RESUMEN
Systemic sclerosis (SSc) is an autoimmune disease characterized by microvascular compromise and fibrosis. Pulmonary fibrosis, a prominent pulmonary complication in SSc, results in impaired lung function due to excessive accumulation of extracellular matrix components. This study aimed to investigate the effects of coadministration of 3'5-dimaleamylbenzoic acid (AD) and quercetin (Q) on key events in the development and maintenance of pulmonary fibrosis in a bleomycin (BLM)-induced SSc mouse model. The model was induced in CD1 mice through BLM administration using osmotic mini pumps. Subsequently, mice were treated with AD (6 mg/kg) plus Q (10 mg/kg) and sacrificed at 21 and 28 days post BLM administration. Histopathological analysis was performed by hematoxylin and eosin staining and Masson's trichrome staining. Immunohistochemistry was used to determine the expression of proliferation, proinflammatory, profibrotic and oxidative stress markers. The coadministration of AD and Q during the fibrotic phase of the BLM-induced SSc model led to attenuated histological alterations and pulmonary fibrosis, reflected in the recovery of alveolar spaces (30 %, p < 0.01) and decreased collagen deposits (50 %, p < 0.001). This effect was achieved by decreasing the expression of the proliferative markers cyclin D1 (87 %, p < 0.0001) and PCNA (43 %, p < 0.0001), inflammatory markers COX-2 (71 %, p < 0.0001) and iNOS (84 %, p < 0.0001), profibrotic markers α-SMA (80 %, p < 0.0001) and TGF-ß (81 %, p < 0.0001) and the lipid peroxidation marker 4-HNE (43 %, p < 0.01). The antifibrotic effect of this combined therapy is associated with the regulation of proliferation, inflammation and oxidative stress, mechanisms involved in the development and progression of the fibrotic process. Our novel therapeutic strategy is the first approach to propose the use of the combination of prooxidant and antioxidant compounds as a potential strategy for SSc-associated pulmonary fibrosis.
Asunto(s)
Fibrosis Pulmonar , Esclerodermia Sistémica , Ratones , Animales , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/tratamiento farmacológico , Fibrosis Pulmonar/metabolismo , Quercetina/uso terapéutico , Quercetina/farmacología , Fibrosis , Colágeno/metabolismo , Bleomicina/efectos adversos , Esclerodermia Sistémica/metabolismo , Modelos Animales de Enfermedad , Pulmón/patologíaRESUMEN
Although several cytokines and chemokines have been investigated as possible mediators of fibrosis in systemic sclerosis (SSc), specific correlation between cytokines and organ involvement have not been found yet, and a cytokine profile characteristic of SSc is far to be identified. We studied the profile of antifibrotic and profibrotic transcripts involved in skin of SSc patients. The mRNA expression was detected by fluorescence-based quantitative real-time PCR (qPCR) in skin's biopsies from 14 patients with SSc and 5 healthy controls. PDGF-A, CTGF, CCL3, IL-6, IL-13, IL-7, IFNγ, IL-17, IL-22 and RORc were analysed in these samples. CCL3, IL-7, IL-13 and IFN-γ were more expressed in skin's biopsy of patients with SSc (P = 0.0002, P = 0.0082, P = 0.0243, P = 0.0335, respectively) when compared with healthy controls. We also found a positive correlation between CCL3 and IL-7 transcripts (P = 0.0050 r = 0.7187). Furthermore, we observed that patients with lung involvement had lower expression of PDGF-A (P = 0.0385). We found an increase in IL-7, IFN-γ, CCL3 and IL-13 relative mRNA expressions on the skin's biopsy of patients with SSc, and a positive correlation between IL-7 and CCL3. These molecules are involved in the pathogenesis of SSc, and how their interactions occur should be the subject of further studies.
Asunto(s)
Quimiocina CCL3/biosíntesis , Interferón gamma/biosíntesis , Interleucina-13/biosíntesis , Interleucina-7/biosíntesis , Adulto , Anciano , Biopsia , Quimiocina CCL3/genética , Femenino , Fibrosis , Regulación de la Expresión Génica , Humanos , Inmunosupresores/uso terapéutico , Interferón gamma/genética , Interleucina-13/genética , Interleucina-7/genética , Pulmón/patología , Masculino , Persona de Mediana Edad , Factor de Crecimiento Derivado de Plaquetas/biosíntesis , Factor de Crecimiento Derivado de Plaquetas/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Esclerodermia Sistémica/tratamiento farmacológico , Esclerodermia Sistémica/genética , Esclerodermia Sistémica/metabolismo , Esclerodermia Sistémica/patología , Transcripción Genética , Regulación hacia ArribaRESUMEN
Glucocorticoids (GC) are widely used in the treatment of SSc, although there is not much evidence to prove the benefits offered by these drugs in this disease. In this study, we evaluated the effects of a GC on cytokine production in peripheral blood mononuclear cells (PBMC) of SSc patients. The effect of dexamethasone (DEX) was evaluated in PBMC of 21 SSc patients and 10 healthy volunteers after stimulation of cells with anti-CD3 and anti-CD28. Cytokines IL-2, IL-4, IL-6, IL-10, IL-17A, IL-17F, IFN-γ, TNF, and IL-1ß were quantified in the culture supernatant by CBA or ELISA. Of the patients evaluated in this study, 8 (38%) were taking corticosteroids, and esophageal dysfunction was more frequent in these patients when compared to those who did not take corticosteroids. DEX (1.000 nM) treatment in PBMC of SSc patients stimulated with anti-CD3 and anti-CD28 promoted a significant reduction in IL-2, IL-4, IL-6, IL-10, IL-17A, IFN-γ, TNF, IL-1ß (p < 0.001 for all), and IL-17F (p = 0.023) cytokines levels. We did not observe differences in response to in vitro treatment with DEX between groups of patients taking or not taking corticosteroids. In PBMC from healthy volunteers, we observed that DEX treatment significantly reduced IL-4, IFN-γ (p = 0.003 for both), IL-6, IL-10, IL-17A, and TNF (p = 0.002 for all) cytokines. These results show that DEX treatment in PBMC of SSc patients reduced the production of important cytokines involved in the pathogenesis of the disease, suggesting a possible mechanism of action of the CG in the treatment of SSc.
Asunto(s)
Citocinas/metabolismo , Dexametasona/farmacología , Leucocitos Mononucleares/efectos de los fármacos , Esclerodermia Sistémica/tratamiento farmacológico , Corticoesteroides/metabolismo , Adulto , Anciano , Estudios de Casos y Controles , Femenino , Humanos , Leucocitos Mononucleares/metabolismo , Masculino , Persona de Mediana Edad , Esclerodermia Sistémica/metabolismo , Adulto JovenRESUMEN
The aim of this study was to evaluate the enantioselective pharmacokinetics of cyclophosphamide and its metabolites 4-hydroxycyclophosphamide and carboxyethylphosphoramide mustard in patients with systemic or multiple sclerosis. Patients with systemic sclerosis (n = 10) or multiple sclerosis (n = 10), genotyped for the allelic variants of CYP2C9*2 and CYP2C9*3 and of the CYP2B6 G516T polymorphism, were treated with 50 mg cyclophosphamide/kg daily for 4 days. Serial blood samples were collected up to 24 hours after administration of the last cyclophosphamide dose. Cyclophosphamide, 4-hydroxycyclophosphamide, and carboxyethylphosphoramide enantiomers were analyzed in plasma samples using liquid chromatography-tandem mass spectrometry coupled to chiral column Chiralcel OD-R or Chiralpak AD-RH. Cytokines IL-2, IL-4, IL-6, IL-8, IL-10, IL- 12p70, IL-17, TNF-α, and INT-δ in the plasma samples collected before cyclophosphamide infusion were analyzed by Milliplex MAP human cytokine/chemokine. Pharmacokinetic parameters showed higher plasma concentrations of (S)-(-)-cyclophosphamide (AUC 215.0 vs 186.2 µg·h/mL for multiple sclerosis patients and 219.1 vs 179.2 µg·h/mL for systemic sclerosis patients) and (R)-4-hydroxycyclophosphamide (AUC 5.6 vs 3.7 µg·h/mL for multiple sclerosis patients and 6.3 vs 5.6 µg·h/mL for systemic sclerosis patients) when compared to their enantiomers in both groups of patients, whereas the pharmacokinetics of the carboxyethylphosphoramide metabolite was not enantioselective. Cytokines' plasma concentrations were similar between multiple and systemic sclerosis groups. The pharmacokinetics of cyclophosphamide is enantioselective in patients with systemic sclerosis and multiple sclerosis, with higher plasma concentrations of the (S)-(-)-cyclophosphamide enantiomer due to the preferential formation of the (R)-4-hydroxycyclophosphamide metabolite.
Asunto(s)
Ciclofosfamida/análogos & derivados , Ciclofosfamida/farmacocinética , Inmunosupresores/farmacocinética , Esclerosis Múltiple/metabolismo , Esclerodermia Sistémica/metabolismo , Adulto , Ciclofosfamida/sangre , Ciclofosfamida/farmacología , Citocromo P-450 CYP2B6/genética , Citocromo P-450 CYP2C9/genética , Citocinas/sangre , Femenino , Genotipo , Humanos , Inmunosupresores/sangre , Inmunosupresores/farmacología , Masculino , Persona de Mediana Edad , Esclerosis Múltiple/sangre , Esclerosis Múltiple/genética , Esclerodermia Sistémica/sangre , Esclerodermia Sistémica/genética , EstereoisomerismoRESUMEN
INTRODUCTION: Systemic sclerosis (SSc) is a chronic autoimmune disease characterized by microvascular damage, inflammation, and fibrosis. It has become increasingly evident that platelets, beyond regulating hemostasis, are important in inflammation and innate immunity. Platelets may be an important source of proinflammatory and profibrotic cytokines in the vascular microenvironment. In this study, we sought to assess the contribution of platelet-derived factors in patients with SSc to the angiogenesis of human dermal microvascular endothelial cells (DMVECs) in a tubule formation assay and to characterize the secretion of profibrotic and proinflammatory cytokines in these platelets. METHODS: We analyzed platelets obtained from 30 patients with SSc and 12 healthy control subjects. Angiogenesis was evaluated in vitro with a DMVEC tubule formation assay on Matrigel and platelet-derived angiogenic factors such as vascular endothelial growth factor (VEGF), 165b isoform (VEGF165b), and cytokine secretion was evaluated. Platelet serotonin content was also determined. RESULTS: When DMVECs were incubated with SSc platelet releasates, tubule formation was significantly inhibited (p < 0.01, t test), and higher expression of endothelin-1 in these cells was observed compared with control subjects (p < 0.05, Mann-Whitney U test). In SSc platelet releasates, VEGF165b was significantly higher (p < 0.05, t test), and the VEGF165b/VEGF ratio was increased compared with that of control subjects. Higher secretion of transforming growth factor ß (p < 0.01, t test) and CD40L (p < 0.01, t test) was observed compared with control subjects. Also, intraplatelet serotonin levels were lower in platelets obtained from patients with diffuse SSc compared with patients with limited SSc and control subjects (p < 0.05, t test). CONCLUSIONS: Our findings suggest that antiangiogenic factors such as VEGF165b, together with proinflammatory and profibrotic factors secreted by platelets, can contribute to the progression of peripheral microvascular damage, defective vascular repair, and fibrosis in patients with SSc.
Asunto(s)
Inhibidores de la Angiogénesis/metabolismo , Plaquetas/metabolismo , Mediadores de Inflamación/antagonistas & inhibidores , Mediadores de Inflamación/metabolismo , Neovascularización Patológica/metabolismo , Esclerodermia Sistémica/metabolismo , Adulto , Anciano , Células Cultivadas , Femenino , Humanos , Masculino , Persona de Mediana Edad , Neovascularización Patológica/patología , Esclerodermia Sistémica/patologíaRESUMEN
OBJECTIVE: Salvia miltiorrhiza has long been used to treat systemic sclerosis. Tanshinone IIA, one of the phytochemicals derived from the roots of Salvia miltiorrhiza, exhibits multiple biological activities. The present study aimed to investigate whether tanshinone IIA has an effect on the interleukin-17A-induced functional activation of systemic sclerosis patient-derived dermal vascular smooth muscle cells. METHODS: Systemic sclerosis patient-derived dermal vascular smooth muscle cells were incubated with various dosages of tanshinone IIA in the presence of interleukin-17A or the serum of systemic sclerosis patients. Cell proliferation was assessed using Cell Counting Kit-8. The expression of collagen 1 and 3 in cells was evaluated by immunofluorescence. Cell migration was measured using a transwell assay. The expression of phospho-extracellular signal-regulated kinase was detected by Western blotting. RESULTS: Our data demonstrate that tanshinone IIA exerts an inhibitory effect on interleukin-17A-induced systemic sclerosis patient-derived dermal vascular smooth muscle cell proliferation, collagen synthesis and migration. CONCLUSION: These findings suggest that tanshinone IIA might serve as a promising therapeutic agent for the treatment of systemic sclerosis.
Asunto(s)
Abietanos/farmacología , Antiinflamatorios no Esteroideos/farmacología , Quinasas MAP Reguladas por Señal Extracelular/efectos de los fármacos , Interleucina-17/análisis , Músculo Liso Vascular/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos , Esclerodermia Sistémica/tratamiento farmacológico , Abietanos/uso terapéutico , Adulto , Antiinflamatorios no Esteroideos/uso terapéutico , Far-Western Blotting , Ensayos de Migración Celular , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas/efectos de los fármacos , Colágeno Tipo I/análisis , Colágeno Tipo III/análisis , Activación Enzimática/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Interleucina-17/metabolismo , Masculino , Persona de Mediana Edad , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Reproducibilidad de los Resultados , Salvia miltiorrhiza/química , Esclerodermia Sistémica/metabolismo , Estadísticas no Paramétricas , Factores de TiempoAsunto(s)
Femenino , Humanos , Masculino , Persona de Mediana Edad , Fibroblastos/patología , Receptores de Quimiocina/metabolismo , Esclerodermia Sistémica/metabolismo , Esclerodermia Sistémica/patología , Actinas/metabolismo , Comunicación Autocrina/fisiología , Biopsia , Diferenciación Celular/fisiología , Células Cultivadas , /metabolismo , Fibroblastos/metabolismo , Técnicas In Vitro , Fenotipo , Piel/metabolismo , Piel/patología , Regulación hacia ArribaRESUMEN
OBJECTIVE: To study the expression of COX-1 and COX-2 in the remodeled lung in systemic sclerosis (SSc) and idiopathic pulmonary fibrosis (IPF) patients, correlating that expression with patient survival. METHODS: We examined open lung biopsy specimens from 24 SSc patients and 30 IPF patients, using normal lung tissue as a control. The histological patterns included fibrotic nonspecific interstitial pneumonia (NSIP) in SSc patients and usual interstitial pneumonia (UIP) in IPF patients. We used immunohistochemistry and histomorphometry to evaluate the expression of COX-1 and COX-2 in alveolar septa, vessels, and bronchioles. We then correlated that expression with pulmonary function test results and evaluated its impact on patient survival. RESULTS: The expression of COX-1 and COX-2 in alveolar septa was significantly higher in IPF-UIP and SSc-NSIP lung tissue than in the control tissue. No difference was found between IPF-UIP and SSc-NSIP tissue regarding COX-1 and COX-2 expression. Multivariate analysis based on the Cox regression model showed that the factors associated with a low risk of death were younger age, high DLCO/alveolar volume, IPF, and high COX-1 expression in alveolar septa, whereas those associated with a high risk of death were advanced age, low DLCO/alveolar volume, SSc (with NSIP), and low COX-1 expression in alveolar septa. CONCLUSIONS: Our findings suggest that strategies aimed at preventing low COX-1 synthesis will have a greater impact on SSc, whereas those aimed at preventing high COX-2 synthesis will have a greater impact on IPF. However, prospective randomized clinical trials are needed in order to confirm that. .
OBJETIVO: Estudar a expressão de COX-1 e COX-2 em áreas pulmonares remodeladas em pacientes com esclerose sistêmica (ES) ou fibrose pulmonar idiopática (FPI) e correlacioná-la com a sobrevida desses pacientes. MÉTODOS: Examinamos espécimes de biópsia pulmonar a céu aberto de 24 pacientes com ES e de 30 pacientes com FPI, utilizando-se tecido pulmonar normal como controle. Os padrões histológicos incluíram pneumonia intersticial não específica (PINE) fibrótica em pacientes com ES e pneumonia intersticial usual (PIU) nos pacientes com FPI. Imuno-histoquímica e histomorfometria foram usadas para avaliar a expressão celular de COX-1 e COX-2 em septos alveolares, vasos e bronquíolos, sua correlação com provas de função pulmonar e seu impacto na sobrevida. RESULTADOS: A expressão de COX-1 e COX-2 em septos alveolares foi significativamente maior em FPI-PIU e ES-PINE do que no tecido controle. Não houve diferença entre FPI-PIU e ES-PINE quanto à expressão de COX-1 e COX-2. A análise multivariada baseada no modelo de regressão de Cox mostrou que os fatores associados a baixo risco de morte foram ter idade menor, valores elevados de DLCO/volume alveolar, FPI, e alta expressão de COX-1 em septos alveolares, ao passo que os fatores associados a alto risco de morte foram ter idade maior, valores baixos de DLCO/volume alveolar, ES (com PINE) e baixa expressão de COX-1 em septos alveolares. CONCLUSÕES: Nossos resultados sugerem que estratégias de prevenção de baixa síntese de COX-1 terão maior impacto sobre a ES, ao passo que as de prevenção de alta síntese de COX-2 terão maior impacto sobre a FPI. Porém, são necessários ensaios clínicos randomizados prospectivos para confirmar essa hipótese. .
Asunto(s)
Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Remodelación de las Vías Aéreas (Respiratorias) , Ciclooxigenasa 1/análisis , /análisis , Fibrosis Pulmonar Idiopática/metabolismo , Esclerodermia Sistémica/metabolismo , Factores de Edad , Biopsia , Estudios de Seguimiento , Inmunohistoquímica , Fibrosis Pulmonar Idiopática/mortalidad , Fibrosis Pulmonar Idiopática/patología , Pulmón/patología , Análisis Multivariante , Alveolos Pulmonares/fisiopatología , Pruebas de Función Respiratoria , Tasa de Supervivencia , Esclerodermia Sistémica/mortalidad , Esclerodermia Sistémica/patologíaRESUMEN
Because histopathological changes in the lungs of patients with systemic sclerosis (SSc) are consistent with alveolar and vessel cell damage, we presume that this interaction can be characterized by analyzing the expression of proteins regulating nitric oxide (NO) and plasminogen activator inhibitor-1 (PAI-1) synthesis. To validate the importance of alveolar-vascular interactions and to explore the quantitative relationship between these factors and other clinical data, we studied these markers in 23 cases of SSc nonspecific interstitial pneumonia (SSc-NSIP). We used immunohistochemistry and morphometry to evaluate the amount of cells in alveolar septa and vessels staining for NO synthase (NOS) and PAI-1, and the outcomes of our study were cellular and fibrotic NSIP, pulmonary function tests, and survival time until death. General linear model analysis demonstrated that staining for septal inducible NOS (iNOS) related significantly to staining of septal cells for interleukin (IL)-4 and to septal IL-13. In univariate analysis, higher levels of septal and vascular cells staining for iNOS were associated with a smaller percentage of septal and vascular cells expressing fibroblast growth factor and myofibroblast proliferation, respectively. Multivariate Cox model analysis demonstrated that, after controlling for SSc-NSIP histological patterns, just three variables were significantly associated with survival time: septal iNOS (P=0.04), septal IL-13 (P=0.03), and septal basic fibroblast growth factor (bFGF; P=0.02). Augmented NOS, IL-13, and bFGF in SSc-NSIP histological patterns suggest a possible functional role for iNOS in SSc. In addition, the extent of iNOS, PAI-1, and IL-4 staining in alveolar septa and vessels provides a possible independent diagnostic measure for the degree of pulmonary dysfunction and fibrosis with an impact on the survival of patients with SSc.
Asunto(s)
Enfermedades Pulmonares Intersticiales/patología , Óxido Nítrico Sintasa/metabolismo , Inhibidor 1 de Activador Plasminogénico/metabolismo , Esclerodermia Sistémica/patología , Adulto , Biomarcadores/sangre , Citocinas/sangre , Femenino , Humanos , Inmunohistoquímica , Interleucina-13/metabolismo , Interleucina-4/metabolismo , Estimación de Kaplan-Meier , Enfermedades Pulmonares Intersticiales/etiología , Enfermedades Pulmonares Intersticiales/metabolismo , Enfermedades Pulmonares Intersticiales/mortalidad , Persona de Mediana Edad , Óxido Nítrico Sintasa de Tipo II/metabolismo , Isoformas de Proteínas/sangre , Esclerodermia Sistémica/complicaciones , Esclerodermia Sistémica/metabolismo , Esclerodermia Sistémica/mortalidadRESUMEN
Because histopathological changes in the lungs of patients with systemic sclerosis (SSc) are consistent with alveolar and vessel cell damage, we presume that this interaction can be characterized by analyzing the expression of proteins regulating nitric oxide (NO) and plasminogen activator inhibitor-1 (PAI-1) synthesis. To validate the importance of alveolar-vascular interactions and to explore the quantitative relationship between these factors and other clinical data, we studied these markers in 23 cases of SSc nonspecific interstitial pneumonia (SSc-NSIP). We used immunohistochemistry and morphometry to evaluate the amount of cells in alveolar septa and vessels staining for NO synthase (NOS) and PAI-1, and the outcomes of our study were cellular and fibrotic NSIP, pulmonary function tests, and survival time until death. General linear model analysis demonstrated that staining for septal inducible NOS (iNOS) related significantly to staining of septal cells for interleukin (IL)-4 and to septal IL-13. In univariate analysis, higher levels of septal and vascular cells staining for iNOS were associated with a smaller percentage of septal and vascular cells expressing fibroblast growth factor and myofibroblast proliferation, respectively. Multivariate Cox model analysis demonstrated that, after controlling for SSc-NSIP histological patterns, just three variables were significantly associated with survival time: septal iNOS (P=0.04), septal IL-13 (P=0.03), and septal basic fibroblast growth factor (bFGF; P=0.02). Augmented NOS, IL-13, and bFGF in SSc-NSIP histological patterns suggest a possible functional role for iNOS in SSc. In addition, the extent of iNOS, PAI-1, and IL-4 staining in alveolar septa and vessels provides a possible independent diagnostic measure for the degree of pulmonary dysfunction and fibrosis with an impact on the survival of patients with SSc.
Asunto(s)
Adulto , Femenino , Humanos , Persona de Mediana Edad , Enfermedades Pulmonares Intersticiales/patología , Óxido Nítrico Sintasa/metabolismo , Inhibidor 1 de Activador Plasminogénico/metabolismo , Esclerodermia Sistémica/patología , Biomarcadores/sangre , Citocinas/sangre , Inmunohistoquímica , /metabolismo , /metabolismo , Estimación de Kaplan-Meier , Enfermedades Pulmonares Intersticiales/etiología , Enfermedades Pulmonares Intersticiales/metabolismo , Enfermedades Pulmonares Intersticiales/mortalidad , Óxido Nítrico Sintasa de Tipo II/metabolismo , Isoformas de Proteínas/sangre , Esclerodermia Sistémica/complicaciones , Esclerodermia Sistémica/metabolismo , Esclerodermia Sistémica/mortalidadRESUMEN
OBJECTIVE: To study the expression of COX-1 and COX-2 in the remodeled lung in systemic sclerosis (SSc) and idiopathic pulmonary fibrosis (IPF) patients, correlating that expression with patient survival. METHODS: We examined open lung biopsy specimens from 24 SSc patients and 30 IPF patients, using normal lung tissue as a control. The histological patterns included fibrotic nonspecific interstitial pneumonia (NSIP) in SSc patients and usual interstitial pneumonia (UIP) in IPF patients. We used immunohistochemistry and histomorphometry to evaluate the expression of COX-1 and COX-2 in alveolar septa, vessels, and bronchioles. We then correlated that expression with pulmonary function test results and evaluated its impact on patient survival. RESULTS: The expression of COX-1 and COX-2 in alveolar septa was significantly higher in IPF-UIP and SSc-NSIP lung tissue than in the control tissue. No difference was found between IPF-UIP and SSc-NSIP tissue regarding COX-1 and COX-2 expression. Multivariate analysis based on the Cox regression model showed that the factors associated with a low risk of death were younger age, high DLCO/alveolar volume, IPF, and high COX-1 expression in alveolar septa, whereas those associated with a high risk of death were advanced age, low DLCO/alveolar volume, SSc (with NSIP), and low COX-1 expression in alveolar septa. CONCLUSIONS: Our findings suggest that strategies aimed at preventing low COX-1 synthesis will have a greater impact on SSc, whereas those aimed at preventing high COX-2 synthesis will have a greater impact on IPF. However, prospective randomized clinical trials are needed in order to confirm that.
Asunto(s)
Remodelación de las Vías Aéreas (Respiratorias) , Ciclooxigenasa 1/análisis , Ciclooxigenasa 2/análisis , Fibrosis Pulmonar Idiopática/metabolismo , Esclerodermia Sistémica/metabolismo , Adulto , Factores de Edad , Anciano , Biopsia , Femenino , Estudios de Seguimiento , Humanos , Fibrosis Pulmonar Idiopática/mortalidad , Fibrosis Pulmonar Idiopática/patología , Inmunohistoquímica , Pulmón/patología , Masculino , Persona de Mediana Edad , Análisis Multivariante , Alveolos Pulmonares/fisiopatología , Pruebas de Función Respiratoria , Esclerodermia Sistémica/mortalidad , Esclerodermia Sistémica/patología , Tasa de SupervivenciaRESUMEN
Recent studies have suggested that the number of dermal dendritic cells is altered in the skin of patients with scleroderma and that these cells may have an important role in the pathogenesis of this disease. There is also a belief that insufficient blood flow to the affected organs may also be responsible for the disease. Our aim was to quantify CD34+ cells, factor XIIIa cells, and blood vessels in the skin of patients with systemic sclerosis and to correlate these data with fibrosis degree and duration of disease. Paraffin-embedded skin sections from patients with systemic sclerosis and from healthy subjects were immunolabelled with antibodies against CD34+ and factor XIIIa. Cells and blood vessels were quantified in the papillary and reticular dermis. Both, the number of CD34+ cells and factor XIIIa cells in the skin of patients with systemic sclerosis were reduced. The reduction of these cell types preceded the appearance of intense fibrosis, suggesting that fibrosis is not responsible of this phenomenon. Blood vessel volume and surface density were also reduced in the skin of systemic sclerosis patients. This reduction was also noted early in the evolution of the disease. Our results suggest that CD34+ cells and factor XIIIa cells may contribute to normal regulation of extracellular matrix assembly. We confirmed the observation that capillary density is diminished in scleroderma skin.
Asunto(s)
Capilares/patología , Células de Langerhans/patología , Esclerodermia Sistémica/patología , Piel/irrigación sanguínea , Piel/patología , Adulto , Anciano , Antígenos CD34/análisis , Biomarcadores/análisis , Biopsia , Brasil , Capilares/química , Capilares/inmunología , Estudios de Casos y Controles , Factor XIIIa/análisis , Femenino , Fibrosis , Humanos , Inmunohistoquímica , Células de Langerhans/inmunología , Masculino , Persona de Mediana Edad , Adhesión en Parafina , Esclerodermia Sistémica/inmunología , Esclerodermia Sistémica/metabolismo , Índice de Severidad de la Enfermedad , Piel/inmunología , Adulto JovenRESUMEN
OBJECTIVE: The physiological and mechanical properties of the skin, the primary tissue affected by systemic sclerosis, depend on the assembly of collagen types I, III and V, which form heterotypic fibers. Collagen V (COLV) regulates heterotypic fiber diameter, and the maintenance of its properties is important for maintaining normal tissue architecture and function. Based on a COLV-induced experimental SSc model, in which overexpression of abnormal COLV was a prominent feature, we assumed that this abnormality could be present in SSc patients and could be correlated to disease duration, skin thickening and disease activity. METHODS: Skin biopsies from 18 patients (6 early-stage and 12 late-stage) and 10 healthy controls were studied. Skin thickening assessment was performed with the Modified Rodnan Skin Score (MRSS), and activity was calculated using the Valentini Disease Activity Index. Morphology, morphometry of COLV deposition in dermis, as well as, quantitative RT-PCR and 3D-reconstruction of the dermal fibroblast culture were performed. RESULTS: Structurally abnormal COLV was overexpressed in SSc skin, mainly in the early stages of the disease, when compared to normal controls and late-stage. A positive correlation between COLV expression and MRSS and disease activity was observed. Collagen V alpha-1 and alpha-2 mRNA expression levels were higher in SSc. Tridimensional reconstruction of SSc dermal heterotypic fibers confirmed the presence of atypical COLV. CONCLUSION: Increased synthesis of abnormal COLV and its correlation with disease stage, activity and MRSS suggest that this collagen can be a possible trigger involved in the pathogenesis of SSc.
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Colágeno Tipo V/metabolismo , Dermis/metabolismo , Dermis/patología , Esclerodermia Sistémica/metabolismo , Esclerodermia Sistémica/patología , Adulto , Biopsia , Estudios de Casos y Controles , Colágeno/genética , Colágeno/metabolismo , Colágeno Tipo V/genética , Femenino , Fibroblastos/metabolismo , Regulación de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Esclerodermia Sistémica/genética , Índice de Severidad de la Enfermedad , Piel/metabolismo , Piel/patología , Adulto JovenRESUMEN
BACKGROUND: Growth differentiation factor (GDF)-15 is a secreted member of the transforming growth factor-ß cytokine superfamily. GDF-15 levels are elevated in the serum of patients with cardiovascular diseases. We hypothesized that GDF-15 levels would also be increased in the plasma and lung tissue of patients with systemic sclerosis-associated pulmonary arterial hypertension (SSc-PAH). METHODS: GDF-15 levels were measured in plasma in subjects with SSc-PAH (n = 30) and compared with subjects with systemic sclerosis (SSc) without pulmonary arterial hypertension (PAH) (n = 24). Patients with idiopathic PAH (IPAH) (n = 44) and normal individuals (n = 13) served as control subjects. Immunohistochemistry and immunofluorescence assay identified GDF-15 protein in lung tissue from patients with SSc-PAH and IPAH. RESULTS: Patients with SSc-PAH had significantly higher mean circulating levels of GDF-15 in plasma compared with patients with SSc without PAH (422.3 ± 369.5 pg/mL vs 108.1 ± 192.8 pg/mL, P = .004). GDF-15 levels correlated positively with estimated right ventricular systolic pressure on echocardiogram and plasma levels of the amino terminal propeptide form of brain natriuretic peptide. There was an inverse correlation between circulating GDF-15 and diffusing capacity of the lung for carbon monoxide (Dlco) and a positive correlation with the FVC to Dlco ratio on pulmonary function test. GDF-15 levels > 125 pg/mL were associated with reduced survival. GDF-15 protein expression was increased in lung tissue from patients with SSc-PAH. CONCLUSIONS: GDF-15 may be a useful biomarker in PAH associated with SSc. Its presence in lung tissue may suggest a role in the pathology of the disease.
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Factor 15 de Diferenciación de Crecimiento/biosíntesis , Hipertensión Pulmonar/etiología , Hipertensión Pulmonar/metabolismo , Esclerodermia Sistémica/complicaciones , Esclerodermia Sistémica/metabolismo , Femenino , Factor 15 de Diferenciación de Crecimiento/análisis , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Juvenile onset systemic sclerosis (JoSSc) is a rare disease, and there are no studies focusing in bone mineral density and biochemical bone parameters. Ten consecutive patients with JoSSc and 10 controls gender, age, menarche age, and physical activity matched were selected. Clinical data were obtained at the medical visit and chart review. Laboratorial analysis included autoantibodies, 25-hydroxyvitamin D (25OHD), intact parathyroid hormone, calcium, phosphorus, alkaline phosphatase and albumin sera levels. Bone mineral density was analyzed by dual-energy X-ray absorptiometry, and bone mineral apparent density (BMAD) was calculated. A lower BMAD in femoral neck (0.294 ± 0.060 vs. 0.395 ± 0.048 g/cm(3), P = 0.001) and total femur (0.134 ± 0.021 vs. 0.171 ± 0.022 g/cm(3), P = 0.002) was observed in JoSSc compared to controls. Likewise, a trend to lower BMAD in lumbar spine (0.117 ± 0.013 vs. 0.119 ± 0.012 g/cm(3), P = 0.06) was also found in these patients. Serum levels of 25OHD were significantly lower in JoSSc compared to controls (18.1 ± 6.4 vs. 25.1 ± 6.6 ng/mL, P = 0.04), and all patients had vitamin D insufficiency (<20 ng/mL) compared to 40% of controls (P = 0.01). All other biochemical parameters were within normal range and alike in both groups. BMAD in femoral neck and total femur was correlated with 25OHD levels in JoSSc (r = 0.82, P = 0.004; r = 0.707, P = 0.02; respectively). We have identified a remarkable high prevalence of 25OHD insufficiency in JoSSc. Its correlation with hip BMAD suggests a causal effect and reinforces the need to incorporate this hormone evaluation in this disease management.
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Densidad Ósea , Enfermedades Óseas Metabólicas/patología , Calcifediol/deficiencia , Esclerodermia Sistémica/patología , Deficiencia de Vitamina D/patología , Enfermedades Óseas Metabólicas/complicaciones , Enfermedades Óseas Metabólicas/metabolismo , Calcifediol/sangre , Salud de la Familia , Femenino , Cuello Femoral/diagnóstico por imagen , Cuello Femoral/metabolismo , Estado de Salud , Humanos , Masculino , Radiografía , Esclerodermia Sistémica/complicaciones , Esclerodermia Sistémica/metabolismo , Encuestas y Cuestionarios , Deficiencia de Vitamina D/complicaciones , Deficiencia de Vitamina D/metabolismo , Adulto JovenRESUMEN
BACKGROUND: The purpose of this study was to evaluate collagen deposition, mRNA collagen synthesis and TGF-beta expression in the lung tissue in an experimental model of scleroderma after collagen V-induced nasal tolerance. METHODS: Female New Zealand rabbits (N = 12) were immunized with 1 mg/ml of collagen V in Freund's adjuvant (IM). After 150 days, six immunized animals were tolerated by nasal administration of collagen V (25 microg/day) (IM-TOL) daily for 60 days. The collagen content was determined by morphometry, and mRNA expressions of types I, III and V collagen were determined by Real-time PCR. The TGF-beta expression was evaluated by immunostaining and quantified by point counting methods. To statistic analysis ANOVA with Bonferroni test were employed for multiple comparison when appropriate and the level of significance was determined to be p < 0.05. RESULTS: IM-TOL, when compared to IM, showed significant reduction in total collagen content around the vessels (0.371 +/- 0.118 vs. 0.874 +/- 0.282, p < 0.001), bronchioles (0.294 +/- 0.139 vs. 0.646 +/- 0.172, p < 0.001) and in the septal interstitium (0.027 +/- 0.014 vs. 0.067 +/- 0.039, p = 0.026). The lung tissue of IM-TOL, when compared to IM, showed decreased immunostaining of types I, III and V collagen, reduced mRNA expression of types I (0.10 +/- 0.07 vs. 1.0 +/- 0.528, p = 0.002) and V (1.12 +/- 0.42 vs. 4.74 +/- 2.25, p = 0.009) collagen, in addition to decreased TGF-beta expression (p < 0.0001). CONCLUSIONS: Collagen V-induced nasal tolerance in the experimental model of SSc regulated the pulmonary remodeling process, inhibiting collagen deposition and collagen I and V mRNA synthesis. Additionally, it decreased TGF-beta expression, suggesting a promising therapeutic option for scleroderma treatment.
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Colágeno Tipo V , Modelos Animales de Enfermedad , Pulmón/metabolismo , ARN Mensajero/metabolismo , Esclerodermia Sistémica/inducido químicamente , Esclerodermia Sistémica/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Administración Intranasal , Animales , Regulación hacia Abajo/efectos de los fármacos , Femenino , Expresión Génica/efectos de los fármacos , Humanos , ConejosRESUMEN
BACKGROUND: Collagen V shows promise as an inducer of interstitial lung fibrosis in experimental systemic sclerosis (SSc). MATERIALS AND METHODS: Remodelling of the pulmonary interstitium was evaluated based on the clinical data and open lung biopsies from 15 patients with SSc. Normal lung tissues obtained from eight individuals who died of traumatic injuries were used as control group. Immunofluorescence, immunohistochemistry, morphometry, tri-dimensional reconstruction and a real-time polymerase chain reaction were used to evaluate the quantity, structure and molecular chains of collagen V. The impact of these markers was tested on clinical data. RESULTS: The main difference in collagen V content between SSc patients and the control group was an increased, abnormal and distorted fibre deposition in the alveolar septa and the pre-acinar artery wall. The lungs from SSc patients presented [alpha1(V)] and [alpha2(V)] mRNA chain expression increased, but [alpha2(V)] was proportionally increased compared with the control group. High levels of collagen V were inversely associated with vital capacity (r = -0.72; P = 0.002), forced vital capacity (r = -0.76; P < 0.001), forced expiratory volume in 1-s (r = -0.89; P < 0.001) and diffusing capacity for carbon monoxide (r = -0.62; P = 0.04). CONCLUSIONS: Abnormal collagen V fibres are overproduced in lungs from SSc patients and may play an important role in the pathogenesis of the disease as this molecule regulates tissue collagen assembly. The aberrant histoarchitecture observed in SSc can be related to the overexpression of the [alpha2(V)] gene of unknown origin.
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Colágeno/metabolismo , Fibrosis Pulmonar/metabolismo , Esclerodermia Sistémica/complicaciones , Adulto , Biopsia , Colágeno/genética , Colágeno/ultraestructura , Femenino , Fluoroinmunoensayo , Humanos , Inmunohistoquímica , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa/métodos , Fibrosis Pulmonar/genética , Fibrosis Pulmonar/inmunología , Fibrosis Pulmonar/patología , ARN Mensajero/metabolismo , Esclerodermia Sistémica/metabolismo , Esclerodermia Sistémica/patologíaRESUMEN
OBJECTIVE: To determine HLA-G expression in skin biopsies from patients with systemic sclerosis (SSc), and its association with epidemiological, clinical, and laboratory variables and survival. METHODS: Paraffin-embedded skin biopsies obtained from 21 SSc patients (14 limited SSc, 7 diffuse SSc) and from 28 healthy controls were studied. HLA-G expression was evaluated by immunohistochemistry. RESULTS: HLA-G molecules were detected in 57% of skin biopsies from patients with SSc (9 from limited SSc, 3 from diffuse SSc), whereas no control sample expressed HLA-G (p=0.000004). In patients, HLA-G molecules were consistently observed within epidermal and some dermal cells. HLA-G expression was associated with a lower frequency of vascular cutaneous ulcers (p=0.0004), telangiectasias (p=0.008), and inflammatory polyarthralgia (p=0.02). After a 15-year followup, SSc patients who exhibited HLA-G survived longer than patients who did not. CONCLUSION: HLA-G is expressed in skin biopsies from patients with SSc, and this is associated with a better disease prognosis. This suggests a modulatory role of HLA-G in SSc, as observed in other skin disorders.
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Antígenos HLA/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Esclerodermia Sistémica/metabolismo , Piel/metabolismo , Biomarcadores/metabolismo , Brasil/epidemiología , Femenino , Antígenos HLA-G , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Pronóstico , Esclerodermia Sistémica/complicaciones , Esclerodermia Sistémica/mortalidad , Esclerodermia Sistémica/patología , Piel/irrigación sanguínea , Piel/patología , Úlcera Cutánea/etiología , Úlcera Cutánea/metabolismo , Úlcera Cutánea/patología , Tasa de Supervivencia , Telangiectasia/etiología , Telangiectasia/metabolismo , Telangiectasia/patologíaRESUMEN
Sinovitis in Scleroderma (SSc) is rare, usually aggressive and fully resembles rheumatoid arthritis. Experimental models of SSc have been used in an attempt to understand its pathogenesis. Previous studies done in our laboratory had already revealed the presence of a synovial remodeling process in rabbits immunized with collagen V. To validate the importance of collagen type V and to explore the quantitative relationship between this factor and synovia remodeling as well as the relationship between collagen type V and other collagens, we studied the synovial tissue in immunized rabbits. Rabbits (N=10) were immunized with collagen V plus Freund's adjuvant and compared with animals inoculated with adjuvant only (N=10). Synovial tissues were submitted to histological analysis, immunolocalization to collagen I, III and V and biochemical analysis by eletrophoresis, immunoblot and densitometric method. The synovial tissue presented an intense remodeling process with deposits of collagen types I, III and V after 75 and 120 days of immunization, mainly distributed around the vessels and interstitium of synovial extracellular matrix. Densitometric analysis confirmed the increased synthesis of collagen I, III and V chains (407.69+/-80.31; 24.46+/-2.58; 70.51+/-7.66, respectively) in immunized rabbits when compared with animals from control group (164.91+/-15.67; 12.89+/-1.05; 32+/-3.57) (p<0.0001). We conclude that synovial remodeling observed in the experimental model can reflect the articular compromise present in patients with scleroderma. Certainly, this experimental model induced by collagen V immunization will bring new insights in to pathogenic mechanisms and allow the testing of new therapeutic strategies to ameliorate the prognosis for scleroderma patients.
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Colágeno Tipo III/metabolismo , Colágeno Tipo I/metabolismo , Colágeno Tipo V/inmunología , Colágeno Tipo V/metabolismo , Inmunización , Membrana Sinovial/metabolismo , Animales , Anticuerpos Monoclonales/inmunología , Modelos Animales de Enfermedad , Matriz Extracelular/metabolismo , Matriz Extracelular/patología , Femenino , Conejos , Esclerodermia Sistémica/inmunología , Esclerodermia Sistémica/metabolismo , Membrana Sinovial/patologíaRESUMEN
OBJECTIVE: To evaluate the exercise capacity of women with systemic sclerosis (SSc) without pulmonary involvement using a cardiopulmonary stress test. METHODS: Thirteen consecutive female SSc patients [mean age 40.8+/-14 years, mean body mass index (BMI) 25.5+/-3.7 kg/m2] without pulmonary and cardiac involvement and 13 healthy sedentary female controls (mean age 41.6+/-9.1 years, mean BMI 23.7+/-3.8 kg/m2) matched by age and BMI underwent a maximum cardiopulmonary stress test (Bruce protocol). The following parameters were analysed: peak oxygen uptake (VO2peak), anaerobic threshold (AT), respiratory compensation point (RCP) and metabolic equivalent (MET) of the VO2peak. Comparisons between groups were analysed using the Student t-test. RESULTS: Forced vital capacity (FVC; 92.2+/-14.2% predicted) and carbon monoxide diffusion lung capacity (DL CO; 85.8+/-5.8% predicted) were within the normal range in SSc patients. VO2peak of SSc patients was significantly reduced in comparison to the control group (19.8+/-4.6 vs. 23.7+/-4.5 mL/kg/min, p = 0.04). SSc patients also had a significant reduction in MET at peak exercise (5.6+/-1.3 vs. 6.7+/-1.3 MET, p = 0.04) and a significant shorter time interval between AT and RCP compared to the control group (112.6+/-95.6 vs. 164.0+/-65.3 s, p = 0.03). CONCLUSION: SSc patients without pulmonary impairment have reduced exercise capacity. Abnormal vascular response to exercise may account for this finding, as the vascular system is one of the major target organs in this pathological condition.