Eloisa Romano

University of Florence, Florens, Tuscany, Italy

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Publications (39)246.98 Total impact

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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.
    Clinical and experimental rheumatology 06/2015; · 2.97 Impact Factor
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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.
    Annals of the Rheumatic Diseases 02/2015; 74(Suppl 1):A67-A67. DOI:10.1136/annrheumdis-2015-207259.154 · 9.27 Impact Factor
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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.
    Annals of the Rheumatic Diseases 02/2015; 74(Suppl 1):A67-A68. DOI:10.1136/annrheumdis-2015-207259.155 · 9.27 Impact Factor
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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.
    Clinical Rheumatology 01/2015; 34(4). DOI:10.1007/s10067-014-2854-1 · 1.77 Impact Factor
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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.
    Autoimmunity Reviews 12/2014; 14(4). DOI:10.1016/j.autrev.2014.12.001 · 7.10 Impact Factor
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    ABSTRACT: Objective. Rheumatoid arthritis (RA) is characterized by chronic synovial inflammation and hyperplasia. Tumor necrosis factor-alpha (TNF-alpha) 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-alpha 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-alpha 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-alpha-naive patients with RA and controls. After anti-TNF-alpha treatment, serum sFas levels significantly increased in patients with RA compared to both anti-TNF-alpha-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-a treatment. Conclusion. In RA, an increase in sFas levels closely correlates with improvement in disease activity induced by TNF-alpha inhibitors, suggesting their ability to modulate Fas-mediated synoviocyte apoptosis.
    The Journal of Rheumatology 09/2014; 41(10). DOI:10.3899/jrheum.131544 · 3.17 Impact Factor
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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
    Annals of the Rheumatic Diseases 06/2014; 73(Suppl 2):573-573. DOI:10.1136/annrheumdis-2014-eular.3227 · 9.27 Impact Factor
  • Annals of the Rheumatic Diseases 06/2014; 73(Suppl 2):575-575. DOI:10.1136/annrheumdis-2014-eular.1905 · 9.27 Impact Factor
  • Annals of the Rheumatic Diseases 06/2014; 73(Suppl 2):867-868. DOI:10.1136/annrheumdis-2014-eular.2948 · 9.27 Impact Factor
  • Annals of the Rheumatic Diseases 01/2014; 71(Suppl 3):522-522. DOI:10.1136/annrheumdis-2012-eular.3097 · 9.27 Impact Factor
  • Annals of the Rheumatic Diseases 01/2014; 72(Suppl 3):A540-A540. DOI:10.1136/annrheumdis-2013-eular.1616 · 9.27 Impact Factor
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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.
    Haemophilia 11/2013; 20(1). DOI:10.1111/hae.12304 · 2.47 Impact Factor
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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.
    Arthritis research & therapy 10/2013; 15(5):R165. DOI:10.1186/ar4349 · 4.12 Impact Factor
  • Annals of the rheumatic diseases 04/2013; 72(8). DOI:10.1136/annrheumdis-2012-203183 · 9.27 Impact Factor
  • Annals of the rheumatic diseases 02/2013; 72(6). DOI:10.1136/annrheumdis-2012-202846 · 9.27 Impact Factor
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    ABSTRACT: Objective Systemic sclerosis (SSc) is characterized by early perivascular inflammation, microvascular endothelial cell (MVEC) activation/damage, and defective angiogenesis. Junctional adhesion molecules (JAMs) regulate leukocyte recruitment to sites of inflammation and ischemia-reperfusion injury, vascular permeability, and angiogenesis. This study was undertaken to investigate the possible role of JAMs in SSc pathogenesis. Methods JAM-A and JAM-C expression levels in skin biopsy samples from 25 SSc patients and 15 healthy subjects were investigated by immunohistochemistry and Western blotting. Subcellular localization of JAMs in cultured healthy dermal MVECs and SSc MVECs was assessed by confocal microscopy. Serum levels of soluble JAM-A (sJAM-A) and sJAM-C in 64 SSc patients and 32 healthy subjects were examined by enzyme-linked immunosorbent assay. Results In control skin, constitutive JAM-A expression was observed in MVECs and fibroblasts. In early-stage SSc skin, JAM-A expression was strongly increased in MVECs, fibroblasts, and perivascular inflammatory cells. In late-stage SSc, JAM-A expression was decreased compared with controls. JAM-C was weakly expressed in control and late-stage SSc skin, while it was strongly expressed in MVECs, fibroblasts, and inflammatory cells in early-stage SSc. Surface expression of JAM-A was higher in early-stage SSc MVECs and increased in healthy MVECs stimulated with early-stage SSc sera. JAM-C was cytoplasmic in resting healthy MVECs, while it was recruited to the cell surface upon challenge with early-stage SSc sera. Early-stage SSc MVECs exhibited constitutive surface JAM-C expression. In SSc, increased levels of sJAM-A and sJAM-C correlated with early disease and measures of vascular damage. Conclusion Our findings indicate that JAMs may participate in MVEC activation, inflammatory processes, and impaired angiogenesis in different stages of SSc.
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    ABSTRACT: Hemarthrosis triggers hemophilic arthropathy, involving the target joints. The histopathogenesis of blood-induced joint damage remains unclear. The triad of receptor activator of nuclear factor-κB (RANK), RANK ligand (RANKL), and osteoprotegerin (OPG; RANK-RANKL-OPG) controls bone turnover. Our aim was to evaluate RANK-RANKL-OPG expression in the synovium of hemophilic patients with severe arthropathy. Synovial biopsies were obtained from 18 patients with hemophilic arthropathy and 16 with osteoarthritis (OA) who were undergoing total knee replacement and synovectomy. The severity of hemophilic arthropathy was evaluated according to ultrasonography score, the World Federation of Hemophilia (WFH) orthopedic joint scale, and the radiographic Pettersson score. RANK-RANKL-OPG expression was examined by immunohistochemistry and Western blotting. Serum levels of soluble RANKL (sRANKL) and OPG from an extended group of 67 patients with hemophilic arthropathy and 30 healthy controls were measured by ELISA. The mean ultrasonography, WFH orthopedic joint scale, and Pettersson scores in patients with hemophilic arthropathy indicated severe arthropathy. A decreased expression of OPG was found in hemophilic arthropathy synovium compared with patients with OA. RANK and RANKL immunopositivity was strong in the lining and sublining layers in hemophilic arthropathy synovial tissue. Western blotting confirmed the immunohistological findings. Serum levels of sRANKL and OPG in patients with hemophilia were lower than in healthy controls. In hemophilic arthropathy, the synovium highly expressed RANK and RANKL, whereas OPG immunopositivity decreased, suggesting an osteoclastic activation. Low tissue expression of OPG paralleled the serum levels of this protein and the severity of hemophilic arthropathy assessed by ultrasonography, Pettersson, and WFH orthopedic joint scale scores.
    The Journal of Rheumatology 07/2012; 39(8):1678-86. DOI:10.3899/jrheum.120370 · 3.17 Impact Factor
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    ABSTRACT: Caveolin-1 (CAV1) is an inhibitor of tissue fibrosis and has been implicated in the pathogenesis of systemic sclerosis (SSc). The aim of the study was to analyse the possible association of CAV1 gene single nucleotide polymorphisms (SNP) with SSc. A total population of 3974 individuals (1355 SSc patients, 2619 controls) was studied. Genotype data for 23 SNP spanning the CAV1-CAV2 gene locus were obtained from a genome-wide scan conducted in a French population (564 SSc patients, 1776 controls). Three CAV1 SNP (rs926198, rs959173, rs9920) displaying the most significant associations with SSc and/or clinical phenotypes were then genotyped in an Italian population (791 SSc patients, 843 controls). CAV1 protein expression in skin biopsies was investigated by immunohistochemistry and western blotting. In the French population, the CAV1 rs959173 C minor allele showed a significant protective association with susceptibility to SSc (OR 0.71, 95% CI 0.59 to 0.86, p(adjusted)=0.009), and with the subset of patients with limited cutaneous SSc (OR 0.71, 95% CI 0.56 to 0.89, p(adjusted)=0.018). The association was replicated in the Italian population and strengthened in the combined populations through Cochran-Mantel-Haenszel meta-analysis (SSc: pooled OR 0.81, 95% CI 0.71 to 0.92, p=0.0018; limited cutaneous SSc: pooled OR 0.80, 95% CI 0.69 to 0.93, p=0.0053). Genotype/protein expression correlations revealed that the rs959173 C protective allele was associated with increased CAV1 protein expression. These results add CAV1 to the list of SSc susceptibility genes and provide further evidence for the contribution of this pathway in the fibrotic process that characterises SSc pathogenesis.
    Annals of the rheumatic diseases 03/2012; 71(6):1034-41. DOI:10.1136/annrheumdis-2011-200986 · 9.27 Impact Factor
  • Arthritis Research & Therapy 02/2012; 14(1). DOI:10.1186/ar3566 · 4.12 Impact Factor
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    ABSTRACT: To determine serum concentrations and tissue expression of matrix metalloproteinase-12 (MMP-12) and their correlation with clinical features in patients with systemic sclerosis (SSc). Serum MMP-12 levels from 72 patients with SSc and 42 healthy volunteers were examined by ELISA. Immunohistochemical expression of MMP-12 was analysed in skin biopsies from 20 patients with SSc and 13 healthy subjects and lung biopsies from three patients with SSc-related interstitial lung disease (ILD) and five controls. Circulating levels of MMP-12 were significantly increased in patients with SSc compared with healthy controls. Serum MMP-12 levels were significantly higher in both patients with limited cutaneous SSc and those with diffuse cutaneous SSc than in healthy controls, and correlated positively with the extent of skin involvement. MMP-12 levels were raised in SSc patients with ILD compared with patients without ILD, and correlated with severity of lung restriction. Increased serum levels of MMP-12 were also associated with the presence of digital ulcers and severity of nailfold capillary abnormalities. In contrast to almost undetectable MMP-12 expression in healthy skin, MMP-12 was strongly expressed in keratinocytes, dermal endothelial cells, fibroblasts/myofibroblasts and inflammatory cells in the skin of patients with SSc. Affected lung tissue from patients with SSc-related ILD showed strong MMP-12 expression in capillary vessels, inflammatory cells, alveolar macrophages and fibroblasts in the thickened alveolar septa, while faint expression was observed in normal lung tissue. MMP-12 levels are increased in patients with SSc and are associated with severity of skin and pulmonary fibrosis and peripheral vascular damage.
    Annals of the rheumatic diseases 01/2012; 71(6):1064-72. DOI:10.1136/annrheumdis-2011-200837 · 9.27 Impact Factor