ABSTRACT
OBJECTIVE: Dysfunctional angiogenesis is a pathogenetic marker of SSc. Circulating levels of endothelial progenitor cells are reduced, and mesenchymal stromal cells show a reduced differentiation into endothelial cells and capacity to form capillaries. This suggests that pathophysiologically relevant changes may already exist in SSc bone marrow (BM) stromal cells that may affect downstream angiogenesis. The aim of this study is to evaluate, in SSc BM, angiogenesis, cellular immune system and fibrosis. METHODS: Eight SSc patients affected by a severe dcSSc and screened for autologous haematopoietic stem cells transplantation (HSCT) underwent a BM biopsy. BM biopsies were compared with six healthy controls. To evaluate angiogenesis and cellular immunity, the following antibodies were used: vascular endothelial growth factor (VEGF), kinase insert domain-containing receptor/fetal liver kinase-1 (KDR/flk-1), MMP-9 and CD34. To evaluate fibrosis, silver impregnation for reticulum was used. The number of vessels, the mean area of vascularization, the perimeter and microvessel density (MVD) were measured with a multiparametric computerized imaging analysis. RESULTS: A significant reduction in BM vascularity was found, while VEGF expression was much higher in SSc BM samples. Two patients had a Grade 2, whereas another two had a Grade 1 fibrosis. CONCLUSION: In SSc, BM is characterized by a reduction of microvascular density and number of vessels and a significant increase of VEGF. This indicates that BM may be involved in the process of loss of angiogenesis, despite the presence of high local and systemic levels of VEGF.
Subject(s)
Bone Marrow/blood supply , Bone Marrow/pathology , Hematopoietic Stem Cells/pathology , Neovascularization, Pathologic/pathology , Scleroderma, Systemic/pathology , Adult , Antigens, CD34/metabolism , Biopsy , Bone Marrow/immunology , Endothelial Cells/metabolism , Endothelial Cells/pathology , Female , Hematopoietic Stem Cells/metabolism , Humans , Image Processing, Computer-Assisted , Immunohistochemistry , Lymphokines , Male , Matrix Metalloproteinase 9/metabolism , Mesenchymal Stem Cells/metabolism , Mesenchymal Stem Cells/pathology , Microvessels/immunology , Microvessels/metabolism , Microvessels/pathology , Neovascularization, Pathologic/immunology , Scleroderma, Systemic/immunology , Vascular Endothelial Growth Factor A/metabolism , Vascular Endothelial Growth Factor Receptor-2/metabolismABSTRACT
OBJECTIVES: To investigate joint involvement in SSc and its relationship with autoantibody to the hnRNP and to anti-cyclic citrullinated peptide (anti-CCP). METHODS: Sera from 55 SSc patients were investigated. Joint involvement was determined by clinical, radiological and ultrasonographical evaluation. Anti-hnRNP proteins A1 and A2 (anti-hnRNP-A1/A2) antibodies were determined by immunoblotting. Anti-CCP, ACA, anti-topo I (ATA), Sm, U1-RNP, ribosomal RNP, Ro/SSA, La/SSB autoantibody and RF were determined. RESULTS: Six patients were positive for anti-hnRNP-A2 autoantibody and two were anti-A1 positive. Eight patients had joint erosions: seven of the eight patients positive for anti-hnRNP-A2 or A1 presented articular involvement (P < 0.04) and five of the eight erosive patients were positive for either of the two autoantibodies (P < 0.02). Of the four patients positive for anti-CCP, none had anti-hnRNP but three had erosive aspects. ATAs were found in 10 patients, six of which were also positive for anti-hnRNP (P < 0.05). RF was positive in 16 patients and in seven among those with articular involvement (P < 0.04). RF was significantly associated with anti-hnRNP in patients with erosive arthritis (P < 0.02), but not with the presence of anti-hnRNP alone. Epitope mapping of the three strongest anti-hnRNP-A2-positive sera recognized the same major epitope as patients with RA. SSc patients have higher incidence of erosions and anti-hnRNP-A2/A1 positivity. RF test and anti-hnRNP had a statistically significant diagnostic value for articular involvement. CONCLUSIONS: These parameters might suggest that autoantibody to both hnRNP antigens might become a non-specific but useful marker for joint involvement in SSc patients and identify SSc patients prone to develop joint damage.
Subject(s)
Autoantibodies/blood , Heterogeneous-Nuclear Ribonucleoproteins/immunology , Joints/immunology , Scleroderma, Systemic/immunology , Adult , Aged , Arthrography , Autoantibodies/immunology , Female , Heterogeneous Nuclear Ribonucleoprotein A1 , Heterogeneous-Nuclear Ribonucleoprotein Group A-B/immunology , Humans , Joints/pathology , Male , Middle Aged , Peptides, Cyclic/immunology , Probability , Scleroderma, Systemic/diagnostic imaging , UltrasonographyABSTRACT
To examine whether the lack of sufficient neoangiogenesis in systemic sclerosis (SSc) is caused by a decrease in angiogenic factors and/or an increase in angiostatic factors, the potent proangiogenic molecules vascular endothelial growth factor (VEGF) and basic fibroblast growth factor, and the angiostatic factor endostatin were determined in patients with SSc and in healthy controls. Forty-three patients with established SSc and nine patients with pre-SSc were included in the study. Serum levels of VEGF, basic fibroblast growth factor and endostatin were measured by ELISA. Age-matched and sex-matched healthy volunteers were used as controls. Highly significant differences were found in serum levels of VEGF between SSc patients and healthy controls, whereas no differences could be detected for endostatin and basic fibroblast growth factor. Significantly higher levels of VEGF were detected in patients with Scl-70 autoantibodies and in patients with diffuse SSc. Patients with pre-SSc and short disease duration showed significant higher levels of VEGF than healthy controls, indicating that elevated serum levels of VEGF are a feature of the earliest disease stages. Patients without fingertip ulcers were found to have higher levels of VEGF than patients with fingertip ulcers. Levels of endostatin were associated with the presence of giant capillaries in nailfold capillaroscopy, but not with any other clinical parameter. The results show that the concentration of VEGF is already increased in the serum of SSc patients at the earliest stages of the disease. VEGF appears to be protective against ischemic manifestations when concentrations of VEGF exceed a certain threshold level.
Subject(s)
Angiogenesis Inducing Agents/blood , Angiogenesis Inhibitors/blood , Collagen/blood , Endothelial Growth Factors/blood , Fibroblast Growth Factor 2/blood , Intercellular Signaling Peptides and Proteins/blood , Lymphokines/blood , Peptide Fragments/blood , Scleroderma, Systemic/blood , Adult , Aged , Aged, 80 and over , Autoantigens/immunology , Capillaries/growth & development , Capillaries/pathology , DNA Topoisomerases, Type I , Endostatins , Enzyme-Linked Immunosorbent Assay , Female , Fingers , Humans , Male , Microscopic Angioscopy , Middle Aged , Nails/blood supply , Nails/pathology , Nuclear Proteins/immunology , Scleroderma, Systemic/complications , Scleroderma, Systemic/pathology , Skin Ulcer/etiology , Skin Ulcer/pathology , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth FactorsABSTRACT
PURPOSE: Systemic sclerosis is characterized by progressive microvascular occlusion and fibrosis and by an imbalance in the fibrinolytic system. In vivo and in vitro studies suggest that the renin-angiotensin system partly regulates vascular fibrinolytic balance. Angiotensin II increases the production and secretion of plasminogen activator inhibitor-1, while angiotensin-converting enzyme (ACE) contributes to reduced production of tissue plasminogen activator and endothelial nitric oxide synthesis by bradykinin degradation. The aim of our study was to investigate the effects of ACE insertion/deletion (I/D) and endothelial nitric oxide synthase (eNOS) Glu298Asp (G894-->T) and T-786-->C polymorphisms in patients with systemic sclerosis. SUBJECTS AND METHODS: We studied 73 consecutive patients (47 with limited and 26 with diffuse cutaneous systemic sclerosis) and 112 control subjects. ACE I/D and eNOS polymorphisms were genotyped by polymerase chain reaction-restriction fragment length polymorphism analysis. RESULTS: The ACE I/D and the eNOS G894-->T polymorphisms were more common in patients than in controls (for the ACE D allele: odds ratio [OR] = 3.4; 95% confidence interval [CI]: 1.5 to 7.9; P = 0.003; for the eNOS T allele: OR = 1.9; 95% CI: 1.0 to 3.4; P = 0.04). There was no association between the eNOS T-786-->C polymorphism and systemic sclerosis. CONCLUSIONS: Our findings of an increased risk of systemic sclerosis in ACE D and eNOS 894T allele carriers suggest that these polymorphisms may contribute to the pathogenesis of the disease.
Subject(s)
Genetic Predisposition to Disease , Nitric Oxide Synthase/genetics , Peptidyl-Dipeptidase A/genetics , Polymorphism, Genetic , Scleroderma, Systemic/epidemiology , Scleroderma, Systemic/genetics , Adult , Aged , Case-Control Studies , Endothelium, Vascular/metabolism , Female , Humans , Italy/epidemiology , Male , Middle Aged , Nitric Oxide Synthase/metabolism , Peptidyl-Dipeptidase A/blood , Prevalence , RiskABSTRACT
OBJECTIVE: To evaluate, in a pilot study, the efficacy of a short term cyclophosphamide (CYC) pulse regimen on alveolitis in a cohort of patients with systemic sclerosis (SSc). METHODS: Twenty-three patients with SSc (17 diffuse SSc and 6 limited SSc) were selected in 5 centers in Italy, based on the findings of an abnormal bronchoalveolar lavage (BAL) cell analysis in association with altered pulmonary function tests (PFT) or recent deterioration in flow volume curve (FVC). Patients were also evaluated by skin score (Rodnan), esophageal manometry and barium swallow radiography, and electrocardiography and 2-mode echocardiography. The pre-enrolment pulmonary evaluation and after 6 months of therapy included evaluation of the clinical status, PFT (FVC, FEV1, DLCO), BAL. standard chest radiograph, and chest high resolution computed tomography. All patients received i.v. CYC (1000 mg/m2 of body surface monthly for 6 mo) and oral prednisone (25 mg daily for the first month and subsequently 5 mg daily of maintenance dosage for the remaining 5 mo). A complete blood count and urinalysis were obtained at monthly intervals. RESULTS: After 6 months of therapy the values for FVC did not change significantly. Individually, 8 of 23 patients showed an improvement (> 15% increase) in FVC after 6 months, while FVC in 13 cases remained stable. Only 2 patients had an important decline in FVC after 6 months of therapy (17 and 24% decrease). Improvement in DLCO was noted in 15 of 23 patients after 6 months of therapy. Four patients were stable and 4 patients had a worsened DLCO at the end of the study. After therapy the mean value of BAL fluid recovery did not change. There was a reduction in total cell number although this value did not reach statistical significance. The levels of neutrophils, eosinophils, lymphocytes, and macrophages did not change significantly. Scans for patients with grades 1, 2, and 3 did not differ significantly after 6 months of therapy, and 14 patients were stable. Changes in appearance, in relation to changes in extent of disease, were seen in 8 patients and consisted of an extension of reticular pattern and transformation from grade 1 to 2 (6/8 patients). All patients showed a ground-glass appearance indicating an acute alveolitis. Improvement in ground-glass was noted in 10 of 23 patients after 6 mo therapy. At the end of the study, 8 patients were stable and 5 patients had a diffusion of the ground-glass to other segments. No side effects were experienced during the treatment except for mild nausea in 4 patients; no patients discontinued therapy during the study. CONCLUSION: CYC pulse regimen seems to stabilize alveolitis in the majority of cases. The association of CYC pulsed modality with prednisone may be useful in SSc patients to control disease evolution in the lung.