Eloisa Romano

Azienda Ospedaliero Universitaria Careggi, Florens, Tuscany, Italy

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Publications (48)324.61 Total impact

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    Full-text · Dataset · Nov 2015
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    ABSTRACT: Recent evidences suggest that patients with severe hemophilia B may have a less severe disease compared to severe hemophilia A. To investigate clinical, radiological, laboratory and histological differences in the arthropathy of severe hemophilia A and hemophilia B, 70 patients with hemophilia A and 35 with hemophilia B with at least one joint bleeding were consecutively enrolled. Joint bleedings (<10, 10-50, >50), regimen of treatment (prophylaxis/on demand), World Federation of Hemophilia, Pettersson and ultrasound scores, serum soluble RANK ligand and osteoprotegerin were assessed in all patients. RANK, RANK ligand and osteoprotegerin expression was evaluated in synovial tissue from 18 hemophilia A and 4 hemophilia B patients. The percentage of patients with either 10-50 or >50 hemarthrosis was greater in hemophilia A than in hemophilia B (p<0.001 and p=0.03, respectively), while that with <10 hemarthrosis was higher in hemophilia B (p<0.0001). World Federation of Hemophilia (36.6 vs 20.2, p<0.0001) and ultrasound (10.9 vs 4.3, p<0.0001) score mean values were significantly higher in hemophilia A patients. Serum osteoprotegerin and soluble RANK ligand were decreased in hemophilia A versus hemophilia B (p<0.0001 and p=0.006, respectively). Osteoprotegerin expression was markedly reduced in synovial tissue from hemophilia A patients. In conclusion, the reduced number of hemarthrosis, the lower World Federation of Hemophilia and ultrasound scores and higher osteoprotegerin expression in serum and synovial tissue in hemophilia B suggest that hemophilia B is a less severe disease than hemophilia A. Osteoprotegerin reduction seems to play a pivotal role in the progression of arthropathy in hemophilia A.
    Full-text · Article · Oct 2015 · Haematologica
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    ABSTRACT: Objectives: In systemic sclerosis (SSc), vascular involvement is characterised by vascular endothelial growth factor (VEGF)-A/VEGF receptor (VEGFR) system disturbances. Neuropilin-1 (NRP1), a receptor for both class-3 semaphorins (Sema3s) and VEGF-A, is required for optimal VEGF-A/VEGFR-2 signalling. Here, we investigated the possible involvement of Sema3A/NRP1 axis in SSc. Methods: Circulating Sema3A and soluble NRP1 (sNRP1) were measured in patients with SSc and controls. NRP1 and Sema3A expression in skin biopsies was evaluated by immunofluorescence and western blotting. NRP1 expression was assessed in SSc and healthy dermal microvascular endothelial cells (SSc-MVECs and H-MVECs), and in SSc and control endothelial progenitor cell (EPC)-derived endothelial cells (ECs). The possible impact of transcription factor Friend leukaemia integration 1 (Fli1) deficiency on endothelial NRP1 expression was investigated by gene silencing. The binding of Fli1 to NRP1 gene promoter was evaluated using chromatin immunoprecipitation. Capillary morphogenesis was performed on Matrigel. Results: Decreased sNRP1 levels in SSc were associated with active and late nailfold videocapillaroscopy patterns and digital ulcers. No difference in Sema3A was found between patients and controls. NRP1 was significantly decreased in SSc-MVECs both ex vivo and in vitro. NRP1 and Fli1 significantly decreased in H-MVECs challenged with SSc sera, while they were not different in SSc and control EPC-derived ECs. Fli1 occupied the NRP1 gene promoter and Fli1 gene silencing reduced NRP1 expression in H-MVECs. NRP1 gene silencing in H-MVECs resulted in a significantly impaired angiogenic capacity comparable to that of cells treated with SSc sera. Conclusion: In SSc, NRP1 deficiency may be an additional factor in the perturbed VEGF-A/VEGFR-2 system contributing to peripheral microvasculopathy and defective angiogenesis.
    No preview · Article · Sep 2015 · Annals of the rheumatic diseases
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    ABSTRACT: The vascular and nervous systems have several anatomic and molecular mechanism similarities. Emerging evidence suggests that proteins involved in transmitting axonal guidance cues, including members of class III semaphorin (Sema3) family, play a critical role in blood vessel guidance during physiological and pathological vascular development. Sema3E is a natural antiangiogenic molecule that causes filopodial retraction in endothelial cells, inhibiting cell adhesion by disrupting integrin-mediated adhesive structures. The aim of the present study was to investigate whether in systemic sclerosis (SSc) Plexin-D1/Sema3E axis could be involved in the dysregulation of vascular tone control and angiogenesis. Sema3E levels were measured by quantitative colorimetric sandwich ELISA in serum samples from 48 SSc patients, 45 subjects with primary Raynaud's phenomenon (pRP) and 48 age-matched and sex-matched healthy controls. Immunofluorescence staining on skin sections from 14 SSc patients and 12 healthy subjects was performed to evaluate Sema3E and Plexin-D1 expression. Western blotting was used to assess Plexin-D1/Sema3E axis in human SSc and healthy dermal microvascular endothelial cells (SSc-MVECs and H-MVECs, respectively) at basal condition and after stimulation with recombinant human vascular endothelial growth factor (VEGF), SSc and healthy sera. Capillary morphogenesis on Matrigel was performed on H-MVECs treated with healthy, pRP or SSc sera in the presence of Sema3E and Plexin-D1 soluble peptides. Serum Sema3E levels were significantly higher both in pRP subjects and SSc patients than in controls. In SSc, Sema3E levels were significantly increased in patients with early nailfold videocapillaroscopy (NVC) pattern compared to active/late patterns and pRP, and in patients without digital ulcers versus those with ulcers. In SSc skin, Sema3E expression was strongly increased in the microvascular endothelium. Cultured SSc-MVECs showed higher levels of phosphorylated Plexin-D1 and Sema3E expression than H-MVECs, and stimulation with SSc sera increased phosphorylated Plexin-D1 and Sema3E in H-MVECs. The addition of Sema3E-binding Plexin-D1 soluble peptide significantly attenuated the antiangiogenic effect of SSc sera on H-MVECs. Our findings suggest that Plexin-D1/Sema3E axis is triggered in SSc endothelium and may have a role in the dysregulation of angiogenesis and vascular tone control by inducing neuro-vascular mechanism alterations clinically evident in particular in the early disease phases.
    Full-text · Article · Aug 2015 · Arthritis research & therapy
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    ABSTRACT: In systemic sclerosis (SSc), clinical evidence has shown that Bosentan may foster the regeneration of the peripheral microcirculatory network. The aim of this study was to verify in vitro the influence of Bosentan on the angiogenic performance of dermal microvascular endothelial cells (MVECs) and its possible capacity to counteract the antiangiogenic effects of SSc sera. Healthy dermal MVECs were challenged with Bosentan at different concentrations (0.1 μM, 1 μM, 10 μM) or with sera from patients with diffuse cutaneous SSc (n=8) and healthy subjects (n=8), alone or in combination with Bosentan (10 μM). Cell viability and chemoinvasion were determined by WST-1 and Boyden chamber assays, respectively. Angiogenesis was evaluated by capillary morphogenesis on Matrigel. Challenge of dermal MVECs with SSc sera induced a significant reduction in angiogenesis (p<0.005 vs. basal condition; p<0.001 vs. healthy sera). The addition of Bosentan could significantly restore angiogenesis in the presence of SSc sera (p<0.01 vs. SSc sera alone). Healthy sera promoted cell viability which was, instead, significantly reduced with SSc sera (p<0.005 vs. healthy sera). The addition of Bosentan to MVECs challenged with SSc sera significantly increased cell viability (p<0.005 vs. SSc sera alone), reaching levels similar to MVECs treated with healthy sera. Co-incubation of MVECs with Bosentan and SSc sera significantly increased chemoinvasion (p<0.005 vs. SSc sera alone) which was inhibited by SSc sera (<0.001 vs. healthy sera). Bosentan effectively counteracts the antiangiogenic effects of SSc sera on dermal MVECs and fosters the restoration of a proangiogenic environment.
    No preview · Article · Jun 2015 · Clinical and experimental rheumatology
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    ABSTRACT: Background SSc is a vasculopathy characterised by dysregulation of angiogenesis and vascular tone control leading to severe ischemia and loss of capillaries. In the complex system of interconnection between blood vessels and nerves there is an emerging evidence suggesting that proteins involved in transmitting axonal guidance cues, such as class III semaphorins, play a role in the regulation of vascular development. Sema3E has been shown to repel endothelial cells, modulating the cytoskeleton and focal adhesion structures leading to cellular apoptosis. Objectives To investigate if soluble Sema3E could be involved in the dysregulation of vascular tone control in primary Raynaud's phenomenon (pRP) and in secondary RP investigating (Very Early Diagnosis of Systemic Sclerosis) VEDOSS and SSc patients. Methods Sema3E serum levels were measured by quantitative colorimetric sandwich ELISA in 102 pRP serum samples without autoantibodies positivity and without nailfold videocapillaroscopy (NVC) alterations, 100 SSc, 48 VEDOSS patients and 104 age- and sex-matched healthy controls. SSc and VEDOSS patients were evaluated for internal organ damage. Sema3E levels were expressed as median and range and compared by Mann-Whitney U test. p≤0.05 were considered statistically significant. Results Sema3E levels were significantly increased in pRP (median 1.00 ng/mL, range 0.00-5.84 ng/mL), VEDOSS (median 0.79 ng/mL, range 0.00-1.45 ng/mL) and SSc patients (median 0.85 ng/mL; range 0.00-18.01 ng/mL) versus healthy controls (median 0.55 ng/mL; range 0.00-1.47 ng/mL) (p<0.001). Sema3E was significantly higher in pRP than VEDOSS (p=0.025), while no significant differences were detected between pRP and SSc. In SSc with early NVC pattern, Sema3E levels were significantly increased (median 1.16 ng/mL range 0.63-9.53 ng/mL) in comparison to those detected in the late NVC pattern (median 0.75 ng/mL range 0.00-3.78 ng/mL, p=0.028). Moreover, Sema3E levels were significantly increased in SSc patients without ulcers (median 0.98 ng/mL; range 0.00-18.01 ng/mL) in comparison to those with digital ulcers (median 0.79 ng/mL; range 0.00-3.78 ng/mL, p=0.038). Sema3E was significantly higher in pRP compared to late SSc NVC pattern (p=0.04) and to SSc patients with digital ulcers (p=0.031). Conclusions Our data show that Sema3E sera levels are significantly increased in pRP, VEDOSS and SSc patients. Circulating Sema3E levels are higher in SSc patients without digital ulcers and with early NVC pattern. In particular, Sema3E may be involved in the early modifications of neuro-vascular mechanisms leading to dysregulation of vascular tone control. Disclosure of Interest None declared
    No preview · Article · Jun 2015 · Annals of the Rheumatic Diseases
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    ABSTRACT: Background The accumulation of myofibroblasts is responsible for uncontrolled production of extracellular matrix during pathological fibrosis. Myofibroblasts may arise from different sources, such as expansion and activation of resident tissue fibroblasts, transition of epithelial cells into mesenchymal cells, and recruitment of bone marrow-derived circulating fibrocytes. Even endothelial cells (ECs) have an ability to acquire myofibroblastic features, providing proof of principle for the process of EndoMT [1]. EndoMT is a transdifferentiation by which ECs disaggregate, lose polarity, and undergo a change in their typical shape, becoming elongated and acquiring the ability to migrate into the surrounding tissue. It is a phenotypic conversion in which ECs lose their specific markers such as CD31, von Willebrand factor and VE-cadherin, and acquire mesenchymal cell products such as alpha-smooth muscle actin (alpha-SMA), S100A4/fibroblast-specific protein 1 (FSP1) and type I collagen [1]. Previous studies have shown that EndoMT can be induced in vitro by transforming growth factor-beta and may have a role in experimental dermal fibrosis [1,2]. However, whether EndoMT may be involved in the pathogenesis of systemic sclerosis (SSc) remains to be determined. Objectives To investigate whether SSc dermal microvascular endothelial cells (dMVECs) might represent a source of myofibroblasts via EndoMT. Methods dMVECs were isolated from biopsies of the involved forearm skin from 6 patients with diffuse cutaneous SSc and from 6 healthy (H) subjects. CD31-positive cells were subjected to immunomagnetic isolation. The expression of CD31, VE-cadherin, alpha-SMA, S100A4/FSP1, CD90/Thy-1, type I collagen and Snail1 transcription factor was evaluated by immunofluorescence staining and Western blotting. Stress fibres were visualized with Alexa Fluor-488-conjugated phalloidin and cell nuclei were stained with DAPI. Results On light microscopy, H-dMVECs exhibited a typical EC morphology with a large and flattened shape, whereas the majority of SSc-dMVECs had an elongated shape often characterized by many branches. Both H-dMVECs and SSc-dMVECs were homogeneously positive for the pan-EC marker CD31. However, the expression of both CD31 and VE-cadherin was significantly decreased in SSc-dMVECs compared with H-dMVECs (both p<0.001). Conversely, the expression of the mesenchymal cell marker CD90/Thy-1 was significantly increased in SSc-dMVECs (p<0.001 vs H-dMVECs). SSc-dMVECs co-expressed alpha-SMA, S100A4/FSP1 and type I collagen, which were instead undetectable in H-dMVECs. The formation of stress fibres was strongly increased in SSc-dMVECs (p<0.001 vs H-dMVECs). Strong expression and nuclear localization of Snail1 were constitutively found in SSc-dMVECs. Conclusions Here, we provide the first evidence that SSc-dMVECs may undergo EndoMT resulting in an intermediate phenotype combining the expression of EC and myofibroblast markers and acquiring the capacity of type I collagen synthesis. In SSc, EndoMT might take center stage as a critical profibrotic switch and become a potential target for antifibrotic therapies. References Disclosure of Interest None declared
    No preview · Article · Jun 2015 · Annals of the Rheumatic Diseases
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    ABSTRACT: Background and objectives The vascular and nervous systems have several anatomic and molecular mechanism similarities. Emerging evidence suggests that proteins involved in transmitting axonal guidance cues, including class III semaphorin families, also play a critical role in blood vessel guidance during physiological and pathological vessel development. Sema3E is a natural antiangiogenic molecule that causes filopodial retraction in endothelial cells, inhibiting cell adhesion by disrupting integrin-mediated adhesive structures. The aim of the present study was to investigate if Plexin-D1/Sema3E axis could be involved in the dysregulation of vascular tone control (RF) in SSc. Materials and methods Sema3E levels were measured by quantitative colorimetric sandwich ELISA in serum samples from 45 subjects with primary Raynaud’s phenomenon (pRP), 48 SSc patients and 48 age- and sex-matched healthy controls. Western blot was used to evaluate Plexin-D1/Sema3E axis in human SSc and healthy dermal microvascular endothelial cells (SSc-MVEC and H-MVEC, respectively) at basal condition, and after stimulation with recombinant human vascular endothelial growth factor (VEGF), SSc and healthy sera (both n = 3). Immunofluorescence staining on skin sections from 14 SSc patients and 12 healthy subjects was performed in order to evaluate Sema3E and Plexin-D1 expression. Results Serum Sema3E levels were significantly higher both in pRP subjects and SSc patients than in sera controls. In SSc, Sema3E levels were significantly increased in patients with early nailfold videocapillascopy pattern (NVC) compared to active/late pattern and pRP, and in patients without ulcers compared to those with digital ulcers. SSc-MVEC showed higher levels of phosphorylated Plexin-D1 and Sema3E expression compared with H-MVEC, and stimulation with SSc sera increased the expression of phosphorylated Plexin-D1 and Sema3E in H-MVEC. In SSc, Sema3E expression was increased in the dermis, particularly in the vascular endothelium. Conclusions Our findings suggest that Plexin-D1/Sema3E axis is triggered in SSc and may have a role in the dysregulation of vascular tone control by inducing neuro-vascular mechanism alterations clinically evident in particular in the early disease phases.
    No preview · Article · Feb 2015 · Annals of the Rheumatic Diseases
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    ABSTRACT: Background and objectives Systemic Sclerosis (SSc) is an autoimmune connective tissue disease characterised by impairs angiogenesis, alterations of microcirculation, senescence and apoptosis of endothelial cells (ECs). A recent study demonstrated that soluble Klotho (sKl) protein interacts with the vascular endothelial growth factor receptor 2 (VEGFR-2) and with transient receptor potential canonical-1 (TRPC-1) by forming a complex on the surface of endothelial cells. This heterotrimeric complex is internalised in response to vascular endothelial growth factor (VEGF) stimulation, thus regulating VEGFR-2/TRPC-1–mediated Ca2+ influx, to maintain endothelial biological homeostasis. The aim of this study was to evaluate, if soluble Klotho might act as protective humoral factor on SSc Microvascular Endothelial Cells (MVECs) by inhibiting cellular senescence and inducing angiogenesis. Materials and methods Wound healing capacity was performed in healthy and SSc-MVECs under standard conditions and after sKl stimulation. Angiogenesis was evaluated by in vitro capillary morphogenesis on Matrigel in healthy and SSc-MVECs at basal condition, upon challenge with sKl, SSc and healthy sera, and sKl in combination with SSc and healthy sera. Western blot was performed in order to evaluate TRPC-1 and VEGFR-2 expression level in SSc and healthy MVECs at basal condition, after stimulation with sKl, SSc sera (n = 5), healthy sera (n = 5), and with sKl in combination with SSc and healthy sera before and after wound injury. Results Wound healing capacity significantly increased in healthy and SSc-MVECs after sKl challenge in respect to basal condition. Angiogenesis was significantly higher in SSc-MVECs upon challenge with sKl compared to both basal SSc and healthy MVEC. Moreover, angiogenesis was significantly increased in healthy MVECs challenged with SSc sera in combination with sKl respect to MVECs with SSc sera alone. TRPC-1 and VEGFR-2 expression level significantly increased under injury in both healthy and SSc-MVECs cultured with sKl respect to basal condition. Conclusions Our findings suggest that, under vascular injury conditions, sKl increases the wound healing ability and induces blood vessels formation in SSc-MVECs.
    Full-text · Article · Feb 2015 · Annals of the Rheumatic Diseases
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    ABSTRACT: Interstitial lung disease (ILD) is the leading cause of systemic sclerosis (SSc) related morbidity and mortality. LL-37 peptide is the only cathelicidin of the human antimicrobial peptide family with antimicrobial effects and immunomodulatory activity. LL-37 has anti-fibrotic effects and anti-apoptotic effects on SSc dermal fibroblasts. The aim of the study was to investigate the circulating levels of LL-37 in SSc patients and its association with clinical, laboratory, and instrumental parameters. Fifty-eight SSc patients (30 with and 28 without pulmonary involvement) and 28 healthy controls were enrolled in the study. Pulmonary involvement was defined when ILD was found at HRCT (ground glass, reticular, and honeycombing pattern). Circulating LL-37 levels were measured with ELISA test. In SSc patients with ILD serum, LL-37 concentrations were remarkably lower (1.36 mg/ml) than those in SSc patients without ILD (4.62 ng/ml, p = 0.035) and controls (5.53 ng/ml, p = 0.009). In SSc patients without ILD, serum LL-37 levels were not different from controls (p = 0.812). No significant association or correlation was found between LL-37 levels and any other clinical, serological, or instrumental features. Serum LL-37 levels are significantly lower in patients with SSc ILD. Our results may suggest that lower LL-37 levels may be associated with the development of ILD. Whether circulating levels of LL-37 might be used as an indirect marker of ILD remains to be determined in larger SSc cohorts.
    No preview · Article · Jan 2015 · Clinical Rheumatology
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    ABSTRACT: Vascular disease is a hallmark of systemic sclerosis (SSc). It is present in every patient, being responsible both for the earliest clinical manifestations and the major life-threatening complications of the disease, and thus determining important morbidity and mortality. In SSc, progressive vascular injury leads to vascular tone dysfunction and reduced capillary blood flow, with consequent tissue ischemia and chronic hypoxia. These phenomena are often accompanied by abnormal levels of vascular factors. Microangiopathy in SSc may be easily assessed by nailfold videocapillaroscopy. The variety of derangements detected in the nailfold capillaries is accompanied by abnormal levels of different vascular mediators and appears to be the best evaluable predictor of the development of peripheral vascular complications, such as digital ulcers. The purpose of this review is to summarize in SSc the most relevant vascular biomarkers and the main associations between vascular biomarkers and capillaroscopic parameters and/or the presence of digital ulcers. Vascular biomarkers could become useful predictive factors of vascular damage in SSc, allowing an earlier management of vascular complications. Copyright © 2014. Published by Elsevier B.V.
    No preview · Article · Dec 2014 · Autoimmunity Reviews
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    ABSTRACT: Objective: Rheumatoid arthritis (RA) is characterized by chronic synovial inflammation and hyperplasia. Tumor necrosis factor-α (TNF-α) plays a pivotal role in RA by interfering with the Fas-Fas ligand (FasL) proapoptotic pathway. We investigated the circulating levels of soluble Fas (sFas) and soluble FasL (sFasL), and their possible correlation with disease activity and improvement after anti-TNF-α treatment in RA. Methods: Serum levels of sFas and sFasL were measured by quantitative ELISA in 52 patients with RA before and after 3 months of anti-TNF-α treatment (adalimumab, n = 32; infliximab, n = 20). Disease activity measures [Disease Activity Score at 28 joints-erythrocyte sedimentation rate (DAS28-ESR), C-reactive protein (CRP)] were recorded before and after treatment. Forty age-matched and sex-matched healthy subjects served as controls. Results: No significant differences in serum sFas levels were detected between anti-TNF-α-naive patients with RA and controls. After anti-TNF-α treatment, serum sFas levels significantly increased in patients with RA compared to both anti-TNF-α-naive patients and controls. Increased sFas levels inversely correlated with disease activity variables (DAS28-ESR: r = -0.739, CRP: r = -0.636, both p < 0.001). No significant differences in sFasL levels were detected in patients with RA before and after anti-TNF-α treatment. Conclusion: In RA, an increase in sFas levels closely correlates with improvement in disease activity induced by TNF-α inhibitors, suggesting their ability to modulate Fas-mediated synoviocyte apoptosis.
    No preview · Article · Sep 2014 · The Journal of Rheumatology
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    ABSTRACT: Background: The main hallmark of systemic sclerosis (SSc) is vasculopathy characterised by dysregulation of angiogenesis and vascular tone leading to loss of capillaries. The vascular and nervous system have several anatomic similarities that extend to level of the molecular mechanisms. Emerging evidence suggests that proteins involved in transmitting axonal guidance cues, including class III semaphorin families, also play a critical role in blood vessel guidance during physiological and pathological vessel development. Sema3E acts through its receptor plexin-D1 to control endothelial cell positioning and patterning of the developing vasculature. Sema3E is a natural antiangiogenic molecule that causes filopodial retraction in endothelial cells inhibiting cell adhesion by disrupting integrin-mediated adhesive structures. Objectives: The aim of the present study was to investigate if plexin-D1/Sema3E axis could be involved in dysregulated vascular tone control (RF) characteristic of SSc. Methods: Sema3E levels were measured by quantitative colorimetric sandwich ELISA in serum samples from 45 subjects with primary Raynaud's phenomenon (PRF) without ANA, Scl70, ACA positivity, 48 SSc patients and 48 age- and sex-matched healthy controls. Patients were classified according to nailfold videocapillaroscopy (NVC) patterns (early, active and late). Sema3E levels were expressed as median and range and compared by Mann-Whitney U test. Differences were considered significant for p values less than 0.05. Western blot was used to evaluate plexin-D1/Sema3E axis in human dermal microvascular endothelial cells from healthy subjects (H-MVECs) at basal condition and after stimulation with recombinant human VEGF165 (10 ng/ml), lcSSc sera (n=3) and healthy sera (n=3) for 24h. Results: Sema3E sera levels were significantly higher both in PRF subjects (median 0.54 ng/ml) and SSc patients (median 0.67 ng/ml) respect to healthy controls (median 0.19 ng/ml) (both p<0.001). In particular, sema3E levels were significantly higher in SSc patients with early NVC pattern both respect to active/late pattern and PRF (both p<0.05). Moreover, sema3E levels were significantly increased in SSc patients without ulcers compared with patients with digital ulcers (p=0.018). H-MVECs stimulated with SSc sera and SSc-MVECs showed higher levels of the activated plexin-D1 form and sema3E protein expression in respect to basal H-MVECs and healthy sera. No differences were found in plexin-D1/Sema3E axis after challenging with VEGF. Conclusions: Circulating sema3E is significantly increased both in PRF and SSc. Higher sema3E levels are increased in the early stages of SSc without digital ulcers. Our findings suggest that plexin-D1/Sema3E axis might have a role in the dysregulation of vascular tone control. Disclosure of Interest: None declared DOI: 10.1136/annrheumdis-2014-eular.3227 Citation: Ann Rheum Dis 2014;73(Suppl2): 573 Session: Scleroderma, myositis and related syndromes - etiology, pathogenesis and animal models
    No preview · Article · Jun 2014 · Annals of the Rheumatic Diseases
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    ABSTRACT: Background Fibrosis of the skin and visceral organs is the hallmark of SSc and ILD is the leading cause of SSc related morbidity and mortality [1]. LL-37 peptide is the only cathelicidin of the human antimicrobial peptide family with antimicrobial effects and an immunomodulatory activity [2]. LL-37 was shown to decrease with age [3]. Recent data defined its anti-fibrotic effects on dermal fibroblasts and anti-apoptotic effects on SSc dermal fibroblasts [4]. Objectives To investigate the association between SSc related ILD and circulating levels of LL-37. Methods SSc patients and healthy controls aged 18-80, without signs or symptoms of systemic infection. Clinical data, autoantibody panel and internal organ assessment results (echocardiogram, esophageal manometry, chest HRCT, Lung Function Tests, Capillaroscopy). The pulmonary involvement was defined as SSc related ILD appearance on HRCT scans like ground-glass, reticular and honeycomb pattern. For the measurement of the LL-37 (Hycult Biotech) levels from blood samples of the patients ELISA has been used. Statistical analysis was performed with association/correlation test as appropriate. Results 58 SSc patients were enrolled and divided into 1.patients with pulmonary involvement (n=30), 2. patients without pulmonary involvement (n=28). 28 healthy subjects were used as controls. LL-37 concentrations were remarkably lower in SSc patients with ILD vs SSc patients without ILD (1,3575 ng/ml vs 4,6175 ng/ml, p=0,035) and control subjects (1,3575 ng/ml vs 5,5300 ng/ml, p=0,009). In SSc patients without pulmonary involvement, LL-37 serum levels were not different from controls (p=0,812). No association was found between LL37 circulating levels and any of the other clinical, laboratory or instrumental parameter recorded. Conclusions Significantly lower levels of LL-37 were observed in patients with SSc-ILD. Our results suggest that reduction of the levels of the peptide may be associated to the development of ILD. It remains to be tested in larger SSc cohorts if the circulating levels of LL-37 might be used as an indirect marker of ILD. References Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.2948
    No preview · Article · Jun 2014 · Annals of the Rheumatic Diseases
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    ABSTRACT: Background Systemic sclerosis (SSc) is a complex connective tissue disease characterized by extensive fibrosis and vascular abnormalities. Dermal capillaries are progressively reduced in number with consequent chronic tissue hypoxia insufficiently compensated by angiogenesis. In SSc, clinical studies reported that cyclophosphamide (CYC) treatment may significantly improve nailfold capillary damage. Moreover, our group has recently shown that the beneficial effects of CYC treatment in SSc may be due in part to the normalization of the endothelial cell-matrix interactions. Objectives In the present study, we evaluated the effects of sera from naïve or CYC-treated SSc patients on the in vitro capacity of human adult dermal blood microvascular endothelial cells (B-MVECs) to perform angiogenesis and to migrate and proliferate in response to injury. Methods Dermal B-MVECs were challenged with sera from SSc patients (n=21; n=13 limited SSc (lSSc) and n=8 diffuse SSc (dSSc)), treatment-naïve (n=8) or under CYC treatment (n=13), and sera from healthy controls (n=8). Angiogenesis was evaluated after 24 hours of cell seeding on Geltrex (reduced growth factor basement membrane matrix) in endothelial basal medium containing 2% fetal bovine serum and 10% control or SSc serum. The number of branching points was quantified. Wound healing assay was performed on confluent cells grown in 12-well plates and evaluated at 24 hours after wounding. Chemotaxis was assessed by using the Boyden chamber assay. Results Angiogenesis was significantly reduced upon challenge with sera from treatment-naïve SSc patients compared with healthy controls (p<0.005). Moreover, angiogenesis was significantly lower in the presence of naïve dSSc sera compared with naïve lSSc sera (p=0.02). Upon challenging of B-MVECs with sera from CYC-treated SSc patients, the angiogenic capacity was comparable to that of cells treated with healthy sera. Wound healing capacity was significantly decreased upon challenge with sera from both treatment-naïve and CYC-treated SSc patients compared with healthy controls (both p<0.005), with no difference between naïve and CYC-treated SSc sera. The Boyden chamber assay gave similar findings with significantly lower migration of B-MVECs in the presence either of treatment-naïve SSc or CYC-treated SSc sera compared with healthy sera (both p<0.001). Furthermore, both wound healing capacity and chemotaxis were significantly reduced upon challenge with naïve dSSc sera compared with naïve lSSc sera (p<0.001). Conclusions Treatment-naïve SSc sera have significant inhibitory effects on angiogenesis, wound healing capacity and chemotaxis of dermal B-MVECs. Moreover, the inhibitory effects of dSSc sera are significantly stronger than those of lSSc sera. Challenge with CYC-treated SSc sera effectively maintained B-MVEC angiogenesis on Geltrex at levels comparable to those of healthy control sera. Conversely, it was not able to specifically restore B-MVEC wound healing capacity and chemotaxis. Therefore, in SSc patients CYC treatment might foster angiogenesis mainly through the normalization of the endothelial cell invasive capacity and cell-matrix interactions. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.1905
    No preview · Article · Jun 2014 · Annals of the Rheumatic Diseases
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    ABSTRACT: Background In systemic sclerosis (SSc) vascular involvement is a primary event characterized by vascular tone dysfunction and increased circulating levels of vascular endothelial growth factor (VEGF). Neuropilin-1 (NRP1) is a receptor for both class-3 semaphorin (sema) family of axon guidance molecules and VEGF. NRP1 is required for optimal VEGF/VEGFR-2 signaling, and NRP1-deficient mice exhibit vascular defects including disorganized blood vessels, lack of normal branching and missing capillary networks. Sema3a controls physiological and pathological angiogenesis. Objectives In the present study, we investigated the possible involvement of sema3a/NRP1 axis in SSc and its correlation to nailfold videocapillaroscopy (NVC) patterns and digital ulcers. Methods Soluble NRP1 (sNRP1) and sema3a levels were measured by quantitative colorimetric sandwich ELISA in serum samples from SSc patients (n=49) and age- and sex-matched healthy controls (n=39). Patients were classified according to NVC patterns (early, active and late). NRP1 and sema3a protein expression was evaluated by immunofluorescence and western blot in skin biopsies from SSc patients (n=10) and healthy controls (n=8). NRP1 expression was also evaluated in human dermal microvascular endothelial cells from SSc patients (SSc-MVECs) and healthy controls (H-MVECs) at basal level, and in H-MVECs after stimulation for 24 hours with recombinant human VEGF165 (10 ng/ml), SSc sera (n=3) and healthy sera (n=3). Results Circulating sNRP1 levels were significantly reduced in SSc patients (median 0.22 ng/ml) compared with healthy controls (median 0.69 ng/ml) (p=0.005). In particular, sNRP1 levels were significantly lower in either SSc patients with active or late NVC patterns than in controls (both p<0.05). Moreover, sNRP1 levels were significantly decreased in SSc patients with digital ulcers compared both with patients without digital ulcers (p=0.009) and controls (p=0.001). No significant differences in sema3a levels were detected between patients and controls. NRP1 expression was decreased in clinically affected skin biopsies from SSc patients compared with healthy skin, especially in dermal endothelial cells and stromal cells. H-MVECs showed higher NRP1 protein expression compared with SSc-MVECs. Stimulation with recombinant VEGF165 strongly upregulated NRP1 expression in H-MVECs. NRP1 expression in H-MVECs increased after treatment with healthy sera compared with basal condition, while it decreased after challenging with SSc sera (both p<0.005 vs basal H-MVECs). Sema3a expression was not different in skin biopsies from SSc patients compared with controls. Conclusions NRP1 expression is significantly decreased in SSc, and lower sNRP1 levels correlate with the severity of nailfold capillary modifications and presence of digital ulcers. NRP1 might play a role in the vascular damage and in the impairment of the angiogenic process in SSc. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.3662
    No preview · Article · Jun 2014 · Annals of the Rheumatic Diseases
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    ABSTRACT: Background Haemophilic arthropathy (HA) may involve mainly the larger joints (target joint) presenting a spontaneous bleeding, haemarthrosis. Repeated bleeding episodes lead to synovitis implying synovial changes, cartilage and bone damages resulting in disability. The mechanisms and pathways of blood-induced damage in cartilage and bone are still not completely clear. Ultrasonography (US) is helpful to detect bone and cartilage alterations and synovitis. Power Doppler US (PDUS) may identify bleeding also in asymptomatic joints and is able to show different entity of haemarthrosis. Objectives To investigate the expression tissue of molecules involved in bone remodelling (RANK/RANKL/OPG system) in synovial tissue from haemophilic patients (HP) with severe HA scored by sonographic score (US score). Methods Synovial biopsies from 20 patients with HA and from 16 osteoarthritic (OA) patients, obtained during arthroplasty of the knee, were routinally processed for light microscopy. The severity of HA was evaluated according to a) US score, based on a semiquantitative method (score ranging from 0-21 with cut off>5), b) the World Federation of Haemophilia orthopaedic joint scale (WFH) and c) radiographic Petterson method. Synovial sections were stained with haematoxylin and eosin to evaluate pathological changes. The expression of RANK/RANK-L/OPG, involved in bone remodelling, was examined by immunohistochemistry. Moreover, levels of soluble RANKL and OPG in serum from 67 patients with HA and 30 healthy controls were measured by ELISA assay. Results The mean sonographic score in HA patients >5 was 11. The mean WFH clinical score was 39.5 point (range:12-57). The mean Pettersson score was 10.4 points (range: 6-12). Microscopically the synovium from patients with HA showed a large amount of intra- and extracellular haemosiderinic deposits. The lining layer showed mild proliferation and in the sublining area the vessels were characterized by thickened wall, due to a local and chronic inflammatory stimuli. A lower expression of OPG was found in HA synovium compared to OA. RANK and RANK-L positivity was strongly expressed in the lining and sublining both in HA and OA synovial tissue. Serum levels of sRANKL and OPG resulted lower than in controls. Conclusions In HA synovial tissue, the elevation of RANK/RANKL and the reduction of OPG suggests an osteoclastic activation. Tissue expression of OPG correlates with serum level of this protein in HP and with severity of HA by US score. Disclosure of Interest None Declared
    No preview · Article · Jan 2014 · Annals of the Rheumatic Diseases
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    ABSTRACT: Background Rheumatoid Arthritis (RA) is an autoimmune disease characterised by chronic inflammation and synovial hyperplasia caused by influx of inflammatory cells and by a reduced rate of programmed cell death. In RA synoviocytes seem to be resistant against apoptosis and not sensitive to TNF-induced apoptosis; by contrast, TNF induces them to proliferate. In this contest TNF- α strongly interfere with Fas (CD95)–Fas ligand (FasL, CD178) death receptor pathway. Biological drugs seem to be able to induce synovial apoptosis by inhibiting TNF-α functions. With the present study we want to investigate possible links between serum sFas and sFasL and anti-TNF therapy. Objectives to investigate whether sFas and sFasL serum levels can be associated to therapeutic response to TNF antagonists (anti-TNF) Infliximab and Adalimumab in patients with RA; to correlate these values with disease activity variables. Methods circulating levels of sFas and sFasL were investigated by specific immunosorbent assay on serum samples (52 RA patients: 36 female, 16 male; mean age: 45.7±12.2 years). The variables of disease activity (analyzed by DAS28, HAQ, CRP), physical function and sFas/sFasL serum levels were determined before and after 3 months of anti-TNF therapy (32 Adalimumab and 20 Infliximab). All patients were naïve to anti-TNF therapy. 40 (28 female, 12 male) age- and sex-matched healthy controls were used as controls. The sFas/sFasL levels are expressed as means ± standard deviations (SD) and compared by Student’s t-test and Mann-Whitney Test. Spearman rank correlation test (Rs) were employed to examine relationships between serum levels and disease activity variables (analyzed by DAS28, CRP). The differences are considered significant for p values <0.05. Results disease activity of patients was severe (baseline DAS28 5.6±1, HAQ of 3.5 (2.3 to 4.1 interquartile range) CRP 12 mg/l (IQR: 9 to 16). Before anti-TNF therapies sFas and sFasL levels were not different between RA patients and controls (5278.44±271.1 vs 5869.09±256.1 pg/ml for sFas; 56.72±20.32 vs 48.74±12.22 pg/ml for sFasL). After 3 months of Infliximab and Adalimumab therapy, RA patients showed higher concentration of serum sFas (9343.61±2356.6 p<0.01 for Infliximab; 7281±1123.3 p<0.05 for Adalimumab). Clinical response correlated with serum sFas levels after 3 months of anti-TNF treatment (Infliximab/DAS28: Rs= -0,681; Infliximab/PCR: Rs= -0,643; Adalimumab/DAS28: Rs= -0,743; Adalimumab/PCR: Rs= -0,746) as demonstrated by significantly reduction of CRP (0,6 mg/l; IQR 0,3 to 2,8) and DAS28 (2,8±0,5) (p<0,05). No differences were about serum sFasL levels before starting biological treatment and after 3 months of therapies (50.53±33.3 for Infliximab; 51.47±43.5 for Adalimumab). Conclusions after Infliximab and Adalimumab therapy RA patients showed higher serum levels of sFas, but no sFasL. This results also correlate with variables of disease activity. This may suggest that Infliximab and Adalimumab blocking TNF-α functions are able to reduce disease activity at least in part by promoting synovial apoptosis, particularly by interfering with Fas apoptotic pathway, as demonstrated by higher serum levels of sFas of RA patients after biological treatment. These data suggest that in patients receiving anti-TNF treatment higher levels of sFas may serve to predict remission. Disclosure of Interest None Declared
    No preview · Article · Jan 2014 · Annals of the Rheumatic Diseases
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    ABSTRACT: Haemophilic arthropathy (HA) is characterized by chronic proliferative synovitis leading to cartilage destruction and shares some pathological features with rheumatoid arthritis (RA). Apoptosis has been implicated in RA pathogenesis, and an agonistic anti-Fas monoclonal antibody (mAb) was found to induce RA fibroblast-like synoviocyte (FLS) apoptosis and suppress synovial hyperplasia in animal models of RA. The aim of this study was to evaluate the effect of anti-Fas mAb on HA-FLS. FLS were isolated from knee synovial biopsies from six HA patients, six RA patients and six healthy subjects. The expression of Fas in synovial biopsies was investigated by immunohistochemistry. FLS were stimulated with anti-Fas mAb at different concentrations, alone or in combination with tumour necrosis factor-α (TNF-α) and basic fibroblast growth factor (bFGF). Fas expression in FLS was assessed by Western blot. Cell viability was studied with the WST-1 assay. Active caspase-3 levels were measured using ELISA and Western blot. A strong Fas-immunoreactivity was observed in different cells of HA synovium, including FLS, inflammatory cells and endothelial cells. Fas antigen was constitutively overexpressed in cultured HA-FLS. Anti-Fas mAb had a significant cytotoxicity on HA-FLS in a dose-dependent manner, either alone or in combination with TNF-α and bFGF. These cytotoxic effects were due to the ability of anti-Fas to induce HA-FLS apoptosis, as shown by the increased active caspase-3 levels. Anti-Fas mAb exhibited a more pronounced pro-apoptotic effect on HA-FLS than RA-FLS. Fas antigen is highly expressed on HA-FLS and its stimulation by anti-Fas mAb may be an effective strategy to induce HA-FLS apoptosis.
    No preview · Article · Nov 2013 · Haemophilia
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    ABSTRACT: 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.
    Full-text · Article · Oct 2013 · Arthritis research & therapy