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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. [ABSTRACT FROM AUTHOR]

Background: Mesenchymal stem cells can differentiate into endothelial cells and participate in angiogenesis in adults. In experimental models of acute myocardial infarction, mesenchymal stem cells led to the recovery of cardiac function through the formation of a new vascular network. Objective: To describe treatment with intravenous infusions of expanded autologous mesenchymal stem cells in 1 patient with critical limb ischemia due to systemic sclerosis. Design: Case report. Setting: The rheumatology unit at the University of Florence, Florence, Italy. Patient: A woman, aged 34 years, with systemic sclerosis who developed acute gangrene of the upper and lower limbs. Intervention: 3 intravenous pulses of expanded autologous mesenchymal stem cells. Measurements: Angiography, skin histopathology, and immunohistochemistry. Results: Areas of necrotic skin were reduced after the first mesenchymal stem-cell infusion. After the third infusion, angiography showed revascularization of the patient's extremities. Skin section analysis revealed cell clusters with tubelike structures, and angiogenic factors were strongly expressed. Limitation: Causality cannot be established by a single case. Conclusion: In patients with systemic sclerosis who have severe peripheral ischemia, intravenous infusion of expanded autologous mesenchymal stem cells may foster the recovery of the vascular network, restore blood flow, and reduce skin necrosis. Primary Funding Source: Fondazione Cassa di Risparmio di Pistoia e Pescia (partial funding). [ABSTRACT FROM AUTHOR]

Objective: Pregnant women with systemic sclerosis (SSc; scleroderma) have an increased risk of premature delivery and small full-term infants. During placental development, angiogenesis and vascular remodelling are essential for a successful pregnancy outcome. An analysis was made of the pathological changes and expression of angiogenic factors in SSc placentas. Methods: Placenta biopsies were obtained from three patients with SSc and four healthy uncomplicated pregnancies after delivery at 34–38 weeks of gestation. The sections were stained with Masson’s trichrome and phosphotungstic-acid-haematoxylin and immunostained for connective tissue growth factor (CTGF), α-smooth muscle actin (α-SMA), vascular endothelial growth factor (VEGF), placenta growth factor (PlGF) and receptors VEGFR-1 and VEGFR-2. Results: The pathological findings were signs of decidual vasculopathy, increased syncytiotrophoblast knotting, placental infarcts and villous hypoplasia. Severe and diffuse perivascular and stromal fibrosis of decidua and chorionic villi, and extensive deposition of fibrinoid material around decidual vessels and in intervillous spaces were observed. Strong CTGF expression in the vessel wall, decidual cells and fibroblasts and α-SMA+ myofibroblasts were found. VEGF and VEGFR-2 expression was stronger in SSc than in healthy placentas, while VEGFR-1 expression was similar to controls. PlGF immunopositivity was weaker in SSc. Conclusion: In SSc placentas, severe fibrosis and abnormal vascular remodelling were detected. This may result in reduced blood flow leading to deep sufferance of maternal placenta and possible premature delivery. [ABSTRACT FROM PUBLISHER]

Background: Induced sputum (IS) is a noninvasive tool, which can be used to collect cellular and soluble materials from lung airways. Objective: To evaluate if IS may be a useful and safe tool for the detection of airway inflammation in patients with interstitial lung disease (ILD) in systemic sclerosis (SSc). Methods: Sixty-eight patients with SSc and ILD as well as 18 healthy individuals (controls) were selected and submitted to IS examination. In 34 of 68 patients with SSc, bronchoalveolar lavage (BAL) was also performed. Safety of IS was assessed by comparison of forced expiratory volume in the first second (FEV1), FEV1/forced vital capacity ratio and peak expiratory flow before and after the IS procedure. Cell composition in samples collected by BAL and IS was correlated, and IS total and differential cell count in SSc patients and controls were compared. Results: The total number of cells was significantly higher in IS samples of SSc patients compared to those of healthy controls. Mean percentage of neutrophils was also higher in SSc patients (41.79 ± 23.89 vs. 27.37 ± 17.90), as well as lymphocytes (17.42 ± 19.70 vs. 3.13 ± 2.28) and eosinophils (2.35 ± 4.43 vs. 0.41 ± 0.46). On the other hand, mean percentage of macrophages was higher in healthy individuals (69.10 ± 19.15 vs. 36.96 ± 20.68). In fluid recovered by BAL, the most frequent cells were macrophages (67.89% ± 17.26), while neutrophils (14.77 ± 17.18%) and lymphocytes (15.62 ± 13.46%) were less frequent and eosinophils (1.66 ± 2.08%) were rare. A similar pattern of cell composition was found in IS samples (41.15 ± 21.67% of macrophages, 39.72 ± 23.15% of neutrophils, 15.28 ± 19.46% of lymphocytes and 2.56 ± 5.03% of eosinophils). Strength of correlation between BAL and IS was significant for macrophages and neutrophils. After IS procedure was performed, improvement of FEV1 (mean value before IS was 85.09 ± 14.44 and 88.93 ± 16.40 after IS) and FEV1/forced vital capacity (mean value before IS was 98.53 ± 12.11 and 105.22 ± 10.78 after IS) was observed. Conclusion: The IS method may allow a noninvasive assessment of cell composition in airway fluid and may contribute to the better understanding of upper/medium airway inflammation in SSc. Future studies are needed to verify whether IS can replace invasive procedures for the detection and monitoring of lung inflammation in SSc. Copyright © 2008 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Objective: To investigate the possible implication of -3′ polymorphism in systemic sclerosis (SSc) susceptibility or clinical phenotype, or both. Methods: 150 patients with SSc and 150 controls were enrolled. Skin involvement, autoantibodies, interstitial lung disease, pulmonary arterial hypertension (PAH), scleroderma renal crisis, past and/or current skin ulcers were assessed. Genotyping was performed by PCR-RFLP. Results: Genotype distribution and allele frequency were similar in SSc and controls. -3′A allele and -3′GA/AA genotype frequencies were significantly higher in SSc-PAH than in SSc-non-PAH (33.3% vs 18.3%, p = 0.01) and in SSc with skin ulcers than in SSc without ulcers (27.3% vs 16.9%, p = 0.03). The -3′A allele influenced the predisposition to SSc-related PAH (OR = 2.52, 95% CI 1.11 to 5.69, p = 0.02) and skin ulcers (OR = 2.31, 95% CI 1.18 to 4.52, p = 0.01). After adjustment for age and gender, the -3′A allele remained a susceptibility factor for the SSc-related vascular manifestations (PAH: OR = 2.37, 95% CI 1.04 to 5.42, p = 0.04; ulcers: OR = 2.33, 95% CI 1.78 to 4.62, p = 0.01). Conclusion: The -3′A allele is significantly associated with microvascular involvement in SSc. [ABSTRACT FROM PUBLISHER]

OBJECTIVE: In systemic sclerosis (SSc) microvascular endothelial cells (MVECs), angiogenesis is blocked by matrix metalloproteinase 12-dependent cleavage of domain 1 of the urokinase-type plasminogen activator receptor (uPAR). Since integrins are associated with the invasive activity of uPAR in angiogenesis, this study was undertaken to show whether full-size and truncated uPAR are differentially associated with integrins and with motor components of the cytoskeleton. METHODS: SSc and normal MVECs were isolated from human skin biopsy specimens and studied by confocal laser scanning microscopy and immunoprecipitation to assess the mechanisms of association of truncated and full-size uPAR with integrins and the actin cytoskeleton. The integrin composition of the MVECs was studied by reverse transcription-polymerasechain reaction. Cell migration and capillary morphogenesis were studied on fibrinogen substrates. Involvement of Rac and Cdc42 was evaluated by Western blotting. RESULTS: Only full-size uPAR showed a connection with the actin cytoskeleton in ECs. This connection was mediated by the uPAR-associated alphaMu- and alphaX-subunits of beta2 integrin, and was absent from SSc MVECs. The cleaved uPAR was not associated with beta2 integrins or with actin. beta3 integrins were associated with both the full-size and cleaved uPAR at focal contacts. The uncoupling of uPAR from beta2 integrins in SSc MVECs impaired the activation of Rac and Cdc42 (thus inhibiting their mediation of uPAR-dependent cytoskeletal rearrangements and cell motility) and blocked the integrin-engagement-delivered signals to the actin cytoskeleton. Invasion and capillary morphogenesis on fibrinogen-coated substrates indicated that ligation of uPAR by uPA empowers the beta2/beta3 integrin-dependent invasion of fibrinogen, and that this system is impaired in SSc MVECs. CONCLUSION: The reduced angiogenic properties of SSc MVECs can be explained by the effects of uPAR truncation and the subsequent loss of the beta2 integrin-mediated connection of uPAR with the actin cytoskeleton in these ECs. [ABSTRACT FROM AUTHOR]

OBJECTIVE: Systemic sclerosis (SSc) is characterized by early endothelial damage evolving to vascular desertification. Stromal cell-derived factor 1 (SDF-1) and its receptor CXCR4 regulate specific steps in new vessel formation. We undertook this study to determine whether an alteration of the SDF-1/CXCR4 axis might be involved in the pathogenetic mechanisms following ischemic damage during SSc. METHODS: We enrolled 36 SSc patients and 15 controls. Skin biopsy samples were obtained from each subject, and the expression of SDF-1 and CXCR4 was assessed by immunohistochemistry, reverse transcription-polymerase chain reaction (RT-PCR), and Western blot analyses. Furthermore, isolated microvascular endothelial cells (MVECs) from 4 patients with diffuse cutaneous SSc (dcSSc) and 3 controls were analyzed for SDF-1 and CXCR4 by confocal laser scanning microscopy, RT-PCR, and Western blotting. RESULTS: SDF-1 and CXCR4 were up-regulated in the skin of patients with early (edematous) SSc, both in the diffuse and limited cutaneous forms, and progressively decreased, with the lowest expression in the latest phases of both SSc subsets. MVECs from patients with dcSSc expressed significantly higher amounts of both isoforms of SDF-1 in the early stage of disease, with a progressive reduction of SDF-1 and CXCR4 in later stages. On the surface of cultured MVECs from patients with dcSSc, SDF-1 and CXCR4 colocalized in polarized areas, suggesting that they are activated in vivo and that they are under strict genetic control to retain capping function. CONCLUSION: Due to its transient expression, SDF-1 could be considered a future therapeutic target to induce new vessel formation in SSc. [ABSTRACT FROM AUTHOR]

Background: It is well documented that exercise reduces the risk of thromboembolic disease, possibly by increasing the plasma concentration of anticoagulant-antithrombotic compounds. Objectives: As plasma glycosaminoglycans (GAGs) play a role in the anticoagulant-antithrombotic potential of plasma, to examine the concentration and profile of these compounds in well trained, long distance runners and sedentary subjects. Methods: Plasma GAGs were measured in 10 male, long distance runners and 10 sedentary counterparts before and after ergometric tests. GAGs were extracted, purified, and identified by electrophoretic and enzymatic methods, and measured as hexosamine. Results: Plasma GAGs found in sedentary subjects were slow migrating heparan sulphates I and II, keratan sulphate I, and chondroitin 4-6-sulphate. Those found in trained athletes were slow migrating heparan sulphate I, chondroitin 4-6-sulphate (or keratan sulphate I), and fast migrating heparan sulphate. Total plasma concentrations of GAGs were higher in athletes than in sedentary subjects at rest. In sedentary subjects, plasma GAGs did not change after cycle ergometric exercise at 80% of their anaerobic threshold. However, the appearance of a novel band of heparan sulphate migrating faster than fast migrating heparan sulphate was observed in athletes after exercise. Conclusions: Exercise changes the amount and profile of plasma GAGs; these changes may play a role in protecting subjects who practise aerobic sports against developing cardiovascular disease. [ABSTRACT FROM AUTHOR]

We evaluated the changes in sarcoplasmic reticulum (SR) function and the parallel hemodynamic and morphological modifications in a heart subjected to volume overload. We also determined the levels of acylphosphatase, a cytosolic enzyme, that could play a regulatory effect on SR Ca2+ pump by hydrolyzing the phosphorylated intermediate of this transport system. For this, swine hearts were subjected to volume overload by aorta-cava shunt for 1, 2, or 3 months. Changes in heart contractility reflected modifications of SR function, whose reduction after 1 month of overload was followed by a gradual recovery. A decrease in SERCA2a protein and mRNA content was shown from 1 month and remained for the following 2 months. Phospholamban content and its phosphorylation status were not modified. Acylphosphatase was unchanged at 1 month, but at 2 months this enzyme exhibited an increased activity, protein and mRNA expression. Morphological alterations consisting of the cytoskeletal architectures, intermyofibrillar oedema, swollen mithochondria and abnormality of the membrane system (T-tubule and SR cisternae) were particularly evident after 1 month but almost disappeared after 3 months. These results suggest that our overloaded hearts underwent a substantial recovery of their structural and biochemical properties at 3 months after surgery. A possible involvement of acylphosphatase in the modification of SR function is discussed. [ABSTRACT FROM AUTHOR]

Objective Junctional adhesion molecule (JAM)-A and JAM-C regulate leucocyte-endothelial cell (EC) interactions. JAMs also interact at inter-EC junctions regulating vascular permeability and are implicated in pathophysiological processes involving leucocyte transmigration, tight junction assembly and angiogenesis. SSc is characterised by early perivascular inflammatory infiltrates, EC damage and defective angiogenesis. We investigated the expression of JAMs in SSc skin and dermal microvascular ECs (MVECs) challenged with SSc sera. Methods Skin biopsies were obtained from the involved skin of 16 SSc patients (10 early, 6 late phase) and from 10 controls. Skin sections were stained with primary antibodies (Abs) anti-human JAM-A or JAM-C followed by fluorochrome-conjugated secondary Abs. The lymphatic EC specific marker podoplanin (D2-40) was used to differentiate blood (D2-40–) and lymphatic (D2-40+) vessels. Human dermal MVECs were stimulated with VEGF or early SSc (n=5) and control (n=4) sera for 1, 6, 24 h. MVECs were double-immunolabeled for JAM-C and the human tight junction protein zonula occludens-1 (ZO-1), or for JAM-A and the pro-angiogenic αVβ3 integrin. Immunostained tissue sections and cells were examined by confocal laser scanning microscopy. Densitometric analysis of staining intensity was performed using ImageJ software. Results In control skin, constitutive expression of JAM-A was observed in blood and lymphatic ECs, fibroblasts and keratinocytes. In early SSc skin, JAM-A expression was increased in blood and lymphatic vessels and fibroblasts. Moreover, perivascular inflammatory cells showed strong JAM-A positivity. In late SSc, JAM-A expression was weaker than in controls. JAM-C was weakly expressed in controls. In early SSc, JAM-C expression was markedly observed in blood and lymphatic ECs, fibroblasts, and inflammatory cells. Instead, in late SSc JAM-C expression was similar to controls. Stimulation of MVECs with early SSc sera increased the cell surface expression of JAM-A and αVβ3 integrin. JAM-C expression was found in MVEC cytoplasm at basal conditions and under stimulation with control sera. Upon early SSc sera challenging, JAM-C was rapidly recruited to tight junctions where it colocalised with ZO-1, with the maximun effect after 1 h. Similar effects were observed after 1 h VEGF stimulation. Conclusions JAMs are differentially expressed in early and late SSc skin. Early SSc sera affect the expression and subcellular localisation of JAMs in dermal MVECs. In SSc, JAMs may participate in early inflammatory process, EC activation and impaired angiogenesis. [ABSTRACT FROM PUBLISHER]

Background and objective Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease, in which neovascularisation of the synovium is regarded as a crucial step in its development and progression. Lymphatic neovascularisation has been observed in RA joints. Junctional adhesion molecule A (JAM-A) and JAM-C regulate leucocyte-endothelial cell (EC) interactions. JAMs also interact at inter-EC junctions regulating vascular permeability and are implicated in pathophysiological processes involving leukocyte transmigration, tight junction assembly and angiogenesis. The aim of this study was to investigate the role of JAM-A and JAM-C in mediating the lymphoangiogenic process in RA synovium. Materials and methods Synovial biopsies from RA and osteoarthritic (OA) patients, used as control, has been collected during arthroplasty or synovectomy. Synovial sections were stained with primary antibodies (Abs) antihuman JAM-A or JAM-C followed by fluorochrome-conjugated secondary Abs. The lymphatic EC specific marker podoplanin (D2-40) was used to differentiate blood (D2-40−) and lymphatic (D2-40+) vessels. Results Immunohistochemical analysis showed, in RA synovium, the presence of abundant D2-40+ structures, identifiable as lymphatic vessels. Many D2-40+ cells were also found in the intimal lyning layer, specially in the hyperplasic areas. Lymphatic vessels were distributed in the sublining, specially around blood vessels. Clusters of D2-40+ cells were also found in the inflammatory infiltrate, in structures resembling new lymphatic vessels. In all sections from RA, JAM-A and JAM-C were observed on almost all vasculature including the lymphatic (D2-40+) and blood vessels (D2-40−). Initial lymphatic vessels reacted with anti-JAM-A and anti-JAM-C. In the synovial lining, JAM-A and JAM-C were expressed on D2-40+ macrophage-like synoviocites. D2-40+ cells in the inflammatory infiltrate showed positivity for both JAM-A and JAM-C. The immunostaining for both JAMs was weaker in OA than in RA synovium. Conclusion This results confirm the presence of an increased number of lymphatic vessels in RA synovial tissue. Moreover, the authors observed D2-40+ structures, mostly around pre-existing blood vessels, indicating the development of a new lymphatic network in the synovial sublining. The presence of D2-40+ cells in the inflammatory infiltrate and in the macrophage-like synoviocites, lead us to hypothesise that in RA, macrophages are activated and may function as lymphatic precursors. In RA synovium, both JAM-A and JAM-C are expressed in all D2-40+ cells, indicating their participation in the inflammatory process and intercellular junction assembly. Their expression in new lymphatic vessels and in D2-40+ cells stimulated to form new vessels, confirm their involvement in the lymphoangiogenic process occurring in RA. [ABSTRACT FROM PUBLISHER]