genes using a gene-gene interaction network, and place the genetic risk loci in the context of the intrinsic subsets. To identify gene expression modules common to three independent datasets from three different clinical centers, we developed a consensus clustering procedure based on mutual information of partitions, an information theory concept, and performed a meta-analysis of these genome-wide gene expression datasets. We created a gene-gene interaction network of the conserved molecular features across the intrinsic subsets and analyzed their connections with SSc-associated genetic polymorphisms. The network is composed of distinct, but interconnected, components related to interferon activation, M2 macrophages, adaptive immunity, extracellular matrix remodeling, and cell proliferation. The network shows extensive connections between the inflammatory- and fibroproliferative-specific genes. The network also shows connections between these subset-specific genes and 30 SSc-associated polymorphic genes including STAT4, BLK, IRF7, NOTCH4, PLAUR, CSK, IRAK1, and several human leukocyte antigen (HLA) genes. Our analyses suggest that the gene expression changes underlying the SSc subsets may be long-lived, but mechanistically interconnected and related to a patients underlying genetic risk.
Author Summary
Systemic sclerosis (SSc) is a rare autoimmune disease characterized by skin thickening (fibrosis) and progressive organ failure. Previous studies of SSc skin biopsies have identified molecular subsets of SSc based upon gene expression termed the inflammatory, fibroproliferative, normal-like, and limited intrinsic subsets. These gene expression signatures are large and although the biological processes are conserved, the exact list of genes can vary across datasets due to random variation, as well as minor differences in the composition of the study cohorts (e.g. early vs. late disease). We developed a computational tool to identify the consensus genes underlying the subsets across heterogeneous data and characterized the biological role of the consensus genes in SSc in order to obtain a systems level perspective of the SSc subsets. Our analysis reveals a complex network of genes connecting two of the major SSc intrinsic subsets, inflammatory and fibroproliferative. Many genetic loci associated with SSc risk show connections with the consensus genes of the intrinsic subsets, indicating that differential expression of genes defining the subsets may be related to genetic risk for SSc, thus for the first time placing the genetic risk factors in the context of, and showing putative relationships with, the intrinsic gene expression subsets.
PMCID: PMC4288710  PMID: 25569146
19.  Platelet-derived growth factor receptor-β and epidermal growth factor receptor in pulmonary vasculature of systemic sclerosis-associated pulmonary arterial hypertension versus idiopathic pulmonary arterial hypertension and pulmonary veno-occlusive disease: a case-control study 
Systemic sclerosis (SSc) complicated by pulmonary arterial hypertension (PAH) carries a poor prognosis, despite pulmonary vascular dilating therapy. Platelet-derived growth factor receptor-β (PDGFR-β) and epidermal growth factor receptor (EGFR) are potential therapeutic targets for PAH because of their proliferative effects on vessel remodelling. To explore their role in SScPAH, we compared PDGFR- and EGFR-mmunoreactivity in lung tissue specimens from SScPAH. We compared staining patterns with idiopathic PAH (IPAH) and pulmonary veno-occlusive disease (PVOD), as SScPAH vasculopathy differs from IPAH and sometimes displays features of PVOD. Immunoreactivity patterns of phosphorylated PDGFR-β (pPDGFR-β) and the ligand PDGF-B were evaluated to provide more insight into the patterns of PDGFR-b activation.
Lung tissue specimens from five SScPAH, nine IPAH, six PVOD patients and five controls were examined. Immunoreactivity was scored for presence, distribution and intensity.
All SScPAH and three of nine IPAH cases (P = 0.03) showed PDGFR-β-immunoreactivity in small vessels (arterioles/venules); of five SScPAH vs. two of nine IPAH cases (P = 0.02) showed venous immunoreactivity. In small vessels, intensity was stronger in SScPAH vs. IPAH. No differences were found between SScPAH and PVOD. One of five normal controls demonstrated focally mild immunoreactivity. There were no differences in PDGF-ligand and pPDGFR-b-immunoreactivity between patient groups; however, pPDGFR-b-immunoreactivity tended to be more prevalent in SScPAH small vasculature compared to IPAH. Vascular EGFR-immunoreactivity was limited to arterial and arteriolar walls, without differences between groups. No immunoreactivity was observed in vasculature of normals.
PDGFR-β-immunoreactivity in SScPAH is more common and intense in small- and post-capillary vessels than in IPAH and does not differ from PVOD, fitting in with histomorphological distribution of vasculopathy. PDGFR-β immunoreactivity pattern is not paralleled by pPDGFR-β or PDGF-B patterns. PDGFR-β- and EGFR-immunoreactivity of pulmonary vessels distinguishes PAH patients from controls.
PMCID: PMC3132056  PMID: 21492463
20.  Chronic Toll-like receptor 4 stimulation in skin induces inflammation, macrophage activation, transforming growth factor beta signature gene expression, and fibrosis 
Arthritis Research & Therapy  2014;16(4):R136.
The crucial role of innate immunity in the pathogenesis of systemic sclerosis (SSc) is well established, and in the past few years the hypothesis that Toll-like receptor 4 (TLR4) activation induced by endogenous ligands is involved in fibrogenesis has been supported by several studies on skin, liver, and kidney fibrosis. These findings suggest that TLR4 activation can enhance transforming growth factor beta (TGF-β) signaling, providing a potential mechanism for TLR4/Myeloid differentiation factor 88 (MyD88)-dependent fibrosis.
The expression of TLR4, CD14 and MD2 genes was analyzed by real-time polymerase chain reaction from skin biopsies of 24 patients with diffuse cutaneous SSc. In order to investigate the effects of the chronic skin exposure to endotoxin (Lipopolysaccharide (LPS)) in vivo we examined the expression of inflammation, TGF-β signaling and cellular markers genes by nanostring. We also identified cellular subsets by immunohistochemistry and flow cytometry.
We found that TLR4 and its co-receptors, MD2 and CD14, are over-expressed in lesional skin from patients with diffuse cutaneous SSc, and correlate significantly with progressive or regressive skin disease as assessed by the Delta Modified Rodnan Skin Score. In vivo, a model of chronic dermal LPS exposure showed overexpression of proinflammatory chemokines, recruitment and activation of macrophages, and upregulation of TGF-β signature genes.
We delineated the role of MyD88 as necessary for the induction not only for the early phase of inflammation, but also for pro-fibrotic gene expression via activation of macrophages. Chronic LPS exposure might be a model of early stage of SSc when inflammation and macrophage activation are important pathological features of the disease, supporting a role for innate immune activation in SSc skin fibrosis.
PMCID: PMC4227089  PMID: 24984848
21.  Impaired Endothelium-Mesenchymal Stem Cells Cross-talk in Systemic Sclerosis: a Link Between Vascular and Fibrotic Features 
To assess if an impaired cross-talk between endothelial cells (ECs) and perivascular/multipotent mesenchymal stem cells (MSCs) might induce a perturbation of vascular repair and leading to a phenotypic switch of MSC toward myofibroblast in Systemic Sclerosis (SSc).
We investigated different angiogenic and profibrotic molecules in a tridimentional matrigel assay, performing co-cultures with endothelial cells (ECs) and bone marrow derived MSCs from patients and healthy controls (HC). After 48 hours of co-culture, cells were sorted and analyzed for mRNA and protein expression.
ECs-SSc showed a decreased tube formation ability which is not improved by co-cultures with different MSCs. After sorting, we showed: i. an increased production of vascular endothelial growth factor A (VEGF-A) in SSc-MSCs when co-cultured with SSc-ECs; ii. an increased level of transforming growth factor beta (TGF-β) and platelet growth factor BB (PDGF-BB) in SSc-ECs when co-cultured with both HC- and SSc-MSCs; iii. an increase of TGF-β, PDGF-R, alpha smooth muscle actin (α-SMA) and collagen 1 (Col1) in both HC- and SSc-MSCs when co-cultured with SSc-ECs.
We showed that during SSc, the ECs-MSCs crosstalk resulted in an altered expression of different molecules involved in the angiogenic processes, and mainly SSc-ECs seem to modulate the phenotypic switch of perivascular MSCs toward a myofibroblast population, thus supporting the fibrotic process.
PMCID: PMC4206764  PMID: 25248297
22.  Ciprofloxacin has antifibrotic effects in scleroderma fibroblasts via downregulation of Dnmt1 and upregulation of Fli1 
Systemic sclerosis (SSc) is characterized by fibrosis of the skin and internal organs. The present study was undertaken to examine the effects of ciprofloxacin, a fluoroquinolone antibiotic implicated in matrix remodeling, on dermal and lung fibroblasts obtained from SSc patients. Dermal and lung fibroblasts from SSc patients and healthy subjects were treated with ciprofloxacin. Western blotting was used to analyze protein levels and RT-PCR was used to measure mRNA expression. The pharmacologic inhibitor UO126 was used to block Erk1/2 signaling. SSc dermal fibroblasts demonstrated a significant decrease in collagen type I mRNA and protein levels after antibiotic treatment, while healthy dermal fibroblasts were less sensitive to ciprofloxacin, downregulating collagen only at the protein levels. Connective tissue growth factor (CCN2) gene expression was significantly reduced and matrix metalloproteinase 1 (MMP1) levels were enhanced after ciprofloxacin treatment to a similar extent in healthy and SSc fibroblasts. Ciprofloxacin induced Erk1/2 phosphorylation, and Erk1/2 blockade completely prevented MMP1 upregulation. However, Smad1 and Smad3 activation in response to TGFβ was not affected. The expression of friend leukemia integration factor 1 (Fli1), a transcriptional repressor of collagen, was increased after treatment with ciprofloxacin only in SSc fibroblasts, and this was accompanied by a decrease in the levels of DNA methyltransferase 1 (Dnmt1). Similar effects were observed in SSc-interstitial lung disease (ILD) lung fibroblasts. In summary, our study demonstrates that ciprofloxacin has antifibrotic actions in SSc dermal and lung fibroblasts via the downregulation of Dnmt1, the upregulation of Fli1 and induction of MMP1 gene expression via an Erk1/2-dependent mechanism. Thus, our data suggest that ciprofloxacin may be an attractive therapy for SSc skin and lung fibrosis.
PMCID: PMC4035782  PMID: 23041765
systemic sclerosis; fibrosis; ciprofloxacin; collagen; matrix metalloproteinase 1; friend leukemia integration factor 1; DNA methyltransferase 1
23.  The HILDA Complex Coordinates a Conditional Switch in the 3′-Untranslated Region of the VEGFA mRNA 
PLoS Biology  2013;11(8):e1001635.
The HILDA complex coordinates three regulatory elements located in the 3′ UTR of the VEGFA mRNA in a RNA switch that controls translation in response to inflammation and hypoxia.
Cell regulatory circuits integrate diverse, and sometimes conflicting, environmental cues to generate appropriate, condition-dependent responses. Here, we elucidate the components and mechanisms driving a protein-directed RNA switch in the 3′UTR of vascular endothelial growth factor (VEGF)-A. We describe a novel HILDA (hypoxia-inducible hnRNP L–DRBP76–hnRNP A2/B1) complex that coordinates a three-element RNA switch, enabling VEGFA mRNA translation during combined hypoxia and inflammation. In addition to binding the CA-rich element (CARE), heterogeneous nuclear ribonucleoprotein (hnRNP) L regulates switch assembly and function. hnRNP L undergoes two previously unrecognized, condition-dependent posttranslational modifications: IFN-γ induces prolyl hydroxylation and von Hippel-Lindau (VHL)-mediated proteasomal degradation, whereas hypoxia stimulates hnRNP L phosphorylation at Tyr359, inducing binding to hnRNP A2/B1, which stabilizes the protein. Also, phospho-hnRNP L recruits DRBP76 (double-stranded RNA binding protein 76) to the 3′UTR, where it binds an adjacent AU-rich stem-loop (AUSL) element, “flipping” the RNA switch by disrupting the GAIT (interferon-gamma-activated inhibitor of translation) element, preventing GAIT complex binding, and driving robust VEGFA mRNA translation. The signal-dependent, HILDA complex coordinates the function of a trio of neighboring RNA elements, thereby regulating translation of VEGFA and potentially other mRNA targets. The VEGFA RNA switch might function to ensure appropriate angiogenesis and tissue oxygenation during conflicting signals from combined inflammation and hypoxia. We propose the VEGFA RNA switch as an archetype for signal-activated, protein-directed, multi-element RNA switches that regulate posttranscriptional gene expression in complex environments.
Author Summary
Many cells of our body, particularly cells such as monocyte/macrophages involved in host immunity, are exposed to diverse and constantly changing environments. These cells require mechanisms by which they can rapidly respond to multiple, sometimes conflicting, environmental cues to generate appropriate responses. The 3′ untranslated regions (UTRs), i.e. the noncoding tail of messenger RNAs, often contain multiple protein- and RNA-binding elements, thereby making it an ideal setting for receiving multiple such environmental cues, which can then be integrated into a single response that regulates the gene's expression. Monocytic cells exposed to inflammation and hypoxia produce vascular endothelial growth factor (VEGF)-A, a critical factor in blood vessel formation. VEGF-A expression is regulated under these conditions via a complex regulatory mechanism that involves its 3′UTR. Here we show how this regulatory switch works. Inflammation induces formation of a four-protein complex that binds an RNA element present in the VEGFA 3′UTR and blocks translation. Hypoxia, however, triggers the assembly of a completely different three-protein complex (termed “HILDA”) that coordinates the function of three neighboring RNA elements to “flip” the RNA conformation in such a way that prevents the first complex from binding, thereby allowing VEGF-A expression. We hypothesize that the VEGFA switch might function to ensure appropriate angiogenesis and tissue oxygenation when cells are exposed to conflicting signals from combined inflammation and hypoxia conditions.
PMCID: PMC3747992  PMID: 23976881
24.  Egr-1 Induces a Profibrotic Injury/Repair Gene Program Associated with Systemic Sclerosis 
PLoS ONE  2011;6(9):e23082.
Transforming growth factor-ß (TGF-ß) signaling is implicated in the pathogenesis of fibrosis in scleroderma or systemic sclerosis (SSc), but the precise mechanisms are poorly understood. The immediate-early gene Egr-1 is an inducible transcription factor with key roles in mediating fibrotic TGF-ß responses. To elucidate Egr-1 function in SSc-associated fibrosis, we examined change in gene expression induced by Egr-1 in human fibroblasts at the genome-wide level. Using microarray expression analysis, we derived a fibroblast “Egr-1-responsive gene signature” comprising over 600 genes involved in cell proliferation, TGF-ß signaling, wound healing, extracellular matrix synthesis and vascular development. The experimentally derived “Egr-1-responsive gene signature” was then evaluated in an expression microarray dataset comprising skin biopsies from 27 patients with localized and systemic forms of scleroderma and six healthy controls. We found that the “Egr-1 responsive gene signature” was substantially enriched in the “diffuse-proliferation” subset comprising exclusively of patients with diffuse cutaneous SSc (dcSSc) of skin biopsies. A number of Egr-1-regulated genes was also associated with the “inflammatory” intrinsic subset. Only a minority of Egr-1-regulated genes was concordantly regulated by TGF-ß. These results indicate that Egr-1 induces a distinct profibrotic/wound healing gene expression program in fibroblasts that is associated with skin biopsies from SSc patients with diffuse cutaneous disease. These observations suggest that targeting Egr-1 expression or activity might be a novel therapeutic strategy to control fibrosis in specific SSc subsets.
PMCID: PMC3172216  PMID: 21931594
25.  High serum sCD163/sTWEAK ratio is associated with lower risk of digital ulcers but more severe skin disease in patients with systemic sclerosis 
Systemic sclerosis (SSc) is an autoimmune disease characterized by chronic inflammation, vascular injury and excessive fibrosis. CD163 is a scavenger receptor which affects inflammatory response and may contribute to connective tissue remodelling. It has recently been demonstrated that CD163 can bind and neutralize the TNF-like weak inducer of apoptosis (TWEAK), a multifunctional cytokine which regulates inflammation, angiogenesis and tissue remodelling. We aimed to investigate the relationships between serum levels of soluble CD163 (sCD163) and soluble TWEAK (sTWEAK) in relation to disease manifestations in SSc patients.
This study included 89 patients with SSc who had not received immunosuppressive drugs or steroids for at least 6 months and 48 age- and sex-matched healthy controls (HC) from four European centres. Serum concentrations of sTWEAK and sCD163 were measured using commercially available ELISA kits.
The mean serum concentrations of sTWEAK were comparable between SSc patients (mean +/- SD: 270 +/- 171 pg/mL) and HC (294 +/- 147pg/mL, P >0.05). Concentration of sCD163 and sCD163/sTWEAK ratio were significantly greater in SSc patients (984 +/- 420 ng/mL and 4837 +/- 3103, respectively) as compared to HC (823 +/- 331 ng/mL and 3115 +/- 1346 respectively, P <0.05 for both). High sCD163 levels and a high sCD163/sTWEAK ratio (defined as > mean +2SD of HC) were both associated with a lower risk of digital ulcers in SSc patients (OR, 95%CI: 0.09; 0.01, 0.71, and 0.17; 0.06, 0.51, respectively). Accordingly, patients without digital ulcers had a significantly higher sCD163 concentration and sCD163/sTWEAK ratio as compared to SSc patients with digital ulcers (P <0.01 for both) and HC (P <0.05 for both). A high sCD163/sTWEAK ratio, but not high sCD163 levels, was associated with greater skin involvement.
The results of our study indicate that CD163-TWEAK interactions might play a role in the pathogenesis of SSc and that CD163 may protect against the development of digital ulcers in SSc. Further studies are required to reveal whether targeting of the CD163-TWEAK pathway might be a potential strategy for treating vascular disease and/or skin fibrosis in SSc.
PMCID: PMC4060194  PMID: 23800379

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